These authors contributed equally to this work.

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#### **Chapter 3 Provisional chapter**

## *Salmonella enterica***: Latency** *Salmonella enterica***: Latency**

#### Bianca Mendes Maciel, Rachel Passos Rezende and Nammalwar Sriranganathan Bianca Mendes Maciel, Rachel Passos Rezende and Nammalwar Sriranganathan

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67173

#### **Abstract**

Infection caused by more than 1500 serotypes of *Salmonella enterica* subsp. *enterica* is one of the most common food-borne diseases, prevalent worldwide. Concerning public health, *Salmonella* latent carrier animals represent an important source of transmission of the disease. They are responsible for silent introduction of the bacteria into the food chain and the environment. Most pathogenesis studies of salmonellosis are focused on events that lead to clinical disease. Researchers have been unable to clearly discern the interaction between intracellular microorganisms and their resistant hosts in latency. However, understanding this interaction is essential for the proper employment of the control and eradication strategies. Thus, the objective of this article is to present an overview of some important events that occur during the infection cycle of *S. enterica* in latent carriers.

**Keywords:** *Salmonella* asymptomatic carrier animals, pathogen-host interaction, pathogenisis, public health, intracellular bacteria

#### **1. Introduction**

The genus *Salmonella* belongs to family Enterobacteriaceae, and its classification follows the Kauffmann-White scheme, which groups serotypes according to their somatic, flagellar and capsular antigens. Serotyping is essential for investigation of outbreaks of salmonellosis, contributing to epidemiological surveillance. Currently, the genus consists of two species, *S. enterica* and *S. bongori*, the first being subdivided into six subspecies, which are designed by Roman numeral, containing more than 2500 antigenically distinct serotypes. Of these serotypes, around 1500 belong to *Salmonella enterica* subspecies *enterica (I)*, which colonizes the intestinal tract of warm-blooded animals and is responsible for 99% of *Salmonella* infections, while the others pertain to other subspecies: *salamae (II), arizonae (IIIa), diarizonae (IIIb),* 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*houtenae (IV)* and *indica (VI)*. Although *S. bongori* has been determined to be a separate species, it was originally designated as subspecies V, which is commonly found in cold-blooded animals and in the environment [1–3]. After serotyping by Kauffman-White scheme, characterization by pulsed-field gel electrophoresis (PFGE) pattern and phage typing provides further subtyping [3]. Eventually complete genome sequencing will be the norm as the cost of such analysis has come down basically replacing multiple-locus variable-number tandem repeat analysis (MLVA) [4].

Most outbreaks of salmonellosis in humans and in domestic animals are caused by a few serotypes, which are grouped according to their adaptation to the host. The first group consists of a few *host-specific serotypes*, which typically cause systemic disease in a single animal species or a limited number of phylogenetically-related species. Noteworthy examples are *S. enterica* serotype Typhi and Paratyphi of humans, serotypes Pullorum and Gallinarum of birds and serotype Abortusovis of sheep. The second group consists of *host-adapted serotypes* that are associated with one or two animal species that are related to each other; however, they may occasionally cause disease in other hosts. Noteworthy examples are *S. enterica* serotype Dublin and serotype Choleraesuis, which are usually associated with severe systemic disease in ruminants and pigs, respectively. Finally, the third group consists of a large numbers of *ubiquitous serotypes*, which typically cause gastroenteritis in a wide variety of unrelated host species;among these are *S. enterica* serotype Typhimurium and serotype Enteritidis [5], and these are the two most prevalent serotypes in the world [6].

Epidemiologically, infections caused by *Salmonella enterica* subsp. *enterica* correspond to the most prevalent disease transmitted via food worldwide. This high prevalence is associated with the absence of clinical disease in animals that often silently infect herds, contaminate food, the environment and thus cause disease in humans. However, historically, studies on the pathogenesis of salmonellosis are focused on events leading to clinical manifestations, and a few studies are conducted to clarify the interaction between latent microorganisms and their resistant hosts.

Certain animal species may develop asymptomatic persistent infection with intermittent shedding of *Salmonella* in their feces over long periods. These animals are called *latent carriers*. Their impact on public health is that the carriers are natural reservoirs of different *Salmonella* serotypes and may be resistance to multiple antimicrobials. Latent *Salmonella* infections can occur in humans [7], in farm animals such as cattle, sheep, pigs and poultry [5], in pets such as dogs [8] and in wild animals such as reptiles [9, 10].

Latent carrier animals are therefore natural reservoirs of *Salmonella* and are responsible for the silent intermittent introduction of the pathogen into the food chain and the environment, hindering control strategies. Thus, increasing our knowledge regarding the interaction of intracellular pathogen *Salmonella* with their host is essential for the development of an efficient strategy for control. In this mini-review, we present some important events that occur during the infection cycle of *S. enterica* leading to latent carriers, including the mechanisms of invasion of the host cells, bacterial multiplication and persistence in intracellular compartments, and intermittent shedding of the pathogen in the feces.

## **2. Pathogenesis of** *Salmonella enterica***: the role of** *Salmonella* **pathogenicity islands (SPIs)**

*houtenae (IV)* and *indica (VI)*. Although *S. bongori* has been determined to be a separate species, it was originally designated as subspecies V, which is commonly found in cold-blooded animals and in the environment [1–3]. After serotyping by Kauffman-White scheme, characterization by pulsed-field gel electrophoresis (PFGE) pattern and phage typing provides further subtyping [3]. Eventually complete genome sequencing will be the norm as the cost of such analysis has come down basically replacing multiple-locus variable-number tandem repeat

Most outbreaks of salmonellosis in humans and in domestic animals are caused by a few serotypes, which are grouped according to their adaptation to the host. The first group consists of a few *host-specific serotypes*, which typically cause systemic disease in a single animal species or a limited number of phylogenetically-related species. Noteworthy examples are *S. enterica* serotype Typhi and Paratyphi of humans, serotypes Pullorum and Gallinarum of birds and serotype Abortusovis of sheep. The second group consists of *host-adapted serotypes* that are associated with one or two animal species that are related to each other; however, they may occasionally cause disease in other hosts. Noteworthy examples are *S. enterica* serotype Dublin and serotype Choleraesuis, which are usually associated with severe systemic disease in ruminants and pigs, respectively. Finally, the third group consists of a large numbers of *ubiquitous serotypes*, which typically cause gastroenteritis in a wide variety of unrelated host species;among these are *S. enterica* serotype Typhimurium and serotype Enteritidis [5],

Epidemiologically, infections caused by *Salmonella enterica* subsp. *enterica* correspond to the most prevalent disease transmitted via food worldwide. This high prevalence is associated with the absence of clinical disease in animals that often silently infect herds, contaminate food, the environment and thus cause disease in humans. However, historically, studies on the pathogenesis of salmonellosis are focused on events leading to clinical manifestations, and a few studies are conducted to clarify the interaction between latent microorganisms and

Certain animal species may develop asymptomatic persistent infection with intermittent shedding of *Salmonella* in their feces over long periods. These animals are called *latent carriers*. Their impact on public health is that the carriers are natural reservoirs of different *Salmonella* serotypes and may be resistance to multiple antimicrobials. Latent *Salmonella* infections can occur in humans [7], in farm animals such as cattle, sheep, pigs and poultry [5], in pets such

Latent carrier animals are therefore natural reservoirs of *Salmonella* and are responsible for the silent intermittent introduction of the pathogen into the food chain and the environment, hindering control strategies. Thus, increasing our knowledge regarding the interaction of intracellular pathogen *Salmonella* with their host is essential for the development of an efficient strategy for control. In this mini-review, we present some important events that occur during the infection cycle of *S. enterica* leading to latent carriers, including the mechanisms of invasion of the host cells, bacterial multiplication and persistence in intracellular compartments,

and these are the two most prevalent serotypes in the world [6].

as dogs [8] and in wild animals such as reptiles [9, 10].

and intermittent shedding of the pathogen in the feces.

analysis (MLVA) [4].

42 Current Topics in Salmonella and Salmonellosis

their resistant hosts.

The pathogenesis of salmonellosis depends on a combination of several factors, including the components of bacterial virulence, the infective dose, route of infection, the genetic makeup and the immune status of the host [11]. All of these variables can influence the immunological responses of the host, resulting in different degrees of inflammation that confer an acute, moderate, chronic or even asymptomatic nature to the disease [12].

Infection by *S. enterica* has the following characteristics: the ability to interact with enterocytes leading to diarrhea (*Salmonella*-induced enteritis), the invasion of non-phagocytic cells and the ability to survive and proliferate within the phagocytes, resulting in systemic disease [13]. These characteristics are determined by multiple virulence factors encoded in *Salmonella* pathogenicity islands (SPIs) comprising large and unstable segments of the bacterial genome of pathogenic organisms. These SPIs are absent in related non-pathogenic organisms and that were acquired by horizontal gene transfer as SPIs G + C content is lower than *Salmonella* genes [14]. SPIs are conserved in several strains; differences may have implications in host specificity [15]. Currently, 16 pathogenicity island of *Salmonella* encoding distinct virulence factors are described, according to pathogenicity island database, PAI DB (http://www.paidb.re.kr), with different distributions among the various *Salmonella* species, subspecies and serotypes. SPI-1 and SPI-2 (both are about 40 kb in length) are the most studied and are present in all subspecies of *S. enterica* [13, 14, 16]. SPI-1 contains the genes responsible for the bacterial invasion of the host epithelium [17, 18], whereas SPI-2 is responsible for bacterial survival and multiplication within eukaryotic cells, including macrophages [19, 20].

Studies of SPIs help in understanding the mechanisms of bacterial virulence, and they may also be useful to clarify the phylogenetic relationships among species [21, 22]. Phylogenetic studies indicated that the gene sequences present in SPI-1 were acquired by lateral gene transfer before the diversification between *S. enterica* and *S. bongori*. In turn, the acquisition of the SPI-2 genes present in *S. enterica* occurred after speciation but before the diversification of the groups (I, II, IIIa, IIIb, IV, VI and VII); therefore, SPI-2 is present in all *S. enterica* subspecies but is absent in *S. bongori* species [22].

The virulence mechanisms of *Salmonella* serotypes are studied in different animal models, depending on the type of clinical manifestation. To study the pathogenesis of typhoid fever (a systemic disease), strains of susceptible mice (*e.g*., Balb/c) experimentally infected with serotype Typhimurium are used. However, in this experimental model, the mice do not develop diarrhea, and therefore, mice are not used to study the pathogenesis of enteritis. In contrast, the experimental infection of calves with the same serotype results in enteric disease, and therefore, this experimental model is used to study *Salmonella*-induced enteritis [23].

According to the animal model, the virulence genes required for systemic infection differ from those genes responsible for the enteritis caused by *Salmonella*. This result is observed by analyzing mutant phenotypes of serotype Typhimurium in experimental infection of mice and calves, which are used to study systemic and enteric infections, respectively. Mutations in SPI-2 result in a significant attenuation of systemic disease in mice, while in calves, the severity of intestinal lesions shows only modest attenuation. In contrast, mutations that prevent the expression of the SPI-1 type III secretion system (T3SS) or of effector proteins translocated by the system result in an avirulent strain with consequent the absence of diarrhea in calves [23].

#### **2.1. SPI-1-mediated invasion of host cells**

After oral infection, a proportion of the *Salmonella* organisms survives the low stomach pH and reaches the distal ileum and cecum, where they invade the epithelial cells and M cells, mediated by a T3SS encoded by the SPI-1 [24, 25]. The T3SS allows some of the enteropathogenic bacteria to adhere to the epithelial surface and inject effector proteins that cross the membrane of the host cells, causing cellular injury [26]. Through this system, *Salmonella* translocates effector proteins encoded by genes present in the SPI-1 as well as genes in independent *loci* of the SPI-1 that promote a chain of events in the host cell to allow pathogen invasion [13]. Another function of the SPI-1 is related to hydroelectrolyte imbalance caused by the effector protein SopB, which stimulates the secretion of chloride ions (Cl− ) through its inositol phosphatase activity, thereby leading to loss of fluid into the intestinal lumen [27] (**Figure 1**).

Once in contact with the intestinal epithelium, the effector proteins SopE, SopE2 and SopB (encoded by genes outside of SPI-1) are translocated to the interiors of enterocytes and M cells via the SPI-1 T3SS. These proteins activate certain GTPases within the host cell, such as Cdc42, Rac-1 and Rho, causing a rearrangement of the actin cytoskeleton called membrane ruffling [28], which is stabilized by the SipA and SipC effector proteins. Furthermore, they also activate the MAP kinase (mitogen-activated protein kinase) pathway, thereby destabilizing tight junctions. Consequently, bacteria can penetrate into the host cell through the apical membrane in a process called macropinocytosis or cross the intercellular space until reaching the lamina propria. This destabilization of tight junctions also allows for the transmigration of polymorphonuclear cells (PMNs) from the basolateral space to the apical surface. However, this transmigration can occur independently from the destabilization of tight junctions when mediated by the bacterial protein SopA [29]. Once inside the cell, the effector protein SptP modulates the inactivation of the GTPases Cdc42 and Rac-1, thus resulting in the end of the membrane ruffling [30].

Signaling via MAP kinase, in addition to promoting the destabilization of tight junctions, also activates the transcription factors AP-1 (activator protein-1) and NF-κB (nuclear factor- κB), which leads to the synthesis of pro-inflammatory interleukin (IL)-8 by PMN leukocytes, thus acting as a chemotactic factor for neutrophils [29].

During the invasion of macrophages, the bacterium injects the effector protein SipB, which is encoded by SPI-1, inducing the intracellular activation of caspase-1 by resident macrophages. Caspase-1 induces apoptosis of infected macrophages resulting in *Salmonella* escape from these cells. Caspase-1 also cleaves the pro-inflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines that further enhance the local inflammatory response, causing infiltration by PMN phagocytes and internalization of the bacterium by these cells [31, 32]. The intracellular medium provides a favorable environment for the bacteria to multiply [33], and once the invasion process is concluded, the bacteria are transported from the gastrointestinal tract to systemic organs.

**Figure 1.** Effector proteins (gray arrows) ejected by type III secretion system encoded in SPI-1 and their actions for *Salmonella* invasion of host cells. *Salmonella* penetrates at the apical space causing the membrane ruffling. It is mediated by SopE, SopE2 and SopB proteins, which promote activation of host GTPases, causing a rearrangement of the actin cytoskeleton that is stabilized by SipA and SipC proteins. *Salmonella* can also cross the basolateral space through destabilization of the tight junctions, also mediated by SopE, SopE2 and SopB proteins (by activation of MAP kinase pathway) and by SopA protein. These events contribute to the activation of chemotactic factors of neutrophils. Once inside the cell, *Salmonella* promotes the end of the membrane ruffling by inactivation of host GTPases by SptP protein. During the invasion of resident macrophages, SipB protein induces the intracellular activation of caspase-1, causing apoptosis and enhancing the local inflammatory response. This event contributes to the escape of *Salmonella* from the macrophages and internalization of the bacteria in PMN phagocytes. The hydroelectrolyte imbalance is caused by SopB protein through inositol phosphatase activity which stimulates the secretion of chloride ions (Cl− ).

There is an alternative SPI-1-independent invasion mechanism in which *S. enterica* does not interact with M cells but is engulfed by dendritic cells that open the tight junctions between epithelial cells, thereby carrying the bacteria to systemic organs [34].

#### **2.2. SPI-2-mediated intracellular multiplication**

SPI-2 result in a significant attenuation of systemic disease in mice, while in calves, the severity of intestinal lesions shows only modest attenuation. In contrast, mutations that prevent the expression of the SPI-1 type III secretion system (T3SS) or of effector proteins translocated by the system result in an avirulent strain with consequent the absence of diarrhea in calves [23].

After oral infection, a proportion of the *Salmonella* organisms survives the low stomach pH and reaches the distal ileum and cecum, where they invade the epithelial cells and M cells, mediated by a T3SS encoded by the SPI-1 [24, 25]. The T3SS allows some of the enteropathogenic bacteria to adhere to the epithelial surface and inject effector proteins that cross the membrane of the host cells, causing cellular injury [26]. Through this system, *Salmonella* translocates effector proteins encoded by genes present in the SPI-1 as well as genes in independent *loci* of the SPI-1 that promote a chain of events in the host cell to allow pathogen invasion [13]. Another function of the SPI-1 is related to hydroelectrolyte imbalance caused by the effector

phatase activity, thereby leading to loss of fluid into the intestinal lumen [27] (**Figure 1**).

the GTPases Cdc42 and Rac-1, thus resulting in the end of the membrane ruffling [30].

Signaling via MAP kinase, in addition to promoting the destabilization of tight junctions, also activates the transcription factors AP-1 (activator protein-1) and NF-κB (nuclear factor- κB), which leads to the synthesis of pro-inflammatory interleukin (IL)-8 by PMN leukocytes, thus

During the invasion of macrophages, the bacterium injects the effector protein SipB, which is encoded by SPI-1, inducing the intracellular activation of caspase-1 by resident macrophages. Caspase-1 induces apoptosis of infected macrophages resulting in *Salmonella* escape from these cells. Caspase-1 also cleaves the pro-inflammatory cytokines IL-1β and IL-18 to produce bioactive cytokines that further enhance the local inflammatory response, causing infiltration by PMN phagocytes and internalization of the bacterium by these cells [31, 32]. The intracellular medium provides a favorable environment for the bacteria to multiply [33], and once the invasion process is concluded, the bacteria are transported from the gastrointestinal tract

Once in contact with the intestinal epithelium, the effector proteins SopE, SopE2 and SopB (encoded by genes outside of SPI-1) are translocated to the interiors of enterocytes and M cells via the SPI-1 T3SS. These proteins activate certain GTPases within the host cell, such as Cdc42, Rac-1 and Rho, causing a rearrangement of the actin cytoskeleton called membrane ruffling [28], which is stabilized by the SipA and SipC effector proteins. Furthermore, they also activate the MAP kinase (mitogen-activated protein kinase) pathway, thereby destabilizing tight junctions. Consequently, bacteria can penetrate into the host cell through the apical membrane in a process called macropinocytosis or cross the intercellular space until reaching the lamina propria. This destabilization of tight junctions also allows for the transmigration of polymorphonuclear cells (PMNs) from the basolateral space to the apical surface. However, this transmigration can occur independently from the destabilization of tight junctions when mediated by the bacterial protein SopA [29]. Once inside the cell, the effector protein SptP modulates the inactivation of

) through its inositol phos-

**2.1. SPI-1-mediated invasion of host cells**

44 Current Topics in Salmonella and Salmonellosis

protein SopB, which stimulates the secretion of chloride ions (Cl−

acting as a chemotactic factor for neutrophils [29].

to systemic organs.

The ability of *S. enterica* to survive inside phagocytes and to replicate in *Salmonella*-containing vacuoles (SCV) in a variety of eukaryotic cells is dependent on another T3SS that is encoded by SPI-2 [22, 35, 36]. This characteristic can lead to systemic infection [20].

Soon after entry by means of macropinocytosis, *Salmonella* is internalized into a phagosome formed by the membrane ruffling that later fuses with lysosomes, thereby originating the SCV [29]. Inside of the SCV, the T3SS encoded by SPI-2 is activated using luminal acid pH, translocating the effector proteins across the phagosome membrane (**Figure 2**). The effector protein SipC prevents the fusion of the SCV with vesicles containing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and inducible nitric oxide synthase (iNOS), hindering the action of reactive oxygen intermediates (ROS) and reactive nitrogen intermediates (RNS) [13]. The effector proteins SifA and PipB2 contribute to the formation of *Salmonella*-induced filaments (SIF) along microtubules, while the effector proteins SseF and SseG aggregate the SCV-adjacent microtubules. In addition, an accumulation of actin occurs around the SCV that is mediated by the SspH2, SpvB and SseI proteins. These events contribute to the maturation and stabilization of SCV [29]. As a consequence, *S. enterica* becomes even more protected against RNS and ROS and against the potent antimicrobial activity of peroxynitrite, which is generated by the RNS and ROS reactions. These mechanisms represent a specific adaptation of *S. enterica* to the intracellular environment, especially phagocytes. Thus, the bacteria can multiply inside the phagocytic cells, transported via circulation and cause systemic infection [14].

**Figure 2.** Effector proteins (gray circles) ejected by type III secretion system encoded in SPI-2 and their actions for *Salmonella* survival inside of phagocytes and its replication in *Salmonella*-containing vacuoles (SCV). The translocation of SipC protein avoids the antimicrobial activities of reactive oxygen intermediates and reactive nitrogen intermediates by prevention of fusion of NADPH oxidase and iNOS vesicles. This antimicrobial activity by the host cell is stronger but prevented by the accumulation of actin around the SCV promoted by SspH2, SpvB and Ssel proteins. These events contribute to maturation of SCV. SifA and PipA proteins contribute to the tubular structures known as *Salmoniella*induced filaments formed along the microtubule motors; in addition, SseF and SseG cause microtubules aggregation adjacent to SCV. These events interfere the molecular motors that drive the cellular trafficking, which transport vesicles and organelles within the cell.

## **3. Natural resistance mechanism to infection by** *S. enterica***: the role of Nramp1 glycoprotein**

[29]. Inside of the SCV, the T3SS encoded by SPI-2 is activated using luminal acid pH, translocating the effector proteins across the phagosome membrane (**Figure 2**). The effector protein SipC prevents the fusion of the SCV with vesicles containing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and inducible nitric oxide synthase (iNOS), hindering the action of reactive oxygen intermediates (ROS) and reactive nitrogen intermediates (RNS) [13]. The effector proteins SifA and PipB2 contribute to the formation of *Salmonella*-induced filaments (SIF) along microtubules, while the effector proteins SseF and SseG aggregate the SCV-adjacent microtubules. In addition, an accumulation of actin occurs around the SCV that is mediated by the SspH2, SpvB and SseI proteins. These events contribute to the maturation and stabilization of SCV [29]. As a consequence, *S. enterica* becomes even more protected against RNS and ROS and against the potent antimicrobial activity of peroxynitrite, which is generated by the RNS and ROS reactions. These mechanisms represent a specific adaptation of *S. enterica* to the intracellular environment, especially phagocytes. Thus, the bacteria can multiply inside the phagocytic cells, transported via circulation and cause systemic infection [14].

46 Current Topics in Salmonella and Salmonellosis

**Figure 2.** Effector proteins (gray circles) ejected by type III secretion system encoded in SPI-2 and their actions for *Salmonella* survival inside of phagocytes and its replication in *Salmonella*-containing vacuoles (SCV). The translocation of SipC protein avoids the antimicrobial activities of reactive oxygen intermediates and reactive nitrogen intermediates by prevention of fusion of NADPH oxidase and iNOS vesicles. This antimicrobial activity by the host cell is stronger but prevented by the accumulation of actin around the SCV promoted by SspH2, SpvB and Ssel proteins. These events contribute to maturation of SCV. SifA and PipA proteins contribute to the tubular structures known as *Salmoniella*induced filaments formed along the microtubule motors; in addition, SseF and SseG cause microtubules aggregation adjacent to SCV. These events interfere the molecular motors that drive the cellular trafficking, which transport vesicles

and organelles within the cell.

The resistance mechanisms of host to infection by *S. enterica* are multigenic. Studies in mice have emphasized the *locus* encoding glycoprotein natural resistance-associated macrophage protein-1 (Nramp1), which has been considered the key for the innate host response to intracellular pathogens [37]. This protein belongs to a family of proteins highly conserved in evolution, with homology among mammals, insects and bacteria suggesting an important role in all living organisms [38].

Nramp1 is a transmembrane glycoprotein and divalent metal ion symporter that deprives intracellular pathogens of these metals by removing mainly Fe++ and Mn++ from the luminal space of the phagosomal and lysosomal vesicles. Because iron and other divalent cations are cofactors for vital enzymes, *S. enterica* expresses a series of carriers that compete with the host cell for traces of these divalent metals within the phagosomes [39]. This Nramp1 glycoprotein is encoded by the gene *Slc11a1* (*Solute carrier family 11 member 1*, first named as *Ity* gene), on chromosome 1 in mice [37]. A single substitution of glycine for aspartate at position 169 results in susceptibility to systemic infection by *S. enterica* in the mice [40]. Consequently, mice that have two *Slc11a1 Asp 169* alleles are significantly more susceptible to lethal *Salmonella* infections and are therefore being used in studies to clarify the host-pathogen relationships in acute systemic infection. In turn, mice carrying the wild-type *locus Slc11a1+/+* can be used to study the pathogenesis of chronic infections that are often asymptomatic [41].

The interaction between the surface receptors of macrophages and microbial ligands results in the internalization of the microorganism into a phagosome. However, this young phagosome is not able to digest its contents, thus requiring a maturation process involving fusion and fission events with endosomes and lysosomes. During the maturation process, phagosomes containing *S. enterica* acquire vacuolar ATPases that acidify the phagosome lumen. In an acidic pH, Nramp1 removes Fe++ and other divalent cations from the inside of phagosomes. Concomitantly, in the presence of functional protein Nramp1, the host cell expresses the mannose-6-phosphate receptor (M6PR), which is responsible for interacting with vesicles containing NADPH oxidase and iNOS. This interaction generates positive feedback for the transcription of high levels of iNOS mRNA [39]. In susceptible mice (*Slc11a1Asp 169*), the phagosomes containing *S. enterica* are negative for M6PR receptors, and therefore, the production of iNOS is lower than in hosts that have the wild-type *locus Slc11a1+/+* [36]. Thus, Nramp1 has proven to be very important to control the exponential growth of *Salmonella* during the early stages of systemic infection [23, 42].

## **4. Infection cycle of** *S. enterica* **in latent carriers**

In asymptomatic carrier animals, the study of the infection cycle of *Salmonella* was described using C57Bl/6-Bcgr (*Slc11a1+/+*) mice as a resistant mouse model inoculated orally with a high dose of *Salmonella* serotype Enteritidis [43]. The animals developed an intermittent infection cycle in the gastrointestinal tract during 4 weeks of study, with interspersed periods of intra- and extracellular spread of the infection, which featured three distinct stages over the course of the cycle (**Figure 3**): *(I) the initial stage* represented by intracellular invasion and bacterial multiplication in the intestine, inducing transient damage to the intestinal mucosa and shedding of the pathogen in the feces. A rapid clearance of a large fraction of the inoculums was observed during the first 48 h postinoculation (PI); (*II) the intermediate stage*, the initial period of bacterial sequestration by the mononuclear phagocyte system (MPS) in which the pathogen was detected only within intracellular compartments. In this period, a transient exponential growth of the remaining intracellular bacteria occurred 2–4 d PI followed by a suppression of bacterial growth, establishing a plateau phase until 15 d PI. The intracellular multiplication in the MPS coincided with the IFNγ production; and finally (*III) the intermittent shedding stage*, the *Salmonella* persists sub-clinically in the tissues (spleen and cecum) with recurrence of intracellular bacterial growth that coincided with the intermittent excretion in feces, characterizing a latent infection.

**Figure 3.** Distribution of *S. enteritidis* in feces (fecal and ileo-cecal content) and tissues (blood, spleen, liver, mesenteric lymph nodes and different parts of the intestine—jejunum, ileum and cecum) at different times after intragastric inoculation with 5 × 108 cfu in C57Bl/6-Bcgr (Slc11a1+/+) mice as a resistant mouse model. These numbers are represented as mean ± SD of three animals (in duplicate). (I) Initial stage of infection, when *Salmonella* invades the intestinal mucosa and it is also eliminated in feces. (II) Intermediate stage marks the initial period of mononuclear phagocyte system (MPS) sequestration. *Salmonella* is found intracellular in the intestine but it is not being eliminated to the environment through feces. (III) Intermittent elimination stage of *Salmonella*, common in a resistant animal model, based on [43].

In pigs, by applying a Markov statistical model, Ivanek et al. [44] were able to distinguish different stages during the dynamic shedding of *Salmonella* in feces and their immune response. In this model, the intermittent shedding of the pathogen was clear. The authors characterized the following stages: (i) *latency*, when pigs were negative for the shedding of *Salmonella* immediately after the challenge; (ii) *continuous shedding*, with continuous shedding of the pathogen in the feces; (iii) *non-intermittent shedding*—when *Salmonella* was not being shed in the feces; (iv) *intermittent shedding*—when the bacteria were again shed in the feces; and (v) *recovery*. The authors observed that the stages could vary depending on the infecting dose and the serotype involved in the infection.

intra- and extracellular spread of the infection, which featured three distinct stages over the course of the cycle (**Figure 3**): *(I) the initial stage* represented by intracellular invasion and bacterial multiplication in the intestine, inducing transient damage to the intestinal mucosa and shedding of the pathogen in the feces. A rapid clearance of a large fraction of the inoculums was observed during the first 48 h postinoculation (PI); (*II) the intermediate stage*, the initial period of bacterial sequestration by the mononuclear phagocyte system (MPS) in which the pathogen was detected only within intracellular compartments. In this period, a transient exponential growth of the remaining intracellular bacteria occurred 2–4 d PI followed by a suppression of bacterial growth, establishing a plateau phase until 15 d PI. The intracellular multiplication in the MPS coincided with the IFNγ production; and finally (*III) the intermittent shedding stage*, the *Salmonella* persists sub-clinically in the tissues (spleen and cecum) with recurrence of intracellular bacterial growth that coincided with the intermittent excretion in

**Figure 3.** Distribution of *S. enteritidis* in feces (fecal and ileo-cecal content) and tissues (blood, spleen, liver, mesenteric lymph nodes and different parts of the intestine—jejunum, ileum and cecum) at different times after intragastric inoculation with 5 × 108 cfu in C57Bl/6-Bcgr (Slc11a1+/+) mice as a resistant mouse model. These numbers are represented as mean ± SD of three animals (in duplicate). (I) Initial stage of infection, when *Salmonella* invades the intestinal mucosa and it is also eliminated in feces. (II) Intermediate stage marks the initial period of mononuclear phagocyte system (MPS) sequestration. *Salmonella* is found intracellular in the intestine but it is not being eliminated to the environment through

feces. (III) Intermittent elimination stage of *Salmonella*, common in a resistant animal model, based on [43].

feces, characterizing a latent infection.

48 Current Topics in Salmonella and Salmonellosis

Thus, independent of the animal model, in latent carriers, there is a period during which *Salmonella* stays hidden in an intracellular compartment, and it is not being eliminated. It can mask the diagnosis of the positive animals. This "*Salmonella'*s hiding-place" may function as a strategic site of bacteria multiplication and, consequently, elimination of high numbers of pathogens in the environment. So, it is very important to identify the sites of bacterial colonization in different latent carriers.

The site of bacterial colonization in persistent infections varies according to serotype and host species. In humans, serotype Typhi expresses proteins encoded by SPI-7 that inhibit the detection of pathogens by the innate immune system of the host. Thus, the bacteria can spread systemically, colonizing macrophages in the liver, spleen and bone marrow. In the liver, *Salmonella* serotype Typhi can be found latent in the gallbladder, making the host an asymptomatic carrier. Intermittently, the bacteria are transported from the gallbladder into the small intestine through the bile and excreted in the feces [7]. In mice, the mesenteric lymph nodes are the colonization site of serotype Typhimurium [41]. In birds, *Salmonella* serotype Pullorum can be found latent in the spleen, ovary and oviduct of chickens [45], and *S*. Enteritidis can infect the ovaries of healthy hens, contaminating the eggs prior to shell formation [46]. In snakes, there is strong evidence that different serotypes of *Salmonella* also colonize the ovary, spreading bacteria to their offspring vertically [47].

In asymptomatic animals, the cecum plays an important role as a reservoir for longer periods of shedding [48–51]. Research using resistant mice orally challenged with high doses of *Salmonella* serotype Enteritidis [43], and we demonstrate that bacteria reach the cecum in the early stages of infection (12 h to 2 days PI) and remain for long periods from 5 days PI, functioning as a reservoir of bacterial multiplication, causing the shedding of *Salmonella* in the intestinal lumen intermittently. The small intestine does not have this reservoir role, since the bacterial colonization in jejune and ileum occurred only in 1–4 days PI. Spleen is another site of *Salmonella* reservoir; from the moment that bacteria reached the MPS, they stayed in spleen for long periods (**Figure 4**).

In chickens, the cecum is also a site for long-lasting carriage of *S*. Enteritidis, both in susceptible and resistant animals [52]. In asymptomatic carriers, it represents a public health and food protection concerns because the cecum may function as a "strategic site" of *Salmonella* proliferation, releasing bacteria to the environment intermittently.

**Figure 4.** Course of *S. enteritidis* in C57Bl/6-Bcgr (Slc11a1+/+), a resistant mouse model. *Salmonella* rapidly reaches the cecum in the early stage of the infection between 12 and 48 h postinoculation (PI) and remains in this organ as an important reservoir for 5 days PI, with increasing bacteria multiplication. The presence of bacteria in the cecum seems to be associated with its extracellular multiplication in the intestinal content and intermittent shedding in the feces. The colonization of the small intestine occurs during the first 4 days PI. In this period, *Salmonella* penetrates the intestinal mucosa, causing different degrees of degeneration of the microvilli, which is reversible (membrane ruffling). This mechanism is mediated by effector proteins translocated by T3SS encoded in SPI-1. Intracellular multiplication of the bacteria in mononuclear phagocyte system (MPS) occurs from 3 days PI. The exact route of *Salmonella* dissemination from intestine to MPS is unclear, but from the moment that bacteria reach the MPS, they remain in spleen, causing splenomegaly by 10 days PI. The intracellular multiplication in MPS coincides with the production of IFNγ, which restricts the replication of intracellular *Salmonella*.

The mechanism of persistence of *Salmonella* in the cecum is not well established. Probably, it is associated with the physiological environment and less peristalsis of this part of the intestine. Upon entry into the large intestine, the bacteria remain longer in the cecum due to fewer peristaltic movements. Despite the production of short-chain fatty acids by resident microbiota due to the intense local fermentation, the pH in the cecal environment remains above 6.3, higher than the inhibitory level for *Salmonella* multiplication [53].

#### **5. Role of IFNγ in controlling of** *S. enterica* **growth**

During intestinal infection, *Salmonella*-host interactions result in different degrees of inflammation related to the levels of cytokines produced [12], which may trigger changes in the composition of the intestinal microbiota. A reduction in symbionts or an increase in pathobionts is usually observed during inflammatory processes, reflecting the diversity of the intestinal microbiota [54]. In gastroenteritis caused by *Salmonella* in susceptible hosts, the production of interferon gamma (IFNγ) in the early stage of intestinal inflammation may alter the lumen conditions, causing an imbalance in the ecology of the resident microbiota that favors competition for pathogen growth and intestinal colonization [55–57]. In latent carriers, however, *S. enterica* can invade the intestinal mucosa and colonize the intestine without triggering a strong immune response, remaining in equilibrium with the resident microbiota [58].

IFNγ plays a crucial role in resistance to systemic infection by *S. enterica*. This cytokine controls the growth of pathogens both in the initial [59, 60] and late stages of the disease [41], and its absence results in septicemia. High levels of IFNγ as well as of its mediator IL-12 contribute to resistance to infection in different animal species [61]. Mice with chronic asymptomatic infection by *Salmonella* serotype Typhimurium develop symptoms after treatment with anti-IFNγ antibodies [41]. In birds, the IFNγ gene expression is lower in susceptible animals than in resistant animals [61].

IFNγ is produced specifically in response to systemic infection and correlates with bacteremia and pathogen invasion of the cells of the mononuclear phagocyte system, such as the lymphoid tissue associated with the intestine (mesenteric lymph nodes and Peyer's patches), spleen and liver. Its production is essential to restrict bacterial intracellular multiplication, thereby contributing to the establishment of a plateau phase during the growth cycle of *Salmonella* serotype Enteritidis in asymptomatic mice [43].

When antigen-specific acquired immunity is triggered, the IFNγ titer in serum begins to decrease [11]. However, even in the presence of high titers of specific circulating antibodies, some *Salmonella* serotypes are capable of causing persistent infections in a host for long periods. This adaptive immune response seems to be important to reduce the number of extracellular bacteria; however, bacteria that are present within macrophages survive both the innate and adaptive immune responses, and the host ultimately becomes a latent carrier [41].

## **6. Gene expression in latent** *Salmonella*

The mechanism of persistence of *Salmonella* in the cecum is not well established. Probably, it is associated with the physiological environment and less peristalsis of this part of the intestine. Upon entry into the large intestine, the bacteria remain longer in the cecum due to fewer peristaltic movements. Despite the production of short-chain fatty acids by resident microbiota due to the intense local fermentation, the pH in the cecal environment remains above 6.3,

**Figure 4.** Course of *S. enteritidis* in C57Bl/6-Bcgr (Slc11a1+/+), a resistant mouse model. *Salmonella* rapidly reaches the cecum in the early stage of the infection between 12 and 48 h postinoculation (PI) and remains in this organ as an important reservoir for 5 days PI, with increasing bacteria multiplication. The presence of bacteria in the cecum seems to be associated with its extracellular multiplication in the intestinal content and intermittent shedding in the feces. The colonization of the small intestine occurs during the first 4 days PI. In this period, *Salmonella* penetrates the intestinal mucosa, causing different degrees of degeneration of the microvilli, which is reversible (membrane ruffling). This mechanism is mediated by effector proteins translocated by T3SS encoded in SPI-1. Intracellular multiplication of the bacteria in mononuclear phagocyte system (MPS) occurs from 3 days PI. The exact route of *Salmonella* dissemination from intestine to MPS is unclear, but from the moment that bacteria reach the MPS, they remain in spleen, causing splenomegaly by 10 days PI. The intracellular multiplication in MPS coincides with the production of IFNγ, which

During intestinal infection, *Salmonella*-host interactions result in different degrees of inflammation related to the levels of cytokines produced [12], which may trigger changes in the composition of the intestinal microbiota. A reduction in symbionts or an increase in pathobionts is usually observed during inflammatory processes, reflecting the diversity of the intestinal microbiota [54]. In gastroenteritis caused by *Salmonella* in susceptible hosts, the production of interferon gamma (IFNγ) in the early stage of intestinal inflammation may alter the

higher than the inhibitory level for *Salmonella* multiplication [53].

restricts the replication of intracellular *Salmonella*.

50 Current Topics in Salmonella and Salmonellosis

**5. Role of IFNγ in controlling of** *S. enterica* **growth**

Zoonotic intracellular pathogens that can cause latent carriers pose a unique public health problem. The ability of such carrier animals to shed pathogens without showing any clinical signs of infection can make outbreak control challenging and the potential of transmission to humans a serious public health concern. Before identifying these carriers, we need to understand the mechanism of bacterial invasion of the host cells and follow the process of establishing a persistent state of infection. SPI 1 encodes for genes *hil*A and *inv*F, which allow the bacteria to enter, survive, and replicate within the host cells [62]. Once the pathogen enters the host cells, glycine cleavage protein subunit P (*gcv*P) has been shown to be a potential key player in the transition from acute to chronic infection [63–65]. The activity of *gcv*P has been shown to increase dramatically in other important zoonotic infections like tuberculosis [66, 67] and leishmaniasis [68]. Understanding the pathogenesis of the invasion, intracellular replication, and the transition to latent carrier state in *Salmonella* would potentially lay the groundwork for the development of a control, treatment and eventual eradication strategies. We are just starting to understand potential genes involved in the transition from active to latent stage of infection in case of intracellular pathogens. There is very little information in case of *Salmonella*, but in case of *M. tuberculosis*, glycine dehydrogenase activity increases tenfold upon entering a state of persistence. Another indicator that its metabolism is vital to persistence is the fact that mutants that are deficient in isocitrate lyase, an enzyme involved in the glyoxylate pathway, cannot cause chronic latent infections [67]. We have some preliminary results from our long-term cell culture *Salmonella* infection model (unpublished personal communication). It shows that *Ace*A the gene that codes for isocitrate lyase, which is the first step in the glyoxylate shunt, is over expressed. Even on day 1, the expression levels are elevated, but not significantly more than any of the other genes. However, on day 10 and day 30 post infection,. *Ace*A expression level on day 30 goes up dramatically. This has biological plausibility since it is the first step in the glyoxylate pathway. Such gene expression studies of lymph node biopsies on a herd basis or at slaughter might allow us to detect chronic/persistent *Salmonella* infections.

## **7. Conclusions**

Despite host's activation of anti-inflammatory and antimicrobial responses, *Salmonella* can establish asymptomatic persistent infections, leading to intermittent high-level shedding of the bacteria in feces. This host-pathogen balance leads to serious problems for public health because asymptomatic animals latently carry the infection for long periods with intermittent cycles of shedding of the pathogen in feces. This outcome is epidemiologically important because false-negative *Salmonella* isolation results can be generated if the diagnostic test is performed during the period when the animal is not shedding the pathogen.

#### **Acknowledgements**

The authors thank Universidade Estadual de Santa Cruz-UESC (Bahia State, Brazil) and Fundação de Amparo à Pesquisa do Estado da Bahia-FAPESB (Bahia State, Brazil).

## **Author details**

Bianca Mendes Maciel<sup>1</sup> \*, Rachel Passos Rezende<sup>1</sup> and Nammalwar Sriranganathan2


#### **References**

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tenfold upon entering a state of persistence. Another indicator that its metabolism is vital to persistence is the fact that mutants that are deficient in isocitrate lyase, an enzyme involved in the glyoxylate pathway, cannot cause chronic latent infections [67]. We have some preliminary results from our long-term cell culture *Salmonella* infection model (unpublished personal communication). It shows that *Ace*A the gene that codes for isocitrate lyase, which is the first step in the glyoxylate shunt, is over expressed. Even on day 1, the expression levels are elevated, but not significantly more than any of the other genes. However, on day 10 and day 30 post infection,. *Ace*A expression level on day 30 goes up dramatically. This has biological plausibility since it is the first step in the glyoxylate pathway. Such gene expression studies of lymph node biopsies on a herd basis or at slaughter might allow us to detect chronic/persis-

Despite host's activation of anti-inflammatory and antimicrobial responses, *Salmonella* can establish asymptomatic persistent infections, leading to intermittent high-level shedding of the bacteria in feces. This host-pathogen balance leads to serious problems for public health because asymptomatic animals latently carry the infection for long periods with intermittent cycles of shedding of the pathogen in feces. This outcome is epidemiologically important because false-negative *Salmonella* isolation results can be generated if the diagnostic test is

The authors thank Universidade Estadual de Santa Cruz-UESC (Bahia State, Brazil) and

and Nammalwar Sriranganathan2

Fundação de Amparo à Pesquisa do Estado da Bahia-FAPESB (Bahia State, Brazil).

1 Department of Biological Sciences, Santa Cruz State University, Ilhéus, BA, Brazil

2 Virginia-Maryland College of Veterinary Medicine, Virginia Teach, Blacksburg, VA, USA

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\*, Rachel Passos Rezende<sup>1</sup>

\*Address all correspondence to: caimbi@uol.com.br

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52 Current Topics in Salmonella and Salmonellosis

**Acknowledgements**

**Author details**

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S0923-2508(03)00025-1

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#### *Salmonella* **Fimbriae: What is the Clue to Their Hairdo?** *Salmonella* **Fimbriae: What is the Clue to Their Hairdo?**

Karine Dufresne and France Daigle Karine Dufresne and France DaigleAdditional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67189

#### **Abstract**

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58 Current Topics in Salmonella and Salmonellosis

14200

Fimbriae are important virulence factors for *Salmonella* pathogenesis. They mediate adhesion to host cells (including plants), food, stainless steel and much more. The fimbrial systems are organised in gene clusters of four to fifteen genes that code for structural, assembly and regulatory proteins. There are three kinds of fimbriae depending on their mode of assembly. The chaperone/usher (CU) fimbriae use a dedicated chaperone and usher protein to coordinate the subunit biogenesis on the cell surface. The curli fimbriae are assembled by nucleation/precipitation pathway. The type IV fimbria assembly requires a transmembrane apparatus and ATP to energise the reaction. Several fimbriae are conserved among *Salmonella* serovars, while some are present in a limited set or only specific serovars. Expression and regulation of fimbrial genes are not well understood, and most *Salmonella* fimbriae are poorly expressed during in vitro culture, which further complicates research concerning their regulation and role during infection. However, *Salmonella fim* gene cluster, coding for type-1 fimbriae, was widely studied and presents its own set of regulators. Investigating fimbrial distribution, expression and regulation will further elucidate their roles in bacterial pathogenesis and host specificity. Furthermore, fimbriae are important for developing efficient diagnostic tests and antimicrobial strategies against *Salmonella*.

**Keywords:** fimbriae, adhesion, chaperone/usher, curli, type IV fimbria, fimbriome, *fim*

#### **1. Introduction**

Multiple virulence factors are implicated in *Salmonella* pathogenesis. These factors include type 3 secretion systems (T3SS) encoded in *Salmonella* Pathogenicity Islands (SPI)-1 and SPI-2, other SPIs, flagella, capsule, plasmids and adhesion systems [1, 2]. Among those factors, fimbriae represent a major player in pathogenesis and a source of diversity for *Salmonella*

and reproduction in any medium, provided the original work is properly cited.

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, serovars. Fimbriae are the most common adhesion systems and are differentially expressed and found in a specific pattern among each serovar [3, 4].

Historically, the first observation of fimbriae was described in 1901 in *Bacillus anthracis* by Hinterberger and Reitman which hypothesised that the filaments were implicated in nutrients acquisition [5]. Then, in 1949, Anderson suggested that the filaments were artefacts due to sample preparation for electron microscopy [6]. However, many other studies contradicted Anderson and confirmed the presence of non-flagellar appendages on the bacterial surface. In 1950, Houwink and Van Iterson observed the appendages and described them as shorter and more rigid filaments than the flagella from *Escherichia coli* and suggested that the fibres were implicated in attachment to surface [7]. The name fimbria (Latin word for fibres) was suggested in 1955 by Duguid et al. to describe the filamentous structures [7, 8]. The term fimbria is preferable to use to describe non-flagellar filaments than pili, which is used to designate structures implicated in conjugation [9, 10]. In 1966, Duguid et al. classified fimbriae in seven types (types 1–6 and F) according to the morphology and haemagglutination patterns. However, another classification, based on serology, better predicted genetic relatedness of fimbrial antigens. Nowadays, fimbriae are designated by the mode of assembly of the fibril [8].

A specific fimbrial gene cluster (FGC) encodes for the structural, assembly and sometime regulatory proteins required for the production of the filamentous adhesive appendage on the bacterial surface. FGCs are usually composed of four to fifteen genes [10, 11]. An average of 12 FGCs by strains was observed in *S. enterica*. Despite that all *Salmonella* genome harbours multiple FGCs, very few are characterised so far. Most fimbriae are poorly expressed under laboratory conditions, and the functional redundancy complicates their studies [10]. However, fimbriae are implicated during infection and in a variety of other roles, like biofilm formation, seroconversion, haemagglutination, cellular invasion and macrophage interactions [2, 7, 12–16]. In mice model, *S*. Typhimurium fimbriae demonstrate a role in intestinal cells attachment, caecum colonisation and persistence in gut [17–19]. Moreover, fimbriae are important determinants of host adaptation by *Salmonella* [20].

In this chapter, an overview of *Salmonella* fimbriae is presented. First, the three pathways for fimbrial biogenesis (CU, precipitation/nucleation, type IV fimbriae) are described. Second, the distribution of fimbrial genes among *Salmonella* subspecies and serovars is presented. Third, the regulation of fimbrial genes is described and *fim* FGC regulation is detailed. Finally, the use of fimbriae as diagnostic and therapeutic tools is discussed.

## **2. Fimbrial biogenesis pathways**

Three pathways for fimbrial assembly exist in *Salmonella*, the chaperone/usher (CU), the nucleation/precipitation and the type IV pathway [21]. Fimbriae of the CU pathway employ dedicated chaperones and ushers for the fimbrial assembly. The nucleation/precipitation pathway forms an aggregative fibre by precipitation of the subunits in the presence of the nucleator in the extracellular environment. Finally, the type IV fimbrial pathway uses complex machinery for the fimbriae formation and needs ATP to drive the reaction of assembly. Furthermore, the type IV fimbriae can retract and reverse its assembly [21].

The three pathways produce quite different fimbriae. CU fimbriae have the classic fimbrial shape with the repetition of major subunits emerging from the usher inserted in the outer membrane. The major subunits can be accompanied by minor subunits and/or adhesins [8]. The fimbriae produced by the nucleation/precipitation pathway have an aggregated shape, due to the precipitation of major subunits together. This kind of fimbriae is highly stable and hardly depolymerised [22]. The type IV fimbriae anchor in the inner membrane and are prolonged by the repetition of the major subunit (pilin) through the periplasm and the outer membrane reaching the extracellular medium [23]. Here, the three fimbrial assembly mechanisms will be detailed.

#### **2.1. Chaperone/usher pathway**

serovars. Fimbriae are the most common adhesion systems and are differentially expressed

Historically, the first observation of fimbriae was described in 1901 in *Bacillus anthracis* by Hinterberger and Reitman which hypothesised that the filaments were implicated in nutrients acquisition [5]. Then, in 1949, Anderson suggested that the filaments were artefacts due to sample preparation for electron microscopy [6]. However, many other studies contradicted Anderson and confirmed the presence of non-flagellar appendages on the bacterial surface. In 1950, Houwink and Van Iterson observed the appendages and described them as shorter and more rigid filaments than the flagella from *Escherichia coli* and suggested that the fibres were implicated in attachment to surface [7]. The name fimbria (Latin word for fibres) was suggested in 1955 by Duguid et al. to describe the filamentous structures [7, 8]. The term fimbria is preferable to use to describe non-flagellar filaments than pili, which is used to designate structures implicated in conjugation [9, 10]. In 1966, Duguid et al. classified fimbriae in seven types (types 1–6 and F) according to the morphology and haemagglutination patterns. However, another classification, based on serology, better predicted genetic relatedness of fimbrial antigens. Nowadays, fimbriae are designated by the mode of

A specific fimbrial gene cluster (FGC) encodes for the structural, assembly and sometime regulatory proteins required for the production of the filamentous adhesive appendage on the bacterial surface. FGCs are usually composed of four to fifteen genes [10, 11]. An average of 12 FGCs by strains was observed in *S. enterica*. Despite that all *Salmonella* genome harbours multiple FGCs, very few are characterised so far. Most fimbriae are poorly expressed under laboratory conditions, and the functional redundancy complicates their studies [10]. However, fimbriae are implicated during infection and in a variety of other roles, like biofilm formation, seroconversion, haemagglutination, cellular invasion and macrophage interactions [2, 7, 12–16]. In mice model, *S*. Typhimurium fimbriae demonstrate a role in intestinal cells attachment, caecum colonisation and persistence in gut [17–19]. Moreover, fimbriae are

In this chapter, an overview of *Salmonella* fimbriae is presented. First, the three pathways for fimbrial biogenesis (CU, precipitation/nucleation, type IV fimbriae) are described. Second, the distribution of fimbrial genes among *Salmonella* subspecies and serovars is presented. Third, the regulation of fimbrial genes is described and *fim* FGC regulation is detailed. Finally, the

Three pathways for fimbrial assembly exist in *Salmonella*, the chaperone/usher (CU), the nucleation/precipitation and the type IV pathway [21]. Fimbriae of the CU pathway employ dedicated chaperones and ushers for the fimbrial assembly. The nucleation/precipitation pathway forms an aggregative fibre by precipitation of the subunits in the presence of the nucleator in the extracellular environment. Finally, the type IV fimbrial pathway uses complex machinery

and found in a specific pattern among each serovar [3, 4].

60 Current Topics in Salmonella and Salmonellosis

important determinants of host adaptation by *Salmonella* [20].

use of fimbriae as diagnostic and therapeutic tools is discussed.

**2. Fimbrial biogenesis pathways**

assembly of the fibril [8].

The CU fimbriae represent the largest and most diversified class of adhesion systems [24, 25]. Multiple CU fimbriae are present in *Salmonella* suggesting a functional redundancy [23, 26]. The assembly is characterised by an interaction between the subunits, a periplasmic chaperone and an outer membrane usher in order to form a mature fibre (**Figure 1**) [27]. Each fimbria produced by this pathway has its own unique and specific chaperone and usher [11]. Usher sequence is a good discrimination tool and is used to subdivide the CU fimbriae into six phylogenetic clades (α, κ, π, σ, γ, β) [10, 26].

**Figure 1.** Chaperone/usher pathway. The subunit proteins are synthesised in the cytoplasm and translocated through the periplasm via SecYEG implying also SecDF/YajC inner membrane proteins. When the signal peptide is cleaved from the subunit, the chaperone protein complements the missing strand of the subunit in a process called donor strand complementation. The energy from the folding of the subunit is preserved by the chaperone. The chaperone drives the subunit to the usher and exchanges the donor strand. The subunit is then translocated by the usher to the extracellular medium and added to other subunits to form the fibril. IM = inner membrane; OM = outer membrane.

The biogenesis of the CU fimbriae begins with the production of the subunits in the cytoplasm and their export through the inner membrane by the general secretory pathway (GSP) [23, 27, 28]. It consists in a post-translational translocation implying the SecYEG complex and SecDF/YajC proteins. When the pre-protein is produced, it can be targeted directly to the accessory factor SecA or transported to SecA by the general chaperone SecB. Then, SecA catalyses the hydrolysis of ATP to energise the translocation through SecYEG. Use of ATP, in combination with proton-motive force, triggers the transport of the pre-protein to the periplasm. During the translocation across the inner membrane, the N-terminal signal peptide is cleaved by periplasmic peptidases [27, 29]. To prevent early folding of the subunits, the fimbrial chaperone instantly forms a complex with the translocated subunit in the periplasm [30].

Fimbrial chaperone shares conserved structural features with the general periplasmic chaperones [30]. They are formed of two β-sheet domains oriented to produce an L-shaped molecule and together form a β-barrel. Each domain has an immunoglobulin-like fold and is composed of seven primary β-strands [30–32]. Hydrophobic residues are alternated in the seven strands, facing the internal part of the barrel. These residues form the hydrophobic core of the domain that is implicated in the binding of the subunit. The fimbrial chaperones have an extended loop that lies at the extremity of one arm of the L-shaped molecule. This loop contains a conserved motif that is involved in the complex formation between the chaperone and subunits [30]. The subunit and the chaperone have a similar structure, but the subunit is missing the seventh β-strand of the C-terminal extremity [28]. The chaperone transfers the missing β-strand to the subunit to complete its structure: this mechanism is called the donor strand complementation [25]. The chaperone preserves the folding energy of the subunit to drive the last steps of the assembly due to lack of energy source (ATP) in the periplasmic space [33]. The chaperone also prevents premature fimbrial formation in the periplasm and primes the assembly through the usher [30, 34].

Then, the uncapping of the chaperone by the usher exposes the interactive surface of the subunit to the outer membrane usher and assembly of subunits at the surface can occur [33]. The transfer of the subunit from the chaperone to the usher happens very rapidly in vivo. In the absence of the usher in vitro, only a slow and inefficient assembly was observed. This suggests that the uncapping of the chaperone is important for the efficiency of mature fimbriae assembly [28, 30]. An interaction between the usher and the subunit and also between the usher and the chaperone is required [31]. This triangular interaction is important for the usher to discriminate subunit-loaded from unloaded chaperone [33]. Fimbrial usher forms a ring in the outer membrane with a transient twin-pore of 2–3 nm diameter to allow passage of subunits to the extracellular environment [35]. The usher catalyses fimbrial polymerisation by involving donor strand exchange where the N-terminal sequence of the subunit is replaced by a short sequence of the last subunit in the polymerised fibril with a zip-in-zip-out mechanism [33]. This step is triggered in part by the chaperone required for the strand exchange between the new subunit and the forming fimbria. The quaternary structure of the subunit is achieved when the protein passes through the pore. The final morphology and structure (rigid or flexible), the length (1–3 μm) and width (2–10 nm) of the fibre of the CU fimbriae depend on the subunits composition and the interactions between subunits [10, 33].

#### **2.2. Nucleation/precipitation pathway**

The biogenesis of the CU fimbriae begins with the production of the subunits in the cytoplasm and their export through the inner membrane by the general secretory pathway (GSP) [23, 27, 28]. It consists in a post-translational translocation implying the SecYEG complex and SecDF/YajC proteins. When the pre-protein is produced, it can be targeted directly to the accessory factor SecA or transported to SecA by the general chaperone SecB. Then, SecA catalyses the hydrolysis of ATP to energise the translocation through SecYEG. Use of ATP, in combination with proton-motive force, triggers the transport of the pre-protein to the periplasm. During the translocation across the inner membrane, the N-terminal signal peptide is cleaved by periplasmic peptidases [27, 29]. To prevent early folding of the subunits, the fimbrial chaperone instantly forms a complex with the translocated subunit in

Fimbrial chaperone shares conserved structural features with the general periplasmic chaperones [30]. They are formed of two β-sheet domains oriented to produce an L-shaped molecule and together form a β-barrel. Each domain has an immunoglobulin-like fold and is composed of seven primary β-strands [30–32]. Hydrophobic residues are alternated in the seven strands, facing the internal part of the barrel. These residues form the hydrophobic core of the domain that is implicated in the binding of the subunit. The fimbrial chaperones have an extended loop that lies at the extremity of one arm of the L-shaped molecule. This loop contains a conserved motif that is involved in the complex formation between the chaperone and subunits [30]. The subunit and the chaperone have a similar structure, but the subunit is missing the seventh β-strand of the C-terminal extremity [28]. The chaperone transfers the missing β-strand to the subunit to complete its structure: this mechanism is called the donor strand complementation [25]. The chaperone preserves the folding energy of the subunit to drive the last steps of the assembly due to lack of energy source (ATP) in the periplasmic space [33]. The chaperone also prevents premature fimbrial formation in the periplasm and primes the

Then, the uncapping of the chaperone by the usher exposes the interactive surface of the subunit to the outer membrane usher and assembly of subunits at the surface can occur [33]. The transfer of the subunit from the chaperone to the usher happens very rapidly in vivo. In the absence of the usher in vitro, only a slow and inefficient assembly was observed. This suggests that the uncapping of the chaperone is important for the efficiency of mature fimbriae assembly [28, 30]. An interaction between the usher and the subunit and also between the usher and the chaperone is required [31]. This triangular interaction is important for the usher to discriminate subunit-loaded from unloaded chaperone [33]. Fimbrial usher forms a ring in the outer membrane with a transient twin-pore of 2–3 nm diameter to allow passage of subunits to the extracellular environment [35]. The usher catalyses fimbrial polymerisation by involving donor strand exchange where the N-terminal sequence of the subunit is replaced by a short sequence of the last subunit in the polymerised fibril with a zip-in-zip-out mechanism [33]. This step is triggered in part by the chaperone required for the strand exchange between the new subunit and the forming fimbria. The quaternary structure of the subunit is achieved when the protein passes through the pore. The final morphology and structure (rigid or flexible), the length (1–3 μm) and width (2–10 nm)

the periplasm [30].

62 Current Topics in Salmonella and Salmonellosis

assembly through the usher [30, 34].

Curli fimbriae were initially discovered in *Escherichia coli* and are very conserved among the *Enterobacteriaceae* family, compared to any other types of FGC. The amyloid fibrils are particularly known for their role in biofilm formation and its recognition by the immune system [36]. The FGC for curli is named *csg* (curli subunit gene) for *E. coli* and *agf* (thin aggregative fimbriae) for *Salmonella*, but the term *csg* is now commonly used for *Salmonella*. Curli formation depends on two divergent operons, *csgBAC* and *csgDEFG*. The *csgBAC* genes encode for CsgA, the major subunit, CsgB, the nucleator, and CsgC, an oxidoreductase of unknown function. The *csgDEFG* genes encode for the transcription regulator of the operon (CsgD) and for the assembly proteins located in the periplasm (CsgE) or in the outer membrane (CsgG and CsgF) [37].

The curli assembly mechanism is characterised by the exportation of the subunits and their precipitation to each other in the presence of a nucleator that fixes the fibril on the bacterial surface. Exportation of curli proteins also uses the GSP to pass through the inner membrane to the periplasm. Then, the CsgA and CsgB proteins are secreted by the lipoprotein CsgG. CsgG is composed of nine anticodon-binding domain-like units that form a 36-stranded β-barrel complex that is inserted in the outer membrane. CsgG forms a pore in the outer membrane that permits the passage of the subunits and the nucleator. CsgG is accompanied by the accessory proteins CsgE and CsgF. CsgE is a specificity factor that forms a nonameric adaptor that binds to CsgG and closes the periplasmic space. The presence of CsgE optimises the uptake of CsgA by CsgG and translocation of CsgA [38]. CsgF helps the nucleation activity of CsgB. It was suggested that CsgF has a role in specific localisation and/or chaperoning of the nucleator, so CsgB will reach its full activity. Moreover, CsgF depends on CsgG and CsgE for its stability [39].

Once at the bacterial surface, the nucleator polymerises the subunits together into thin aggregative fimbriae (fibrils). This process happens only in the extracellular environment and requires the presence of the nucleator CsgB to polymerise CsgA into a filament. CsgA proteins fold into an insoluble cross β-sheet molecules [26]. CsgB anchors the curli fimbriae on the surface of the bacterial cell (**Figure 2**). In *E. coli*, it was observed that CsgB, in addition to its role of nucleator, is also part of the fimbriae with the CsgA subunits. A structurally different fibril made of CsgB subunits can be formed in the absence of CsgA [40]. CsgA and CsgB share 30% of sequence identity and have the same predicted length [37]. In *E. coli*, interbacterial complementation between a nucleator mutant and a subunit mutant is possible. However, in *Salmonella*, this complementation cannot happen, suggesting that the curli fimbriae are different in their nucleation process. However, the interbacterial complementation was observed in *Salmonella* when a lipopolysaccharide O-antigen mutant was used [41]. The nucleation/ precipitation pathway is still poorly understood, and research is actually performed on the different aspects of the curli fimbrial formation.

**Figure 2.** Nucleation/precipitation pathway. The subunit CsgA is synthesised in the cytoplasm and translocated by the GSP. CsgA passes through the periplasm and is translocated in the extracellular medium by CsgG, helped by CsgE. The nucleator CsgB is also translocated by CsgG and supported by CsgF for its stability on the bacterial surface. When CsgA is in the presence of the nucleator in the extracellular environment, the subunits precipitate in an aggregated fibril. CsgC is an oxidoreductase, but its specific role is still undiscovered. IM, inner membrane; OM, outer membrane.

#### **2.3. Type IV fimbriae**

Type IV fimbriae are usually from 1 to 5 μm long and are composed of repeated subunits of a single pilin. Type IV fimbria is subdivided into two groups based on homology of the major subunits: type IVa and type IVb fimbriae [26]. The difference between the two types is in the length of the peptide sequence and the mature major pilin sequence. Specific mechanism of assembly of type IVb fimbriae from *Salmonella* has not been characterised yet [42].

Type IV fimbriae pathway has the most complex machinery. They form an apparatus, composed of various proteins, that goes through the inner and outer membranes allowing the anchor of the fibre and energy accessibility for fimbrial assembly. The gene cluster also encodes numerous proteins with diverse functions, as the fibril is not only assembled but also disassembled. Type IV fimbriae are frequently compared to the type II secretion system (T2SS) which possesses similar structure and mechanism of assembly. Type IV fimbriae are implicated in adherence and twitching motility [11].

Type IV fimbriae are present in a variety of organisms including human pathogens such as *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Pseudomonas aeruginosa* and *Vibrio cholerae*. For *Salmonella*, they are found in *S. bongori*, *S. enterica* serovars Heidelberg, Paratyphi B and Typhi [42]. *S. bongori* type IV fimbria is encoded by the *sbe* operon that remains uncharacterised and is located on a plasmid, as well as in *S*. Paratyphi B, while the type IVb gene cluster is located on the chromosome of *S*. Heidelberg and *S*. Typhi [26].

For *S*. Typhi, the PilS subunits are produced in the cytoplasm and translocated to the periplasm by the GSP. In the periplasm, the N-terminal sequence of PilS is cleaved by PilU, a prepilin peptidase [23]. The mature pilins are then anchored to the inner membrane on platform proteins and linked together into a fibril (**Figure 3**) [26, 43]. The N-terminal domain of the mature subunits is highly hydrophobic, which permits the PilS proteins to group into a helical structure [22, 42]. The pilins are added one by one, but at three sites simultaneously, each corresponding to a strand to form a three-helix bundle [44]. An ATPase inserted in the inner membrane supplies the energy required for the assembly of the type IV fimbriae. The secretin proteins are inserted in the outer membrane and form a channel that permits the passage of the intact pilus through the bacterial surface [26]. These proteins form complexes that are then assembled in a cage-like final structure [44]. Other proteins are also involved in the assembly/disassembly mechanisms, such as another ATPase dedicated for the disassembly of the fimbriae, lipoproteins of the secretin complex (pilotins), inner membrane proteins or gene products involved in peptidoglycan remodelling to permit the passage of the fibril through the periplasm [22, 44, 45]. This assembly pathway is less understood and requires further investigations [44].

**Figure 3.** Type IV pathway. The pre-pilins are transported and translocated through the inner membrane (IM) to the periplasm by GSP. A peptidase cleaves the signal peptide of the pre-pilin, and the pilin can be assembled on the platform proteins. An ATPase triggers the reaction. The pilins form a three-helix structure that passes through the outer membrane (OM) by a secretin supported by pilotin. The type IV fimbriae can also retract depending on the environmental conditions.

#### **3.** *Salmonella* **fimbriome**

**2.3. Type IV fimbriae**

64 Current Topics in Salmonella and Salmonellosis

Type IV fimbriae are usually from 1 to 5 μm long and are composed of repeated subunits of a single pilin. Type IV fimbria is subdivided into two groups based on homology of the major subunits: type IVa and type IVb fimbriae [26]. The difference between the two types is in the length of the peptide sequence and the mature major pilin sequence. Specific mechanism of

**Figure 2.** Nucleation/precipitation pathway. The subunit CsgA is synthesised in the cytoplasm and translocated by the GSP. CsgA passes through the periplasm and is translocated in the extracellular medium by CsgG, helped by CsgE. The nucleator CsgB is also translocated by CsgG and supported by CsgF for its stability on the bacterial surface. When CsgA is in the presence of the nucleator in the extracellular environment, the subunits precipitate in an aggregated fibril. CsgC

Type IV fimbriae pathway has the most complex machinery. They form an apparatus, composed of various proteins, that goes through the inner and outer membranes allowing the anchor of the fibre and energy accessibility for fimbrial assembly. The gene cluster also encodes numerous proteins with diverse functions, as the fibril is not only assembled but also disassembled. Type IV fimbriae are frequently compared to the type II secretion system (T2SS) which possesses similar structure and mechanism of assembly. Type IV fimbriae are

Type IV fimbriae are present in a variety of organisms including human pathogens such as *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Pseudomonas aeruginosa* and *Vibrio cholerae*. For *Salmonella*, they are found in *S. bongori*, *S. enterica* serovars Heidelberg, Paratyphi B and Typhi [42]. *S. bongori* type IV fimbria is encoded by the *sbe* operon that remains uncharacterised and is located on a plasmid, as well as in *S*. Paratyphi B, while the type IVb gene cluster

For *S*. Typhi, the PilS subunits are produced in the cytoplasm and translocated to the periplasm by the GSP. In the periplasm, the N-terminal sequence of PilS is cleaved by PilU, a prepilin pep-

assembly of type IVb fimbriae from *Salmonella* has not been characterised yet [42].

is an oxidoreductase, but its specific role is still undiscovered. IM, inner membrane; OM, outer membrane.

implicated in adherence and twitching motility [11].

is located on the chromosome of *S*. Heidelberg and *S*. Typhi [26].

Each fimbrial pathway described above is present in *Salmonella* creating a great element of genetic diversity. CU fimbriae are the most common fimbriae detected in the *Salmonella* genome. Curli (*csg*) is found in all *Salmonella* genome, whereas only a few serovars have the type IV fimbriae. There are 38 unique FGCs identified so far in 111 sequenced genomes from 34 different serovars (**Table 1**) [46, 47]. Each serovar has its own repertoire of FGCs, but there are seven FGCs that are highly conserved in most *Salmonella* strains forming the core of *Salmonella* FGCs. Most of the FGCs are sporadic or found only in a few strains constituting the signature of each serovar.


**Table 1.** *Salmonella* fimbriome.

Each *Salmonella* strain contains 5–14 different CU fimbriae with an average of 12 fimbriae in *S. enterica*. Representatives from all the six phylogenetic clades are present in *Salmonella* (**Table 2**) [26]. The γ-fimbriae constitute the largest clade with 22 FGCs and include the highly conserved FGCs (*bcf, fim* and *sth*) that belong to the clade γ-1. The most diverse clade is γ-4, with the conserved *stb* and *stc* or *peg* (*stc-peg*) and many of the new sporadic FGCs, while the α clade (for alternate CU), also known as class 5 fimbriae, has one FGC, *tcf*, which is found in several serovars. The σ clade also had only one FGC representative, *sdc*, that was only found in *S. enterica* subspecies IIIa (*arizonae*).

The distribution of the 38 FGCs gave a signature for each species, subspecies and serovars (**Table 3**). Seven FGCs, curli and the CU *fim, bcf, sth, stb*, *stc-peg* and *std*, represent the conserved (core) fimbriae of *Salmonella* (positive in more than 90% of strains). The *fim* fimbriae were found in all *S. enterica* strains, only missing in *S. bongori*. The *bcf* cluster was only missing in *S. enterica* ssp. IV (*houtenae*), and the *sth* cluster was only missing in *S. enterica* ssp. IIIa and IIIb. The *stb* cluster was present in *S. enterica* ssp. I, II, IIIb and the *std* cluster was not detected in *S. enterica* serovar Gallinarum, ssp. II, IIIA and *S. bongori*. The FGC *stc* and *peg* had probably emerged from a common ancestor: they belong to the same clade (γ-4) and are inserted at the same position in the genome (between *thiM* and *mrp*); their distribution is mutually exclusive; and either one is present in the majority of *Salmonella* strains.


**Table 2.** *Salmonella* fimbrial type.

Each *Salmonella* strain contains 5–14 different CU fimbriae with an average of 12 fimbriae in *S. enterica*. Representatives from all the six phylogenetic clades are present in *Salmonella* (**Table 2**) [26]. The γ-fimbriae constitute the largest clade with 22 FGCs and include the highly conserved FGCs (*bcf, fim* and *sth*) that belong to the clade γ-1. The most diverse clade is γ-4, with the conserved *stb* and *stc* or *peg* (*stc-peg*) and many of the new sporadic FGCs, while the α clade (for alternate CU), also known as class 5 fimbriae, has one FGC, *tcf*, which is found in several serovars. The σ clade also had only one FGC representative, *sdc*, that was only found

**Fimbriae CU clade Prevalence Distribution Fimbriae CU clade Prevalence Distribution** *bcf* γ1 Core Absent in IV *sdj* γ4 Sporadic IIIb *diarizonae csg* curli Core All *Salmonella sdk/sfi* π Sporadic IIIb, VI

*lpf* γ1 Conserved Absent in ID *sef* γ3 Sporadic IB, D (pseudo)

*sdl* π Sporadic IIIb *diarizonae*

*sib* β Sporadic VI *indica*

*fae/skf* κ Sporadic IB, IE

*sta* γ4 Sporadic ID

*ssf* γ4 Sporadic II *salamae*

*stb* γ4 Core I, II, IIIb;

missing in Gallinarum

IA, IE

ID, IE

and IIIb

*bongori*

Montevideo

and *bongori*

Montevideo

*saf* γ3 Conserved ssp. I *stc* γ4 Conserved IA, IB, ID *sba* γ4 Sporadic *bongori std* π Core II, IIIa,

*sbb/sbf* π Sporadic *bongori ste* π Conserved Missing in

*sbc/spf* κ Sporadic IV, VI, *bongori stf* π Conserved Missing in

*sbs* β Sporadic II *salamae stg* γ1 Sporadic ID, bongori *sdc/sas* σ Sporadic IIIa *arizonae sth* γ1 Core Missing IIIa

*sdd/smf* γ1 Sporadic IE, II, IIIa, IV *sti* γ1 Conserved Missing in ID

*sde* γ3 Sporadic Tennessee (IE) *stj* β Sporadic IA, IE *sdh* γ4 Sporadic IE *stk* γ4 Sporadic IE *sdi/sdf* γ4 Sporadic IIIb *diarizonae tcf* α Sporadic IC, ID, IE

*bongori*

*bongori*

in *S. enterica* subspecies IIIa (*arizonae*).

**Table 1.** *Salmonella* fimbriome.

*fim* γ1 Core Absent in

66 Current Topics in Salmonella and Salmonellosis

*mrk* γ4 Sporadic Only in

*peh* γ4 Sporadic Only in

*pef* κ Sporadic Only in IA, IC

*peg* γ4 Conserved IB, IC, IIIa, VI,

*pil* Sporadic Type IV; ID, IE,

Most cases of salmonellosis in humans are caused by *S. enterica* ssp. I, and many of the sequenced serovars were from ssp. I. Thus, 27 out of the 38 FGCs are found in ssp. I. The ssp. I was divided into five classes using previous phylogenetic analysis [46, 47] (**Table 3**). The class IA contains broad host range serovars involved in gastroenteritis, mainly serovar Typhimurium. The class IB is formed by serovars Dublin, Enteritidis, Pullorum and Gallinarum, all sharing similar O-antigens and FGCs. The class IC contains serovars Choleraesuis and Paratyphi C and class ID contains the human-specific serovars Typhi and Paratyphi A. A separate branch of class IA, including serovars Heidelberg, Virchow and Hadar, that had the highest number of FGCs, as well as serovars Montevideo, Schwarzengrund, Welterveden, Javiana, Kentucky and Tennessee, was commonly isolated in association with edible plants and constitutes the class IE.

In addition to the seven core FGCs, five highly conserved FGCs (*saf, ste, stf*, *sti* and *lpf*) were associated with *S*. *enterica* ssp. I (**Table 3**). The *sti*, *lpf* and *stf* clusters are missing in humanspecific serovars (class ID). The *ste* cluster is missing in class IA serovars and in some of the class IE serovars. Thus, *S. enterica* ssp. I harbours the core FGCs (*fim, bcf, sth, stb*, *stc-peg* and *std*), the conserved FGCs (*saf, ste, stf*, *sti* and *lpf*) and some sporadic FGCs unique to each serovar. Many FGCs of *Salmonella* are sporadic and form the unique repertoire in each serovar.

Despite the presence of many FGCs, extensive gene degradation was observed in most of the host-restricted and warm-blooded host-adapted serovars, mainly Gallinarum, Choleraesuis, Paratyphi A and Typhi. Genome degradation of FGCs may correspond to the loss of genes rendered unnecessary by niche specialisation or by selective pressure in order to diminish antigen presentation at the bacterial surface during systemic disease. Intriguingly, most of FGCs were intact in Paratyphi B.

There are 11 FGCs that are not in ssp. I, with only *sbc* and *sdk* that are shared by more than one serovars. The low numbers of FGCs might be specific for cold-blooded animals' colonisation. A conserved signature specific for each subspecies was observed. As more diverse strains will be sequenced, new FGCs probably be discovered.


**Table 3.** Fimbrial distribution.

## **4. Fimbrial regulation**

*Salmonella* fimbriae are usually not expressed constitutively and rarely expressed under laboratory condition, except for Fim fimbriae, a type-1 fimbria [3]. Fimbriae are important during infection [19, 48, 49], suggesting that their expression is tightly regulated. Little is known about the regulation mechanisms that promote fimbrial expression. In general, fimbrial expression is positively or negatively regulated at the genetic level. Some regulators are unique to a specific fimbriae, like the regulation of curli by CsgD, while others are global, like Dam, H-NS and Lrp (leucine-responsive regulatory protein) [50]. These mechanisms include regulatory proteins, DNA methylation, cyclic di-GMP and small RNAs [50]. In *Salmonella*, a regulation network exists between the virulence factors. Here, we present the regulation of fimbrial genes including the interaction with motility and invasion. Then, we propose an example of regulation of the *fim* FGC expression in *S*. Typhimurium, the most characterised fimbriae of *Salmonella*.

#### **4.1. General regulation of fimbrial genes**

Paratyphi A and Typhi. Genome degradation of FGCs may correspond to the loss of genes rendered unnecessary by niche specialisation or by selective pressure in order to diminish antigen presentation at the bacterial surface during systemic disease. Intriguingly, most of

There are 11 FGCs that are not in ssp. I, with only *sbc* and *sdk* that are shared by more than one serovars. The low numbers of FGCs might be specific for cold-blooded animals' colonisation. A conserved signature specific for each subspecies was observed. As more diverse strains will

**Subspecies Core Conserved Accessory Absent**

VI. *indica sbc, sdk, sib stb* II. *salamae sdd, ssf, sbs std* IV. *houtenae sbc, sdd bcf, stb* IIIb. *diarizonae sdi, sdj***,** *sdk*, *sdl sth*

IIIa. *arizonae sdc, sdd sth, stb, std*

*lpf sba, sbb, sbc, sbe,* 

A *pef, stj ste*

D *sef, sta, stg, tcf, pil sti, lpf, stf*

*lpf, ste, stf*

*fim, stb, std*

E *fae, mrk, peh, sdd, sde, sdh, stj, stk, pil*

*stg(sbd)*

B *fae, sef* C *pef, tcf*,

*sti,, saf, ste, stf, lpf*

*Salmonella* fimbriae are usually not expressed constitutively and rarely expressed under laboratory condition, except for Fim fimbriae, a type-1 fimbria [3]. Fimbriae are important during infection [19, 48, 49], suggesting that their expression is tightly regulated. Little is known about the regulation mechanisms that promote fimbrial expression. In general, fimbrial expression is positively or negatively regulated at the genetic level. Some regulators are unique to a specific fimbriae, like the regulation of curli by CsgD, while others are global, like Dam, H-NS and Lrp (leucine-responsive regulatory protein) [50]. These mechanisms include regulatory proteins, DNA methylation, cyclic di-GMP and small RNAs [50]. In *Salmonella*, a regulation network exists between the virulence factors. Here, we present the regulation of fimbrial genes including the interaction with motility and invasion. Then, we propose an example of regulation of the *fim* FGC expression in *S*. Typhimurium, the most characterised

FGCs were intact in Paratyphi B.

68 Current Topics in Salmonella and Salmonellosis

*Salmonella enterica*

*Salmonella bongori*

**4. Fimbrial regulation**

**Table 3.** Fimbrial distribution.

fimbriae of *Salmonella*.

be sequenced, new FGCs probably be discovered.

I. *enterica bcf, csg, fim, sth, stb, std stc-peg*

Genes implicated in different aspects of virulence including motility, adhesion, invasion of host cells and intestinal persistence are all regulated during infection. It was proposed that there is a temporal hierarchy between the T3SS of SPI-1 (invasion), flagellar and fimbrial genes, where SPI-1 is first activated, followed by flagellar genes and then type-1 fimbrial genes (*fim*). The crosstalk between these systems seems to be critical for bacterial pathogenesis [51]. Each element of virulence is related to a large regulation network that is not completely understood. DNA adenine methylation (Dam) regulates many virulence genes in *Salmonella* [52]: it is required for SPI-1 and *pef* expression, but it also represses many genes, including the *std, csg* and flagellar genes [52–54]. It was also shown that fimbrial FGCs are repressed by the Rcs phosphorelay, a sensor of outer membrane stress [55]. Another example of regulation interaction between motility and fimbrial expression was observed by a deletion of *ydiV* in *S*. Typhimurium that results in the derepression of curli fimbriae (*csgAB*), causing an increase in swimming motility and a decrease in swarming [56].

Crosstalk regulation also occurs between the capsule and the type IVb fimbriae in *S*. Typhi. Both virulence factors are encoded on SPI-7 and facilitate invasion of monocytes, suggesting a regulation overlapped. However, the exact regulation elements that act on those two systems are unknown [57].

One of the post-transcriptional regulation mechanisms uses the binding of small RNAs and the Hfq chaperone. In an *hfq* mutant strain, the expression of fimbrial gene *sefA* was activated when most of the other fimbrial subunit genes were repressed in *S*. Enteritidis. Overall, the *hfq* deletion decreased adherence compared to wild-type strain. Thus, Hfq seems to regulate fimbrial expression of most fimbrial genes from *S*. Enteritidis [58]. There is probably more sRNAs regulation of fimbrial gene expression awaiting to be discovered.

Phase variation is a transcriptional mechanism that controls the switch between fimbriated (ON) and afimbriated (OFF) cells within a bacterial population. In *Salmonella*, expression of *lpf* and *pef* was shown to be controlled by phase variation. The regulators of this mechanism are various and depend on the FGCs concerned [54, 59].

The secondary messenger cyclic-di-GMP controls virulence and biofilm formation in *Salmonella* [60]. In *Salmonella*, curli expression was activated by AdrA, a GGDEF-domain protein that increases intracellular level of cyclic-di-GMP [61]. Fimbrial production regulated by the cyclic-di-GMP level was also observed in other species such as *Klebsiella pneumonia*, *E. coli* and *P. aeruginosa* [62].

In spite of all those known elements of regulation, how *Salmonella* passes from being afimbriated in vitro to a fimbriated form in vivo is still unknown.

#### **4.2. Regulation of** *fim* **in** *S***. Typhimurium**

The *fim* FGC codes for six genes (*fimAICDHF*). This cluster is the most studied and one of the most conserved fimbriae of *Salmonella enterica* and was mainly characterised in *S*. Typhimurium. These fimbriae have a binding specificity for mannose residues [63]. The fim fimbria of *Salmonella* is not homologue with its homonym from *E. coli*, except for sharing some morphological and mechanistic features [64, 65]. Regulation and amino acid sequences of fimbrial proteins are divergent between the two species. The transition from afimbriated to fimbriated stage occurs for *fim*, but there is no inversion of the promoter region as observed for *E. coli* phase variation [66]. For *S*. Typhimurium, the major subunit FimA is accompanied by two other subunits, FimI and FimF, and by the adhesin, FimH. The *fimC* and *fimD* genes encode respectively the chaperone and the usher [67]. Ancillary genes, *fimZYW*, and a rare arginine transfer RNA (tRNA) *fimU* and *STM0551*, inserted between *fimY* and *fimW*, directly regulate *fim* expression [62].

Ancillary genes *fimZYW* regulate the expression of *fimA* [50, 68–70]. FimZ, a sensor DNAbinding protein, is the principal positive regulator of *fimA* [71]. FimY upregulates *fimZ* expression by binding to the *fimZ* promoter. FimY and FimZ then form a complex that activates the *fimA* promoter [63, 69]; *fimY* is itself regulated by the arginine tRNA *fimU* [72]. Lrp is another regulator that binds and activates the *fimZ* promoter, probably by antagonising the binding of the global repressor protein H-NS to this promoter region [67, 70, 73]. H-NS has a high affinity for AT-rich DNA region, and *fimZ* gene has an unusual AT-rich sequence [73].

At the opposite, FimW repressed directly *fimA* expression and indirectly by lowering FimZ availability by its degradation caused by FimW binding [74]. It is also suggested that STM0551, an EAL domain protein, is a negative regulator of *fim* expression by lowering the c-di-GMP level [62].

FimZ is also used as a regulator relay by two-component system for expression of *hilA*, the principal regulator of SPI-1 (invasion). The two-component system PhoBR induces *fimZ* expression, and PhoPQ activates *hilE*, one of the negative regulators of *hilA*, by a FimYZdependant manner. FimZ also downregulates *flhDC*, genes implicated in flagella expression [75]. Flagellar gene *fliZ* also represses *fimZ* on a post-transcriptional manner, reinforcing the fact that there is an alternated expression of flagella (motility) and fimbriae (adhesion) [51] and confirming a regulation network between SPI-1 (invasion) and fimbrial expression [76]. Thus, a combination of factors directly implicated in fimbrial genes regulation can also impact on other virulence systems of *Salmonella* (**Figure 4**).

**Figure 4.** *fim* regulation. The fim FGC is activated and inhibited by diverse regulators. FimZ, accompanied by FimY, is the principal activator of *fimA*. Lrp and c-di-GMP also activate *fimA* at the promoter level. H-NS and FimW inhibit *fimA* expression by linking its promoter. FimW also reduces *fimA* expression by linking FimZ and decreases the availability of this activator. The gene *fimZ* is activated by FimZ itself and by FimY, but is repressed by FliZ in a post-transcriptional manner. The tRNA *fimU* regulates fimY. FimZ downregulates *flhCD*, genes implicated in flagellar expression.

## **5. Fimbriae as a tool**

mechanistic features [64, 65]. Regulation and amino acid sequences of fimbrial proteins are divergent between the two species. The transition from afimbriated to fimbriated stage occurs for *fim*, but there is no inversion of the promoter region as observed for *E. coli* phase variation [66]. For *S*. Typhimurium, the major subunit FimA is accompanied by two other subunits, FimI and FimF, and by the adhesin, FimH. The *fimC* and *fimD* genes encode respectively the chaperone and the usher [67]. Ancillary genes, *fimZYW*, and a rare arginine transfer RNA (tRNA) *fimU*

Ancillary genes *fimZYW* regulate the expression of *fimA* [50, 68–70]. FimZ, a sensor DNAbinding protein, is the principal positive regulator of *fimA* [71]. FimY upregulates *fimZ* expression by binding to the *fimZ* promoter. FimY and FimZ then form a complex that activates the *fimA* promoter [63, 69]; *fimY* is itself regulated by the arginine tRNA *fimU* [72]. Lrp is another regulator that binds and activates the *fimZ* promoter, probably by antagonising the binding of the global repressor protein H-NS to this promoter region [67, 70, 73]. H-NS has a high affinity

At the opposite, FimW repressed directly *fimA* expression and indirectly by lowering FimZ availability by its degradation caused by FimW binding [74]. It is also suggested that STM0551, an EAL domain protein, is a negative regulator of *fim* expression by lowering the c-di-GMP level [62].

FimZ is also used as a regulator relay by two-component system for expression of *hilA*, the principal regulator of SPI-1 (invasion). The two-component system PhoBR induces *fimZ* expression, and PhoPQ activates *hilE*, one of the negative regulators of *hilA*, by a FimYZdependant manner. FimZ also downregulates *flhDC*, genes implicated in flagella expression [75]. Flagellar gene *fliZ* also represses *fimZ* on a post-transcriptional manner, reinforcing the fact that there is an alternated expression of flagella (motility) and fimbriae (adhesion) [51] and confirming a regulation network between SPI-1 (invasion) and fimbrial expression [76]. Thus, a combination of factors directly implicated in fimbrial genes regulation can also impact

**Figure 4.** *fim* regulation. The fim FGC is activated and inhibited by diverse regulators. FimZ, accompanied by FimY, is the principal activator of *fimA*. Lrp and c-di-GMP also activate *fimA* at the promoter level. H-NS and FimW inhibit *fimA* expression by linking its promoter. FimW also reduces *fimA* expression by linking FimZ and decreases the availability of this activator. The gene *fimZ* is activated by FimZ itself and by FimY, but is repressed by FliZ in a post-transcriptional manner. The tRNA *fimU* regulates fimY. FimZ downregulates *flhCD*, genes implicated in flagellar expression.

and *STM0551*, inserted between *fimY* and *fimW*, directly regulate *fim* expression [62].

for AT-rich DNA region, and *fimZ* gene has an unusual AT-rich sequence [73].

on other virulence systems of *Salmonella* (**Figure 4**).

70 Current Topics in Salmonella and Salmonellosis

*Salmonella* infections are a major concern for public and animal health. Some serovars are host specific, while others are broad-spectrum pathogens and can be transmitted from foodborne animals to humans. On the other hand, animals can develop health problems and will not be suitable for consumption. To prevent those issues, it is critical to develop ways to detect *Salmonella* and protect potential hosts against infection. The importance of fimbriae for detection of *Salmonella* by molecular techniques and for vaccine development is presented in this section [77].

#### **5.1.** *Salmonella* **detection using fimbrial genes**

*Salmonella*-specific tests were performed since the end of the 1980s and mainly targeted surface antigens. Those tests include agglutination tests and ELISA (enzyme-linked immunosorbent assays) [77–79]. In 1993, Doran et al. presented a DNA-based test that targets *csgA* (*agfA)*, offering a faster and more precise test for genus identification [80]. Then, in early 2000s, PCR (polymerisation chain reaction) tests using fimbrial genes, like *sef* or *csgA* (*agfA)*, in combination with other virulence genes were developed to differentiate *Salmonella* strains from each other [81]. Different PCR tests (multiplex, nested and direct PCR) were elaborated for detection of *Salmonella*. Several of those tests integrated detection of fimbrial genes (i.e. *staA*, *fimW*) to discriminate between serovars [81–83]. Recently, a loop-mediated isothermal amplification (LAMP) assay was developed to detect *Salmonella* by targeting *bcfD*, a gene that belongs to the core of FGC. In isothermal conditions, the reaction occurs in an hour permitting rapid detection of *Salmonella* [84].

*Salmonella*-specific tests evolved from detecting antigens, which can be long and expensive to perform, to detecting specific genes in less than an hour by sensitive methods. Fimbrial genes are tools of choice for detection of *Salmonella*. The presence of conserved fimbrial genes allows the discrimination between *Salmonella* and non-*Salmonella* species. On the other hand, the presence of a specific pattern of fimbrial genes enables the discrimination between serovars.

#### **5.2. Vaccines development**

As surface structures, fimbriae constitute antigens of choice for the development of vaccines against *Salmonella* [85]. Fimbriae are difficult to study because they are poorly expressed under laboratory conditions and are redundant. The most interesting fimbriae are the ones expressed during infection. Targeting those fimbriae will confer higher chances to be recognised by the immune system in key moments of infection.

More than 20 fimbrial antigens were detected in typhoid fever patient's blood by transcriptomic analysis: SteD, StaACD, BcfDE, SafBC, TcfBCD, StbBC, FimAIDH, StdBC, StgACD and SthA [86]. Antibodies against immunogenic fimbrial proteins TcfB, StbD and CsgEFG were identified in the blood of typhoid fever patients [12]. Immunoreactive antibodies against SthDA and BcfA were found in lymphocytes supernatant (ALS) of patients with typhoid fever [87].

SefA, a protein from the SEF14 fimbriae of *S*. Enteritidis, was used as an antigen associated with liposomes for oral immunisation of chickens [88]. The immunisation of chickens by fimbrial antigens was efficient for IgG and IgA responses and reduced *Salmonella* colonisation. Four weeks after immunisation, the bacterial excretion from the intestinal tract was significantly reduced [88]. The liposome-associated immunisation was also performed with fimbrial antigen from SEF21 and resulted in a similar efficiency [89]. SefD, another antigen from SEF14, was also used to vaccinate animals in a bacterin preparation, a vaccine prepared from inactivated bacteria. This vaccine was efficient to reduce the presence of *Salmonella* from the spleens of hens [90].

As factors implicated in the first stages of infection, fimbriae are an interesting target for vaccine development [91]. Fimbrial antigens are important for the development of new anti-*Salmonella* therapies [85, 86]. However, a better understanding of their expression pattern in vivo is needed to optimise the therapeutic effects of fimbrial-targeted vaccines. Fimbrial antigens may be combined with other immunogenic proteins to increase the immune response [91].

## **6. Conclusion**

Fimbriae are diverse proteinaceous surface structures. They diverge by their assembly mechanisms and result in different filamentous structures with roles in pathogenesis. However, their roles are not completely understood. They were first known for adherence to cells and inert surfaces, but they seem to be implicated in so much more functions during infection.

The multiplicity of adhesion systems is also an enigma. Most of the *Salmonella* serovars possess 12 fimbrial gene clusters. Some fimbriae are specific to certain serovars and may play a role in these bacteria that do not need to be fulfilled in other serovars. At the opposite, there is a core of fimbrial genes that are present in most of the serovars. Fimbriae are one of the keys to understand *Salmonella* pathogenesis. The specific pattern of each serovar, with further investigations on the sporadic fimbriae, may also bring insights into our understanding of *Salmonella* pathogenesis.

Regulation of fimbrial genes is a complex network that is tightly related to invasion and motility. Virulence factors are finely regulated, and a temporal expression hierarchy allows the success of *Salmonella* infection. General regulators are already known to regulate fimbrial genes such as stress sensor Rcs relay or the Hfq factor. Phase variation from a fimbriated to afimbriated status occurs in *Salmonella*. However, this phenomenon is not from a promoter inversion of the Fim cluster, but from the regulation by ancillary genes related to *fim* gene cluster. These ancillary genes are themselves precisely regulated by a variety of regulators known for their role in other bacterial processes.

The actual understanding of fimbrial expression opens a new area on human health prevention. Some conserved fimbrial genes, in combination with other virulence genes, are precious markers for *Salmonella* detection. These tools could permit a faster diagnostic for human patients, but also a rapid detection of contaminated food or infected animals. Fimbrial proteins can serve as good immunogens in vaccine preparation against *Salmonella* infection.

A better understanding of fimbrial expression, production and regulation processes becomes important for prevention of *Salmonella* infection. It will also enlighten the importance of fimbriae in other human pathogens, as fimbrial systems are part of virulence factors in many bacteria.

## **Author details**

SefA, a protein from the SEF14 fimbriae of *S*. Enteritidis, was used as an antigen associated with liposomes for oral immunisation of chickens [88]. The immunisation of chickens by fimbrial antigens was efficient for IgG and IgA responses and reduced *Salmonella* colonisation. Four weeks after immunisation, the bacterial excretion from the intestinal tract was significantly reduced [88]. The liposome-associated immunisation was also performed with fimbrial antigen from SEF21 and resulted in a similar efficiency [89]. SefD, another antigen from SEF14, was also used to vaccinate animals in a bacterin preparation, a vaccine prepared from inactivated bacteria. This vaccine was efficient to reduce the presence of *Salmonella* from the

As factors implicated in the first stages of infection, fimbriae are an interesting target for vaccine development [91]. Fimbrial antigens are important for the development of new anti-*Salmonella* therapies [85, 86]. However, a better understanding of their expression pattern in vivo is needed to optimise the therapeutic effects of fimbrial-targeted vaccines. Fimbrial antigens may be combined with other immunogenic proteins to increase the immune response [91].

Fimbriae are diverse proteinaceous surface structures. They diverge by their assembly mechanisms and result in different filamentous structures with roles in pathogenesis. However, their roles are not completely understood. They were first known for adherence to cells and inert surfaces, but they seem to be implicated in so much more functions during infection.

The multiplicity of adhesion systems is also an enigma. Most of the *Salmonella* serovars possess 12 fimbrial gene clusters. Some fimbriae are specific to certain serovars and may play a role in these bacteria that do not need to be fulfilled in other serovars. At the opposite, there is a core of fimbrial genes that are present in most of the serovars. Fimbriae are one of the keys to understand *Salmonella* pathogenesis. The specific pattern of each serovar, with further investigations on the sporadic fimbriae, may also bring insights into our understanding of

Regulation of fimbrial genes is a complex network that is tightly related to invasion and motility. Virulence factors are finely regulated, and a temporal expression hierarchy allows the success of *Salmonella* infection. General regulators are already known to regulate fimbrial genes such as stress sensor Rcs relay or the Hfq factor. Phase variation from a fimbriated to afimbriated status occurs in *Salmonella*. However, this phenomenon is not from a promoter inversion of the Fim cluster, but from the regulation by ancillary genes related to *fim* gene cluster. These ancillary genes are themselves precisely regulated by a variety of regulators

The actual understanding of fimbrial expression opens a new area on human health prevention. Some conserved fimbrial genes, in combination with other virulence genes, are precious markers for *Salmonella* detection. These tools could permit a faster diagnostic for human patients, but also a rapid detection of contaminated food or infected animals. Fimbrial proteins can serve as good immunogens in vaccine preparation against *Salmonella* infection.

spleens of hens [90].

72 Current Topics in Salmonella and Salmonellosis

**6. Conclusion**

*Salmonella* pathogenesis.

known for their role in other bacterial processes.

Karine Dufresne and France Daigle\*

\*Address all correspondence to: france.daigle@umontreal.ca

Department of Microbiology, Infectiology and Immunology, Université de Montréal, Montréal, Canada

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**Salmonella Detection and Antibiotic Resistance**

**Provisional chapter**

## **Current and Emerging Innovations for Detection of Food-Borne** *Salmonella* **Food-Borne** *Salmonella*

**Current and Emerging Innovations for Detection of** 

Wei Wu and Lingwen Zeng Wei Wu and Lingwen Zeng Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67264

#### **Abstract**

*Salmonella* is one of the leading causes of food-borne illnesses worldwide, and one of the main contributors to salmonellosis is the consumption of contaminated egg, poultry, pork, beef, and milk products. Since deleterious effects of *Salmonella* on public health and the economy continue to occur, improving safety of food products by early detection of food-borne pathogens would be considered an important component for limiting exposure to *Salmonella* contamination. Therefore, there is an ongoing need to develop more advanced detection methods that can identify *Salmonella* accurately and rapidly in foods before they reach consumers. In the past three decades, there have been increasing efforts toward developing and improving rapid pathogen detection and characterization methodologies for application to food products. In this chapter, we discuss molecular methods for detection, identification, and genetic characterization of *Salmonella* in food. In addition, the advantages and disadvantages of the established and emerging rapid detection methods are addressed here. The methods with potential application to the industry are highlighted in this chapter.

**Keywords:** *Salmonella*, food-borne pathogens, rapid detection, molecular methods, aptamer, antibody

#### **1. Introduction**

Food-borne disease is one of the major public health problems for the food industry, especially in developing countries [1]. Failure to detect food-borne pathogens may lead to a dreadful effect. The World Health Organization (WHO) reported that in 2010 alone 1.8 million people died from diarrheal diseases, a great proportion of these cases can be attributed to contaminated food and drinking water [2]. The Centers for Disease Control and Prevention

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(CDC) have estimated that 48 million cases of food-borne illnesses occur in the United States (US) annually, approximately 128,000 cases require hospitalization, and 3,000 cases result in death [3]. The CDC reported that viruses, bacteria, and parasites are major causative agents for food-borne illnesses. Among these, bacterial agents including *Salmonella*, *Listeria monocytogenes*, and *Escherichia coli* are associated with these cases, being responsible for most of the hospitalizations (63.9%) and deaths (63.7%). Especially, *Salmonella* species were considered as the leading cause for these more severe cases resulting in 35% of the hospitalizations and 28% of the deaths [4]. *Salmonella*, belonging to the family of *Enterobacteriaceae*, are Gram-negative, facultative anaerobic, and nonspore-forming bacilli. The genus *Salmonella* is consisted of two species, *enterica* and *bongori*, with six subspecies of *S*. *enterica*. The different serotypes are divided based on the specific surface molecules O-antigen (O-Ag) and H-antigen (H-Ag) [5]. Collectively, there are over 2500 serotypes of salmonellae capable of causing disease in humans. Most serotypes of the salmonellae could cause gastroenteritis, while a few serotypes of salmonellae would cause severe disease enteric fever, which was characterized as the onset of high fever accompanied with abdominal pain and malaise without diarrhea or vomiting [6]. Commonly, salmonellosis is self-limiting, resolving in about a week. Occasionally, however, the infection becomes systemic, a much more severe disease requiring antibiotic interventions [7]. The dose of *Salmonella* causing infection in humans indicated a wide range for the number of cells required to cause disease, ranged from 105 to 1010 cells. In contrast, enumeration of food products indicate much lower numbers of organisms, as low as ten cells, were present to cause illness [8, 9].

Most human salmonellosis cases are associated with consumption of contaminated egg, poultry, pork, beef, and milk products, which are considered one of the most important reservoirs from which *Salmonella* is passed through the food chain and ultimately transmitted to humans [10]. With increasing consumption of these food products, the number of associated salmonellosis continues to be a public health issue all around the world. It is estimated that 95% of *Salmonella* infections are due to the consumption of contaminated foodstuffs, which suggest that salmonellae may be present at low levels in food but still capable of causing a significant number of infections [11]. Yearly, in the United States, it is estimated that *Salmonella* is responsible for over a million illnesses, 19,000 hospitalizations, and almost 400 deaths. This is in part due to their marked ability to persist in a wide range of varying environmental conditions [12]. For example, *Salmonella* strains can grow in foods stored at low (2–4° C) and high (54° C) temperatures [13].

Since *Salmonella* is a major causative agent for food-associated food-borne illnesses, improving safety of poultry products by early detection of food-borne pathogens would be considered an important component for limiting exposure to *Salmonella* contamination. In order to safeguard the food supply and ensure public health, it is essential to establish rapid, reliable, and sensitive method for *Salmonella* detection. In the past two decades, there has been a thrust to develop rapid methods for identifying and detecting *Salmonella* specifically in foodstuffs [14–17]. This chapter will focus on the current culture-dependent and culture-independent methods for the rapid, accurate detection, identification, and subtyping of salmonellae in foodstuffs.

## **2. Methodologies for detection of** *Salmonella*

#### **2.1. Culture-dependent methods**

(CDC) have estimated that 48 million cases of food-borne illnesses occur in the United States (US) annually, approximately 128,000 cases require hospitalization, and 3,000 cases result in death [3]. The CDC reported that viruses, bacteria, and parasites are major causative agents for food-borne illnesses. Among these, bacterial agents including *Salmonella*, *Listeria monocytogenes*, and *Escherichia coli* are associated with these cases, being responsible for most of the hospitalizations (63.9%) and deaths (63.7%). Especially, *Salmonella* species were considered as the leading cause for these more severe cases resulting in 35% of the hospitalizations and 28% of the deaths [4]. *Salmonella*, belonging to the family of *Enterobacteriaceae*, are Gram-negative, facultative anaerobic, and nonspore-forming bacilli. The genus *Salmonella* is consisted of two species, *enterica* and *bongori*, with six subspecies of *S*. *enterica*. The different serotypes are divided based on the specific surface molecules O-antigen (O-Ag) and H-antigen (H-Ag) [5]. Collectively, there are over 2500 serotypes of salmonellae capable of causing disease in humans. Most serotypes of the salmonellae could cause gastroenteritis, while a few serotypes of salmonellae would cause severe disease enteric fever, which was characterized as the onset of high fever accompanied with abdominal pain and malaise without diarrhea or vomiting [6]. Commonly, salmonellosis is self-limiting, resolving in about a week. Occasionally, however, the infection becomes systemic, a much more severe disease requiring antibiotic interventions [7]. The dose of *Salmonella* causing infection in humans indicated a wide range

enumeration of food products indicate much lower numbers of organisms, as low as ten cells,

Most human salmonellosis cases are associated with consumption of contaminated egg, poultry, pork, beef, and milk products, which are considered one of the most important reservoirs from which *Salmonella* is passed through the food chain and ultimately transmitted to humans [10]. With increasing consumption of these food products, the number of associated salmonellosis continues to be a public health issue all around the world. It is estimated that 95% of *Salmonella* infections are due to the consumption of contaminated foodstuffs, which suggest that salmonellae may be present at low levels in food but still capable of causing a significant number of infections [11]. Yearly, in the United States, it is estimated that *Salmonella* is responsible for over a million illnesses, 19,000 hospitalizations, and almost 400 deaths. This is in part due to their marked ability to persist in a wide range of varying environmental conditions

Since *Salmonella* is a major causative agent for food-associated food-borne illnesses, improving safety of poultry products by early detection of food-borne pathogens would be considered an important component for limiting exposure to *Salmonella* contamination. In order to safeguard the food supply and ensure public health, it is essential to establish rapid, reliable, and sensitive method for *Salmonella* detection. In the past two decades, there has been a thrust to develop rapid methods for identifying and detecting *Salmonella* specifically in foodstuffs [14–17]. This chapter will focus on the current culture-dependent and culture-independent methods for the rapid, accurate detection, identification, and subtyping of salmonellae in

to 1010 cells. In contrast,

C) and high (54°

C)

for the number of cells required to cause disease, ranged from 105

[12]. For example, *Salmonella* strains can grow in foods stored at low (2–4°

were present to cause illness [8, 9].

84 Current Topics in Salmonella and Salmonellosis

temperatures [13].

foodstuffs.

Current testing of food samples for the presence of salmonellae can be divided into three steps: (1) detection of pathogen by plate culture, (2) identification of the isolate and its specific serovar designation, and (3) subtyping of the isolate for association with salmonellosis [18, 19]. These methods rely on traditional bacterial culture procedures that apply serial enrichments with increasing selectivity culminating in the isolation of *Salmonella* on selective differential agar plates (**Figure 1**). It always takes up to 5 days to obtain a presumptive positive result. Then traditional biochemical testing of nutrient utilization medium is needed for confirmation, another few days to complete [20]. Although innovative technologies have been applied to subtype salmonellae isolation, at least 24 h is needed for a confirmation of *Salmonella* in multiple analytes. DNA fingerprinting techniques are based on DNA size differences on an agarose gel. The digested genomic DNA of target bacteria is separated on an agarose gel and then hybridized with complementary sequences for identifying the banding pattern. A database of fingerprint species, serovar, and strain identifications is used for comparison [21–23]. The fingerprinting methods include pulsed-field gel electrophoresis (PFGE), ribotyping, and intergenic sequence (IGS) ribotyping. The use of PFGE has greatly increased the ability of track and trace back illness clusters and outbreaks. However, PFGE still requires a pure isolate and a minimum of 3 days to complete [24, 25].

Due to its sensitivity, with a limit of detection of 1 cfu, this analytical schema is considered as the "gold standard" of regulatory agencies (**Figure 1**). The disadvantages of this method are as follows. First, it is time-consuming, taking at least a week for isolation and few more days for serotyping and subtyping. The long time frame hampers its application in many food commodities, especially fresh products, before they are consumed or on hold in warehouses while awaiting test results before they spoil. Second, the operation is tedious; the amount of media and numerous plates are required for each sample. The procedures are labor-consuming and necessitate large areas of space, particularly in many sample detections. Finally, the complex ingredients in foodstuffs, such as indigenous microbiota and antimicrobials, make it notably difficult for traditional microbiological methods [11, 26–29].

#### **2.2. Culture-independent methods**

Recent advances in technology have made the detection of food-borne pathogens more rapid and convenient, while achieving improved sensitivity and specificity in comparison to conventional methods. These methods employing newer technologies are generally referred as "rapid methods," which include nucleic acid-based or antibody-based assays that are modified or improved compared to conventional methods [30–35]. These rapid detection methods can be of high value to the food industry by providing several key advantages such as speed, specificity, sensitivity, cost-efficiency, and labor efficiency.

**Figure 1.** Overview of Bacteriological Analytical Manual (FDA-BAM) workflow for the detection, isolation, and subtyping of *Salmonella*. It takes 5 days for the detection and isolation of *Salmonella*, and a week more for subsequent confirmation and subtyping recent molecular methods, such as MS, WGS, and PCR/qPCR, may shorten the result time [36].

#### *2.2.1. Polymerase chain reaction (PCR)*

**Figure 1.** Overview of Bacteriological Analytical Manual (FDA-BAM) workflow for the detection, isolation, and subtyping of *Salmonella*. It takes 5 days for the detection and isolation of *Salmonella*, and a week more for subsequent confirmation and subtyping recent molecular methods, such as MS, WGS, and PCR/qPCR, may shorten the result time [36].

86 Current Topics in Salmonella and Salmonellosis

The largest advance toward faster detection of salmonellae has been in the realm of molecular biology, where polymerase chain reaction (PCR) and quantitative PCR (qPCR) are predominantly being applied as the methods of choice for the detection. Different protocols targeting different specific genes or gene regions specific to salmonellae have been published. Numerous studies have been conducted to detect and characterize *Salmonella* in poultry, poultry products, and feeds using PCR assays to target selected antibiotic resistance or virulence genes along with genus-, species-, and serotype-specific genes [16, 37–40].

Over the past years, PCR-based methods have advanced to provide high sensitivity for *Salmonella* detection and identification. Aabo et al. used PCR assay for *Salmonella* detection in minced meat and compared this method to a culture-based methodology. The sensitivity of the PCR was 89% (85 out of 96 samples), which was much higher than that of the culture method (50%, 48 out of 96 samples) [41]. Rychlik et al. established nested PCR with high sensitivity, which has a higher annealing temperature than the primers used in the first PCR, to detect *Salmonella* in chicken feces [42].

As we all know, the quality and quantity of target DNA, PCR template, are important factors during the design of a PCR assay. Although well-designed PCR primer and good PCR template can bring high specificity of the target detection, it is still not sufficient to overcome the side effects of PCR inhibitors in samples, such as denatured proteins, organic chemicals, and sucrose. Moreover, the presence of DNA and cells other than those from the targeted organism can affect the efficiency of the PCR methods. To overcome this, an enrichment step is commonly performed to enhance assay sensitivity by ensuring the detection of viable pathogens before PCR reaction. Ferretti et al. reported that PCR with a 6 h nonselective enrichment could detect various *Salmonella* serotypes in salami stuffs as low as 1 cfu in 100 ml of food homogenate [43, 44]. Myint et al. reported a PCR method for *Salmonella* detection in contaminated poultry tissue samples, and false negative results were obtained without enrichment. However, a positive rate of 90% was observed after enrichment. Generally, culture enrichment is recommended in order to distinguish live cells from dead cells before PCR [45]. Maciorowski et al. investigated different enrichment times to detect indigenous *Salmonella* in poultry dietary samples using PCR. It was found that it could not be detectable for *Salmonella* with 7 h enrichment, and the sensitivity for detection was 25 and 50% with 13 h enrichment and 24 h enrichment, respectively [46].

Improvements have also been made on the basic PCR technology as well. In particular, two primary PCR-based methods have emerged over the past several years, such as multiplex PCR and real-time quantitative PCR [47, 48]. The current status of the optimization and development of these PCR applications is summarized in the following.

Multiplex PCR is a modified PCR method that allows for multiple sequence targets to be simultaneously detected within a single reaction. This method has proven useful for the rapid identification of multiple pathogens simultaneously in a given sample. Generally, multiplex PCR amplifies the target samples using multiple primers in a reaction, which can detect and identify several target sequences in *Salmonella*. Sharma employed a multiplex fluorogenic PCR assay for simultaneous detection of *Salmonella* and *E*. *coli* O157:H7, which was capable of detecting as low as 10 cfu/g in meat [49]. Similarly, Kawasaki detected multiple *Salmonella* serotypes, *L*. *monocytogenes*, and *E*. *coli* O157:H7 simultaneously in enriched meat samples using multiplex PCR [48]. Cortez et al. identified *Salmonella* from chicken abattoirs by multiplex PCR. In this paper, 29 out of 288 (~10%) samples were found to be positive for *Salmonella* spp., and 16 (~5.6%) and 7 (~2.4%) samples were characterized as *Salmonella Typhimurium* and *Salmonella enteritidis*, respectively [50]. Kim differentiated the 30 most prevalent *Salmonella* serotypes in the United States by using two five-plex PCR assays. In this study, primer pairs targeting six genetic loci from *S*. *Typhimurium* and four from *S. Typhi* were designed to evaluate various *Salmonella* serotypes [51]. More recently, Salemis et al. also established two fiveplex assays for the detection of the most common *Salmonella* in Tunisia as well [52]. Although multiplex PCR can simultaneously detect several targets, the primary difficulties are uncommitted, in which reaction conditions are needed optimized as high amounts of DNA in the reaction mixture compared to single PCR-based assays. The complex conditions and ingredients in the reaction still increase the difficulty in discrimination between prominent PCR product sizes on traditional agarose gel electrophoresis. In practice, cross-reactivity of primer pairs and sensitivity limitations associated with the procedure make it still quite challenging to routinely use multiplex PCR for reliable simultaneous *Salmonella* serovar detection [53].

With the appearance of fluorescence technology that endows increased sensitivity (e.g., intercalating dyes such as SYBR Green or labeled probes), the limitations of conventional PCR can be overcome, such as the errors associated with end-point analyses and lack of quantification. The "real-time" aspect of real-time PCR, also referred to as qPCR, technology is linked to its ability to label and cumulatively quantify the generated PCR products at each cycle throughout the ongoing amplification process. The qPCR has been widely used to quantify *Salmonella* [54–56]. Daum screened nine foodstuffs associated with a *Salmonella* outbreak in Texas using qPCR. It was reported that only one food item was positive for *Salmonella* [57]. Wang et al. reported a qPCR method to detect *Salmonella* in raw sausage meat with detection limit of 4 cfu/g [58]. He also used this method to quantify *Salmonella* detection limits of 2.5 cfu/25 g for salmon and minced meat, 5 cfu/25 g of chicken meat, and 5 cfu/25 ml for raw milk, respectively [59]. Malorny et al. reported a duplex qPCR assay to detect *S. enteritidis* in whole chicken carcass rinses and eggs, with a detection limit of 3 cfu/50 ml of chicken carcass rinses and 3 cfu/10 ml of homogenized egg content [60]. Bohaychuk used qPCR for *Salmonella* detection in poultry cecal contents and carcasses with reported sensitivities ranging from 97 to 100% for various matrices [61]. Although qPCR is an effective tool to detect *Salmonella* with high sensitivity and specificity, it does have several limitations, which are listed in **Table 1**.

#### *2.2.2. Enzyme-linked immunosorbent assay (ELISA)*

Enzyme-linked immunosorbent assay (ELISA)-based approaches are the most prevalent antibody-based assay for pathogen detection in foods [62]. This immunological approach has been used to detect *Salmonella* in poultry production (poultry feed, feces, litter, carcass rinsing, and water samples) and has provided a better sensitivity and shorter time frame than that of culture-based methods [46]. Improvements by combination with other advanced technologies have been made to the basic ELISA method for *Salmonella* detection. For example, incorporation of monoclonal antibodies can improve the sensitivity of the assay, and it can quantify *Salmonella* among poultry probiotic bacteria such as *Veillonella* [63]. In this study, the detection limit for *S*. *Typhimurium* was determined to be 5.5 × 104 cells/ml in pure culture. Dill combined monoclonal and polyclonal antibodies and a commercial filtering system to detect *S*. *Typhimurium* cells in a chicken rinsate, with detection limit of fewer than 100 *S*. *Typhimurium* cells [64]. As the advantages of ELISA methods for *Salmonella* detection in foods and animal feeds, they are now widely used for detection of *Salmonella* in animal-producing foods [65]. The comparison of ELISA methods with culture-based methods is performed and listed in **Table 1**.

PCR assay for simultaneous detection of *Salmonella* and *E*. *coli* O157:H7, which was capable of detecting as low as 10 cfu/g in meat [49]. Similarly, Kawasaki detected multiple *Salmonella* serotypes, *L*. *monocytogenes*, and *E*. *coli* O157:H7 simultaneously in enriched meat samples using multiplex PCR [48]. Cortez et al. identified *Salmonella* from chicken abattoirs by multiplex PCR. In this paper, 29 out of 288 (~10%) samples were found to be positive for *Salmonella* spp., and 16 (~5.6%) and 7 (~2.4%) samples were characterized as *Salmonella Typhimurium* and *Salmonella enteritidis*, respectively [50]. Kim differentiated the 30 most prevalent *Salmonella* serotypes in the United States by using two five-plex PCR assays. In this study, primer pairs targeting six genetic loci from *S*. *Typhimurium* and four from *S. Typhi* were designed to evaluate various *Salmonella* serotypes [51]. More recently, Salemis et al. also established two fiveplex assays for the detection of the most common *Salmonella* in Tunisia as well [52]. Although multiplex PCR can simultaneously detect several targets, the primary difficulties are uncommitted, in which reaction conditions are needed optimized as high amounts of DNA in the reaction mixture compared to single PCR-based assays. The complex conditions and ingredients in the reaction still increase the difficulty in discrimination between prominent PCR product sizes on traditional agarose gel electrophoresis. In practice, cross-reactivity of primer pairs and sensitivity limitations associated with the procedure make it still quite challenging to routinely use multiplex PCR for reliable simultaneous *Salmonella* serovar detection [53].

With the appearance of fluorescence technology that endows increased sensitivity (e.g., intercalating dyes such as SYBR Green or labeled probes), the limitations of conventional PCR can be overcome, such as the errors associated with end-point analyses and lack of quantification. The "real-time" aspect of real-time PCR, also referred to as qPCR, technology is linked to its ability to label and cumulatively quantify the generated PCR products at each cycle throughout the ongoing amplification process. The qPCR has been widely used to quantify *Salmonella* [54–56]. Daum screened nine foodstuffs associated with a *Salmonella* outbreak in Texas using qPCR. It was reported that only one food item was positive for *Salmonella* [57]. Wang et al. reported a qPCR method to detect *Salmonella* in raw sausage meat with detection limit of 4 cfu/g [58]. He also used this method to quantify *Salmonella* detection limits of 2.5 cfu/25 g for salmon and minced meat, 5 cfu/25 g of chicken meat, and 5 cfu/25 ml for raw milk, respectively [59]. Malorny et al. reported a duplex qPCR assay to detect *S. enteritidis* in whole chicken carcass rinses and eggs, with a detection limit of 3 cfu/50 ml of chicken carcass rinses and 3 cfu/10 ml of homogenized egg content [60]. Bohaychuk used qPCR for *Salmonella* detection in poultry cecal contents and carcasses with reported sensitivities ranging from 97 to 100% for various matrices [61]. Although qPCR is an effective tool to detect *Salmonella* with high sensitivity and specificity, it does have several limitations, which are listed in **Table 1**.

Enzyme-linked immunosorbent assay (ELISA)-based approaches are the most prevalent antibody-based assay for pathogen detection in foods [62]. This immunological approach has been used to detect *Salmonella* in poultry production (poultry feed, feces, litter, carcass rinsing, and water samples) and has provided a better sensitivity and shorter time frame than that of culture-based methods [46]. Improvements by combination with other advanced technologies have been made to the basic ELISA method for

*2.2.2. Enzyme-linked immunosorbent assay (ELISA)*

88 Current Topics in Salmonella and Salmonellosis


**Table 1.** Advantages and disadvantages of detection methods.

#### *2.2.3. Aptamer-based detection assay*

Besides antibodies, other biomolecules have been investigated to selectively capture and enrich *Salmonella* from cultures, among which aptamer is the most prevalent one [66]. Aptamers are single-stranded oligonucleotides, DNA, or RNA that can fold into unique 3D structures based on their primary nucleotide sequence, rendering them capable of binding to specific ligands, like antibody interacting with an antigen [67]. Aptamers offer some advantages over antibodies in that they are relatively inexpensive to synthesize and they provide more batch-to-batch consistency [68]. However, few studies have reported their specific use in detecting *S. Typhimurium* from river water and fecal samples [66, 69]. Bacteriophages have also been explored as a means to capture *Salmonella* cells. Phages may offer some advantages over antibodies given their inherent specificity for host cells, their ease of production in bacteria versus animals or eukaryotic cell culture, and their relative stability in harsh conditions such as pH and temperature extremes [70].

Relative to culture-independent detection, researchers have focused on methods to concentrate whole cells within the sample before the pre-enrichment step. The enriched whole *Salmonella* allows for direct detection from food and environmental samples. The enrichment steps mainly rely on filtering liquids, rinsates, or mechanically disintegrated (i.e., blended or stomached) samples. Therefore, this approach has been widely used in large volumes of water, but the testing of food samples was problematic due to the food particles difficult to go through filter membranes [71]. To overcome this problem, endopeptidases have been added to apply in food samples. These degrade the small, soluble proteins and peptides so that they are unable to clog the filter and pass through with the permeate. The United States has awarded the method with grant prize. The Food and Drug Administration also recommends the method for food safety guard, (http://www.foodsafetychallenge.com), which signified its potential to greatly enhance the detection of *Salmonella* directly from foods.

#### **2.3. Conclusion**

In summary, the mentioned methods here have utility advantages for *Salmonella* detection in the food safety sector. It is important to emphasize that none of the methods will be recommended or even suited for every situation in detecting all food varieties for *Salmonella*. Application to specific food samples will be dictated by method performance. As noted previously, the performance of these methods depends on several factors, such as matrix-driven effects, general specificity and sensitivity, and their technical complexity. Meanwhile, other extrinsic factors would affect the performance, including user skill set and technical prowess, cost of the equipment, and cost per sample. Hence, the systematic validation to evaluate the methods should be considered according to its specific utility and application across the food supply.

In order to meet the current requirement of rapid detection, it is clear that several approaches have emerged including PCR-based, antibody-based, aptamer-based, and other approaches encompassing those stemming from the current genomic era. A clear character of method development direction is moving toward greater automation, cost-saving, and time-saving network integration. It is important to mention that outputs from one approach would serve to strengthen directly or tangentially other approaches. At last, it seems that a suite of tools is emerging for the food safety microbiologist, each with its specific advantages and disadvantages but all with the ability to rapidly and accurately detect *Salmonella* in certain cases and early in its contamination of the human and veterinary food supply.

## **Author details**

*2.2.3. Aptamer-based detection assay*

90 Current Topics in Salmonella and Salmonellosis

such as pH and temperature extremes [70].

**2.3. Conclusion**

supply.

Besides antibodies, other biomolecules have been investigated to selectively capture and enrich *Salmonella* from cultures, among which aptamer is the most prevalent one [66]. Aptamers are single-stranded oligonucleotides, DNA, or RNA that can fold into unique 3D structures based on their primary nucleotide sequence, rendering them capable of binding to specific ligands, like antibody interacting with an antigen [67]. Aptamers offer some advantages over antibodies in that they are relatively inexpensive to synthesize and they provide more batch-to-batch consistency [68]. However, few studies have reported their specific use in detecting *S. Typhimurium* from river water and fecal samples [66, 69]. Bacteriophages have also been explored as a means to capture *Salmonella* cells. Phages may offer some advantages over antibodies given their inherent specificity for host cells, their ease of production in bacteria versus animals or eukaryotic cell culture, and their relative stability in harsh conditions

Relative to culture-independent detection, researchers have focused on methods to concentrate whole cells within the sample before the pre-enrichment step. The enriched whole *Salmonella* allows for direct detection from food and environmental samples. The enrichment steps mainly rely on filtering liquids, rinsates, or mechanically disintegrated (i.e., blended or stomached) samples. Therefore, this approach has been widely used in large volumes of water, but the testing of food samples was problematic due to the food particles difficult to go through filter membranes [71]. To overcome this problem, endopeptidases have been added to apply in food samples. These degrade the small, soluble proteins and peptides so that they are unable to clog the filter and pass through with the permeate. The United States has awarded the method with grant prize. The Food and Drug Administration also recommends the method for food safety guard, (http://www.foodsafetychallenge.com), which signified its

In summary, the mentioned methods here have utility advantages for *Salmonella* detection in the food safety sector. It is important to emphasize that none of the methods will be recommended or even suited for every situation in detecting all food varieties for *Salmonella*. Application to specific food samples will be dictated by method performance. As noted previously, the performance of these methods depends on several factors, such as matrix-driven effects, general specificity and sensitivity, and their technical complexity. Meanwhile, other extrinsic factors would affect the performance, including user skill set and technical prowess, cost of the equipment, and cost per sample. Hence, the systematic validation to evaluate the methods should be considered according to its specific utility and application across the food

In order to meet the current requirement of rapid detection, it is clear that several approaches have emerged including PCR-based, antibody-based, aptamer-based, and other approaches encompassing those stemming from the current genomic era. A clear character of method development direction is moving toward greater automation, cost-saving, and time-saving

potential to greatly enhance the detection of *Salmonella* directly from foods.

Wei Wu<sup>1</sup> and Lingwen Zeng2 \*

\*Address all correspondence to: zeng\_lingwen@gibh.ac.cn

1 Institute for Translational Medicine, Qingdao University, Qingdao, China

2 Institute of Environment and Safety, Wuhan Academy of Agriculture Sciences, Wuhan, China

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**Provisional chapter**

## **Factors Contributing to the Emergence and Spread of Antibiotics Resistance in** *Salmonella* **Species Factors Contributing to the Emergence and Spread of Antibiotics Resistance in** *Salmonella* **Species**

Kabiru Olusegun Akinyemi and Samuel Oluwasegun Ajoseh Oluwasegun Ajoseh Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Kabiru Olusegun Akinyemi and Samuel

http://dx.doi.org/10.5772/67701

#### **Abstract**

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*Salmonella,* a genus of the family *Enterobacteriaceae* with over 2450 species, has been responsible for diseases ranging from non-typhoidal salmonellosis to typhoidal salmonellosis. Several groups of antibiotics such as β-lactams, aminoglycosides, tetracyclines, quinolones, cephalosporins and sulfonamides are used against *Salmonella* species. Many *Salmonella* species had developed resistance to several antibiotics over the years. Two major groups of mechanism of resistance demonstrated by this pathogen are (1) Biochemical Mechanisms; such as enzymatic inactivation, prevention of access to the target site by antibiotics and active efflux pumps. (2) Genetic mechanisms; such as mutation, horizontal gene transfer and vertical gene transfer. Some factors identified to contribute to the emergence and dissemination of antibiotic resistant-*Salmonella* include; miss-used of antibiotics, used of antibiotics in agriculture, unregulated sales of antibiotics, inappropriate prescription and dispensing practices, and poor hygiene practices (external or behavioural factors), the presence of mobile genetic elements in the organisms; plasmid DNA, transposons, integrons etc. The clinical and public health consequences, and the strategies to stem the growing tides associated with drugs resistance in Salmonella species are herein discussed. A more radical approach and commitment from the policy makers in health sector to solving problems emanating from increasing spread of resistant *Salmonella* is advocated.

**Keywords:** *Salmonella*, resistance, antibiotics, factors, chromosomes, plasmid

## **1. Introduction**

*Salmonella* are Gram-negative, facultative anaerobes, rod-shaped bacteria and are trivially known as 'enteric bacteria' [1], with over 2587 serotypes. *Salmonella* are grouped into two basic species namely, *Salmonella enterica* and *Salmonella bongori* [2, 3]. *Salmonella* generally cause a disease

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

termed salmonellosis, which are both typhoidal and non-typhoidal in nature. Moreover, investigation of the molecular mechanisms of *Salmonella* virulence factors have shown that pathogenic *Salmonella* species are distinguished from non-pathogenic relatives by the presence of specific pathogenicity genes, often called pathogenicity island (PIs), which contributes to both natural and acquired resistance in *Salmonella* species [4, 5]. However, the ability of *Salmonella* to cause invasive infection varies with serovars, the age of the patient and environmental factors [6].

Drug resistance among *Salmonella* serotypes has been a public health concerns at global level [7]. This could be intrinsic (natural resistance) as seen in *S. enterica* serotype typhimurium definitive phage type (DT) 104 that contains the chromosomal *Salmonella*, genomic island type 1 (SGI-1), which harbours resistance genes that confer *ACSST* phenotype (resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline) [8, 9]. Also, the intrinsic resistance could be as a result of impermeability of bacterial cell wall to antibiotic of interest [9]. Several factors have been identified to contribute to the acquired resistance in *Salmonella*, which include: inappropriate use of antibiotic (either through over-prescription, incomplete course of treatment or inadequate dosing, etc.), use of antimicrobial agents in agriculture (either as growth promoter or for prophylaxis purposes), hospital, mutation and transferable genetic materials (plasmid, transposons and integron) [1, 9, 10]. These factors are responsible for the acquisition (emergence) and dissemination (spread) of resistance in *Salmonella* species. Various mechanisms of antimicrobial inactivation exist in *Salmonella* spp., which lead to the emergence of multi-drug resistance (MDR) strains [11, 12]. Some of these mechanisms are as follows:


Mobile genetic elements such as plasmids, transposons and integrons play an important role in the evolution (emergence) and dissemination (spread) of multi-drug resistance by either horizontal or vertical gene transfer [13]. The role of integrons in the acquisition and dissemination of resistance in *Salmonella* species is crucial. Integrons are DNA elements that contain collection of genes (gene cassette). Integrons are frequently associated with plasmid, transposon and are therefore easily transferable among *Salmonella* and/or between different bacteria [14].

The presence of virulence encoded plasmid DNA *spv*A, *spv*B and *spv*C in several *Salmonella* serovars had been documented and the outer membrane proteins (OMPs) of *Salmonella* typhimurium have a role in the virulence of the organism and are potent candidate for vaccine development since it is immunogenic, capable of evoking both humoral and cell-mediated immune response. These OMPs indirectly play part in intrinsic resistance and can be disseminated between or among *Salmonella* species [15, 16]. Thus, resistance acquisition or dissemination in *Salmonella* species usually involves several factors [1, 13].

## **2.** *Salmonella* **virulence and mechanisms of resistance**

#### **2.1.** *Salmonella* **and its virulence factors**

termed salmonellosis, which are both typhoidal and non-typhoidal in nature. Moreover, investigation of the molecular mechanisms of *Salmonella* virulence factors have shown that pathogenic *Salmonella* species are distinguished from non-pathogenic relatives by the presence of specific pathogenicity genes, often called pathogenicity island (PIs), which contributes to both natural and acquired resistance in *Salmonella* species [4, 5]. However, the ability of *Salmonella* to cause invasive infection varies with serovars, the age of the patient and environmental factors [6].

Drug resistance among *Salmonella* serotypes has been a public health concerns at global level [7]. This could be intrinsic (natural resistance) as seen in *S. enterica* serotype typhimurium definitive phage type (DT) 104 that contains the chromosomal *Salmonella*, genomic island type 1 (SGI-1), which harbours resistance genes that confer *ACSST* phenotype (resistance to ampicillin, chloramphenicol, streptomycin, sulphonamide and tetracycline) [8, 9]. Also, the intrinsic resistance could be as a result of impermeability of bacterial cell wall to antibiotic of interest [9]. Several factors have been identified to contribute to the acquired resistance in *Salmonella*, which include: inappropriate use of antibiotic (either through over-prescription, incomplete course of treatment or inadequate dosing, etc.), use of antimicrobial agents in agriculture (either as growth promoter or for prophylaxis purposes), hospital, mutation and transferable genetic materials (plasmid, transposons and integron) [1, 9, 10]. These factors are responsible for the acquisition (emergence) and dissemination (spread) of resistance in *Salmonella* species. Various mechanisms of antimicrobial inactivation exist in *Salmonella* spp., which lead to the emergence of multi-drug resistance (MDR) strains [11, 12]. Some of these mechanisms are as follows:

Mobile genetic elements such as plasmids, transposons and integrons play an important role in the evolution (emergence) and dissemination (spread) of multi-drug resistance by either horizontal or vertical gene transfer [13]. The role of integrons in the acquisition and dissemination of resistance in *Salmonella* species is crucial. Integrons are DNA elements that contain collection of genes (gene cassette). Integrons are frequently associated with plasmid, transposon and are therefore easily transferable among *Salmonella* and/or between different bacteria [14]. The presence of virulence encoded plasmid DNA *spv*A, *spv*B and *spv*C in several *Salmonella* serovars had been documented and the outer membrane proteins (OMPs) of *Salmonella* typhimurium have a role in the virulence of the organism and are potent candidate for vaccine development since it is immunogenic, capable of evoking both humoral and cell-mediated immune response. These OMPs indirectly play part in intrinsic resistance and can be

(i) Enzymatic inactivation of the drug (e.g. β-lactamase)

(ii) Prevention of access to the target agent

(iv) Novel penicillin binding protein (PBPs)

(iii) Change or mutation in the target site

(v) Altered membrane permeability

98 Current Topics in Salmonella and Salmonellosis

(viii) Creation of biofilm barriers, etc.

(vi) Active efflux pumps (vii) Ribosome alteration

The ability of *Salmonella* to attach itself to the host, invade and penetrate intestinal epithelial cell is determined by its virulence factor [17]. Adherence of *Salmonella* is often mediated by fimbriae and/or non-fimbrial adhesion (lipopolysaccharide) [11]. Invasion process is not merely a passive consequence of bacterial contact with epithelial cells, but instead requires the active production and transport of secreted effector proteins by a Type III secretion system (T3SS) 1 & 2 and Type 1 secretion systems, which are encoded in *Salmonella* pathogenicity island I & 2 (SPI-I & 2), respectively [17]. In addition, invasion is also induced by flagella/ flagellin since *Salmonella* is a flagellated facultative anaerobe. Many invasion regulators have been described, for examples, *Hil*A [18], *Hil*C [19], InvF [20], PhoP/PhoQ, *Hil*E [21], H-NS [19] and *Inv*A genes, PhoP/PhoQ pair is also essential for the expression of genes in *Salmonella* pathogenicity island 2 (SP1-2), which encodes a second Type III secretory system. SP1-2 is required for intra-macrophage survival, which is a cell-type encountered by *Salmonella* immediately after the invasion of epithelium. PhoP/PhoQ also serves to repress SP1-1 genes, a function mediated by H*il*A [22]. PhoP/PhoQ may thus act as a genetic switch, activating traits required for macrophages survival while repressing those not needed for invasion [17].

Other factors that are involved in *Salmonella* virulence are the MgtC in *Salmonella* typhimurium. This is required for growth at low—Mg2+ concentrations and intra-macrophage survival. Iron acquisition (for iron deprivation survival) this is achieved by the production of two siderophores, which are enterobactin and salmochelin in response to iron deprivation. Superoxide dismutase is used to counteract the reactive oxygen produced through the activity of the phagosome NADPH oxidase (NOX₂) that are required for the killing of intracellular pathogens and enterotoxin (responsible for food-intoxication) [23]. The genes coding for the above-mentioned factors and others are conserved in *Salmonella* pathogenicity island 3 (SP1-3), and is also present in the chromosomes of other *Salmonella enterica* serovars [23]. It has been reported by several researchers [15, 24, 25], that *stn* genes coding for *Salmonella* enterotoxin) *sef* genes for *Salmonella* Enteritidis fimbriae, and *pef* genes for plasmid encoded fimbriae are widely distributed in resistant *Salmonella* strains and are responsible for *Salmonella*-associated diseases in animal and human populations globally.

#### **2.2. Resistance mechanisms exhibited by** *Salmonella* **species to some antibiotics**

Various mechanisms of anti-microbials inactivation have been reported by [11, 26], which invariably lead to the emergence of multidrug resistance in *Salmonella* species. These mechanisms are summarized into two broad groups, namely:


#### *2.2.1. Biochemical mechanisms of antibiotic resistance by Salmonella species*

*Enzymatic inactivation*: This may result into either destruction of antimicrobial agents, such as occurs with the β-lactamases, or lead to a major modification of the antibiotic so that it does not bind to its target as it's seen with the aminoglycoside and chloramphenicol [1]. The major mechanisms of resistance to beta-lactam antibiotics among *Enterobacteriaceae* involve production of β-lactamase or extended spectrum β-lactamase (ESBLs) [1]. ESBLs have traditionally been defined as transmissible β-lactamases that can be inhibited by clavulanic acid, tazobactam or sulbactam. They are group of enzymes that break down antibiotics belonging to the penicillin and cephalosporin groups and render them ineffective [1]. ESBLs are generally encoded by mobile genes that can be exchanged between bacteria [27]. It has been noted that when ESBL strains occur, they often have co-resistance with the aminoglycosides (gentamicin), tetracycline and trimethoprim/sulphamethoxazole [27]. CTX–M ESBLs arise by plasmid acquisition of pre-existing chromosomal ESBL genes; this proved that ESBL can be plasmid mediated and thus capable of spreading to other microorganisms of either related species or genera [28].

*Prevention of access to the target site*: This may be by substitutions, amplifications or modification of the drug target, thereby reducing the affinity of the drug to the target. For example, in *Salmonella*, the outer membrane proteins may be altered such that antibiotics are unable to cross its cell wall [26]. Gram-negative bacteria can regulate membrane permeability by altering expression of outer membrane porin (omp) proteins that form channels for passive diffusion. Loss or reduced levels of ompF has been implicated in anti-microbial resistance in *Salmonella* over the years [29].

*Active efflux pumps*: This involves the expellant of multiple kinds of antibiotics out of the cytoplasm of the microorganism to the external environment [11]. Increased expression of non-specific, energy-dependent efflux systems allow bacteria to prevent the accumulation of effective concentrations of quinolones inside the cell by actively pumping out the drug. In *Escherichia coli*, the AcrAB-TolC efflux pump plays a major role in quinolone efflux and studies suggest that this may be the primary mechanism of fluoroquinolone resistance in *Salmonella* [30]. It is thoughtful that these efflux systems cause low-level resistance to quinolones that can become clinically relevant when combined with mutations in the target enzymes [31].

*Reduced permeability of the antimicrobial agent*: This is a common mechanism of anti-microbial resistance usually exhibited my *S. enterica*. It involves the alteration in membrane permeability, which occurs when new genetic information, change the nature of proteins in the membrane. This alteration changes a membrane transport system pores in the membrane for an anti-microbial agent not to be able to cross the membrane. This form of resistance mechanism has been discovered in *Salmonella* typhi to tetracycline, quinolones and some aminoglycosides and sulphonamide antibiotics [32, 33].

#### *2.2.2. Genetics mechanisms of antibiotics resistance in Salmonella species*

The genes coding for antibiotics resistance and virulence at times share common features of being located in the bacterial chromosome, as well as on plasmid (**Tables 1** and **2**). They are associated in gene clusters to form resistance or pathogenicity island, which are transferred by mobile genetic elements such as integrons, transposons or phage [34].

The major genetics mechanisms are as follows:

• Mutation

*2.2.1. Biochemical mechanisms of antibiotic resistance by Salmonella species*

*Salmonella* over the years [29].

100 Current Topics in Salmonella and Salmonellosis

and sulphonamide antibiotics [32, 33].

*Enzymatic inactivation*: This may result into either destruction of antimicrobial agents, such as occurs with the β-lactamases, or lead to a major modification of the antibiotic so that it does not bind to its target as it's seen with the aminoglycoside and chloramphenicol [1]. The major mechanisms of resistance to beta-lactam antibiotics among *Enterobacteriaceae* involve production of β-lactamase or extended spectrum β-lactamase (ESBLs) [1]. ESBLs have traditionally been defined as transmissible β-lactamases that can be inhibited by clavulanic acid, tazobactam or sulbactam. They are group of enzymes that break down antibiotics belonging to the penicillin and cephalosporin groups and render them ineffective [1]. ESBLs are generally encoded by mobile genes that can be exchanged between bacteria [27]. It has been noted that when ESBL strains occur, they often have co-resistance with the aminoglycosides (gentamicin), tetracycline and trimethoprim/sulphamethoxazole [27]. CTX–M ESBLs arise by plasmid acquisition of pre-existing chromosomal ESBL genes; this proved that ESBL can be plasmid mediated and thus capable of spreading to other microorganisms of either related species or genera [28].

*Prevention of access to the target site*: This may be by substitutions, amplifications or modification of the drug target, thereby reducing the affinity of the drug to the target. For example, in *Salmonella*, the outer membrane proteins may be altered such that antibiotics are unable to cross its cell wall [26]. Gram-negative bacteria can regulate membrane permeability by altering expression of outer membrane porin (omp) proteins that form channels for passive diffusion. Loss or reduced levels of ompF has been implicated in anti-microbial resistance in

*Active efflux pumps*: This involves the expellant of multiple kinds of antibiotics out of the cytoplasm of the microorganism to the external environment [11]. Increased expression of non-specific, energy-dependent efflux systems allow bacteria to prevent the accumulation of effective concentrations of quinolones inside the cell by actively pumping out the drug. In *Escherichia coli*, the AcrAB-TolC efflux pump plays a major role in quinolone efflux and studies suggest that this may be the primary mechanism of fluoroquinolone resistance in *Salmonella* [30]. It is thoughtful that these efflux systems cause low-level resistance to quinolones that can

become clinically relevant when combined with mutations in the target enzymes [31].

*2.2.2. Genetics mechanisms of antibiotics resistance in Salmonella species*

*Reduced permeability of the antimicrobial agent*: This is a common mechanism of anti-microbial resistance usually exhibited my *S. enterica*. It involves the alteration in membrane permeability, which occurs when new genetic information, change the nature of proteins in the membrane. This alteration changes a membrane transport system pores in the membrane for an anti-microbial agent not to be able to cross the membrane. This form of resistance mechanism has been discovered in *Salmonella* typhi to tetracycline, quinolones and some aminoglycosides

The genes coding for antibiotics resistance and virulence at times share common features of being located in the bacterial chromosome, as well as on plasmid (**Tables 1** and **2**). They are


Resistance in enteric bacteria: *Salmonella, E. coli, Shigella*, spp. etc., can be a result of gene mutation (a permanent change in the DNA of an organisms), which had been detected through several research studies globally or transfer of resistance determinant (R-determinants) between the same species or different species (Horizontal gene transfer) or by transfer of resistance genes from parental microorganism to its progeny or offspring (Vertical gene transfer) [13, 35]. Clinically, chromosomal and plasmid-mediated resistance in *Salmonella* to gentamicin and Betalactam antibiotics had been reported in some host animal and humans [35–37].

The role of integrons in the acquisition and dissemination of resistance in enteric bacteria such as *Salmonella* is very crucial. Integrons are genetic elements that capture and incorporate gene cassettes by using a site-specific recommendation mechanism [38]. The class 1 and class 2 integrons are known to play specific role in anti-book resistance in *Salmonella* spp., which usually contain conserved segments. For example, integron class 1 has been reported to carry *aad*A2 , *bla* and *pse*1 cassette [39, 40]. Most of these integrons are located within transposons that contribute to vertical transmission, favouring their mobilization between plasmid and


Adapted from Refs. [1, 40].

**Table 1.** Properties of some common antibacterial drugs commonly used against *Salmonella* species.


**Table 2.** Antibiotic resistance genes and resistance mechanisms found in *Salmonella* species.

the bacterial chromosome by transposition events [14]. They have the ability to integrate stably into regions of other DNA, where they deliver, in a single exchange multiple new genes, particularly for drug resistance [41]. Many of the gene cassettes in resistance integrons, probably originated from super-integron (larger integron structures with hundreds of accessory genes), which encode for resistance against newer antibiotics such as cephalosporin and carbapenems [22].

#### **2.3. Antibiotics commonly used for the treatment of salmonellosis and their mechanisms of actions**

Some groups of antibiotics used in the treatment of salmonellosis globally for public health purposes as shown in **Table 1** include:

*Aminoglycosides: Salmonella* resistance to aminoglycosides is usually by enzymatic modification and binding to the 30S ribosomal subunit, resulting in the inhibition of protein synthesis in the organism aminoglycoside phosphotransferase confirms resistance to Kanamycin and Neomycin. Major, resistance genes include *str*A, *str*B, *aac, aad*, etc. [42, 43].

*Tetracycline*: Tetracycline targets the 30S ribosomal subunit of the bacteria ribosome just as aminoglycosides. Resistance mechanisms include efflux, modification of rRNA and inactivation of the compound [43]. In *Salmonella*, active efflux systems are most commonly observed and it includes *tet*A, B, C, D, G and H [43].

*β-lactams*: β-lactams prevent synthesis and maintenance of the peptidoglycan component of the bacteria cell wall by mimicking one of the building blocks used by enzymes to construct peptidoglycan. Most resistance to β-lactams is confirmed by β-lactamase that enzymatically cleaves the β-lactams ring and prevents it from bonding to and inactivating cell wall enzymes. Furthermore, extended spectrum β-lactamase is an important group of β-lactamases newly discovered not more than one decade ago [44]. However, other resistance mechanisms reported in major regions across the globe include efflux of the β-lactams and modification of porin (e.g. *omp*F and *omp*C) [42, 45].

*Phenicols*: Phenicols, e.g. chloramphenicol and related compound such as florphenicol, inhibit protein synthesis by binding to the 50S ribosome subunit. Resistance to chloramphenicol is highly prevalence in developing countries based on its cheapness and easy accessibility in the counter e.g. Nigeria [10, 44], despite its ban in developed countries, e.g. USA, based on it toxicity [43]. Most resistance mechanisms exhibited by *Enterobacteriaceae* including *Salmonella* are efflux pumps such as *flo*R and *cml*A as well as inactivating enzymes such as chloramphenicol acetyltransferase *cat*1 [46].

*Sulphonamides*: They are also called *folate pathway inhibitors*. These are compounds that compete for substrate of the essential folic acid pathway in bacteria at two different steps, and the sulphanilamide inhibits DHPS (dihydrofolate reductase). Sulphonamides are bacteriostatic when used alone or bactericidal when combined with trimethoprim-sulphamethoxazole [47]. Resistance to both of these antimicrobials occurs by acquisition of gene-encoding enzymes that do not bind these compounds [43], these include, the *sul* genes eg *sul*1, *sul*2 and *sul*3, which encode for insensitive DHPs enzymes, and are found in *Salmonella* globally [46].

#### **2.4. Phenotypic and genotypic detection of resistance in** *Salmonella*

the bacterial chromosome by transposition events [14]. They have the ability to integrate stably into regions of other DNA, where they deliver, in a single exchange multiple new genes, particularly for drug resistance [41]. Many of the gene cassettes in resistance integrons, probably originated from super-integron (larger integron structures with hundreds of accessory genes), which encode for resistance against newer antibiotics such as cephalosporin and car-

Some groups of antibiotics used in the treatment of salmonellosis globally for public health

*Aminoglycosides: Salmonella* resistance to aminoglycosides is usually by enzymatic modification and binding to the 30S ribosomal subunit, resulting in the inhibition of protein synthesis

**2.3. Antibiotics commonly used for the treatment of salmonellosis and their** 

**Resistance genes Resistance genes** 

Aminoglycosides *aacC(3), aacC(3')-*

102 Current Topics in Salmonella and Salmonellosis

*strB*

*parE*,

Tetracycline *tet(A), tet(B), tet(C),* 

*tet(H)*

β-Lactams *omp*C, *omp*F,

Quinolones and floro-quinolones

Phenicol eg Chloramphenicol *IIa, aacC(6'), aacC2, aadA, aadA1, aadA2, aadA12, aadB, ant(3")-Ia, aphAI, aphAI IAB, aph(3)- Ii-iv, aph(3)-IIa, strA,* 

*bla*CMY-2, *bla*PSE-1, *bla*TEM-1, *bla*SHV-1, *bla*OXA-1, *bla*NDM-1

*GryA, GyrB, parC,* 

*tet(D), tet(G)*, and

Sulphanamides *Sul1, sul2 sul*3, *dfr* CH, P Dihydropteroate

*floR, cmlA, cat1* CH, P Efflux pumps

**Table 2.** Antibiotic resistance genes and resistance mechanisms found in *Salmonella* species.

**location(s)**

CH, P Enzymatic modification

CH, P β-Lactamases, ESBL,

CH, P Mutation in the

P Efflux pumps,

*parC, pare*

compound

synthase

(*floR, cmlA*) and chloramphenicol acetyltransferase

and inactivation of aminoglycoside

Modification of porin (*ompF)*, Efflux of β-lactam(*ompC*)

Quinolones Resistance Determining Region (QRDR) *GryA, GyrB,* 

Modification of rRNA target, Inactivation of

**Resistance mechanisms Region(s) References**

Across the Globe [33, 42]

Across the Globe [33, 42, 44]

Across the Globe [33, 42, 45]

Across the Globe [42–44]

Across the Globe [42, 45]

Across the Globe [30]

bapenems [22].

**mechanisms of actions**

purposes as shown in **Table 1** include:

Drug or antibiotic resistance is the decreased sensitivity of microbes to drug or antibiotics that are capable of causing cell death or inhibition of growth [48]. This is determined through antimicrobial sensitivity testing of *Salmonella* species (isolates) in order to determine its susceptibility or resistance to the antibiotics [49]. Resistance in *Salmonella* is encoded by genes that are present on either chromosome or extra-chromosomal DNA (plasmid) or transferable genetic materials (transposons, integrons), which is determined by genetic or molecular method [50]. The most common method is the Kirby-Bauer method [49]. Although resistance may occur due to mutation in key genetic loci in the bacterial genome, but most resistance to antimicrobial agents mediated by genes are acquired via mobile genetic elements such as plasmid and transposons [50]. The identification of resistance genotype is accomplished through detection of novel genetic materials and characterization of mutations in specific genes through polymerase chain reaction (PCR). Several genetic methods including DNA probes, PCR and other amplification techniques are now used in varieties of clinical laboratories for identification and quantification of pathogenic organisms [51, 52].

#### **2.5. Some identified factors for the emergence and spread of resistance in** *Salmonella* **species**

The emergence of resistance is the natural response of microbes to the presence of antimicrobial agents [53, 54]. Several factors contribute to the increase in multi-drug resistance by *Salmonella* species, which can be grouped into two, namely:


#### *2.5.1. Behavioural factors*

These involve attitudinal conduct of the prescribers (Doctors), dispensers (Pharmacist), patients, agriculturists and/or government to prescription, sales, usage and regulation of antibiotics. These are elucidated as follows:

*Inappropriate prescribing and dispensing*: Lack of access to update information makes prescribers to prescribe less rationally [55]. Economic incentive and enticement from pharmaceutical companies further pressure the prescriber to prescribe unnecessarily or inappropriately [56]. Moreover, it is a common practice in many developing countries for antibiotics to be dispensed without a prescription. Also, over the counter sales of antibiotics is common. These practices had been attributed to weak enforcement of laws in such countries with resultant increase in acquisition of drug resistance in *Salmonella* species and high morbidity and mortality of *Salmonella*-associated diseases [10, 56].

*Patients*: Patient attitude contributes to the emergences of resistance through poor compliance to the prescribed course of treatment [1], especially if their symptoms are mild and resolved quickly [57]. The attitudes of self-medication in most patients has seriously contributed to the emergence of MDR in *S*. typhi to most first line drugs such as chloramphenicol, cephalosporin, streptomycin, tetracycline, ampicillin, etc. [10]. More also, poor hygiene practices of handling raw animal product and food in general with inadequate heat treatment has greatly contributed to the spread of antibiotic resistant strains from animal products and food to human [58, 59]. Furthermore, improper cooking methods; re-heating of food by food handlers in restaurants and canteens have been identified as also a major factor responsible for the spread of multidrug resistant *Salmonella* species since most of developing countries (e.g. Nigeria) population live below the average level of \$1 per day meal, hence they resort to patronizing restaurants and canteens of questionable cooking standards [60, 61].

*Hospitals and laboratories* (*medical centres*): Majority of antimicrobials usages occur in the community, of which most intense usage occurs in hospital [64]. The strong selective pressure together with the multitude of opportunities for resistant strains of *Salmonella* to spread from patient to patient is of high increase, this then means that hospital-acquired infections (Nosocomial infection) are mainly caused by multi-drug resistant strains, as seen in *S*. typhi in the case of typhoid fever [10]. Early discharges from hospitals either through changes in practice (e.g. cost reduction in developed countries) or lack of patient's ability to pay (particularly in developing countries) contribute to the emergence and dissemination of multi-drug resistance strains of *Salmonella* species [56]. However, hospitals work based on laboratory diagnosis must of the time not relying on clinical diagnosis alone since the former is the most reliable means of diagnosing a patient. But it is in the other way in developing countries based on exclusive reliance on Widal test as a means of diagnosing typhoid fever, which can be misleading as individuals with pyrexia are assumed and erroneously diagnosed as having typhoid fever based on single Widal agglutination test [62].

*Government*: contributes to the emergence of resistance in *Salmonella* species and other disease causing organisms that are of public health concern in the following perspective:


(PCR). Several genetic methods including DNA probes, PCR and other amplification techniques are now used in varieties of clinical laboratories for identification and quantification of patho-

The emergence of resistance is the natural response of microbes to the presence of antimicrobial agents [53, 54]. Several factors contribute to the increase in multi-drug resistance by

• *Behavioural factors* (external factors): which include misuse of antibiotics, use of antibiotics in agriculture, unregulated sales of antibiotics, inappropriate prescription and dispensing

• *Genetics factors*: the mobile genetic elements, which include plasmids, transposons, inte-

These involve attitudinal conduct of the prescribers (Doctors), dispensers (Pharmacist), patients, agriculturists and/or government to prescription, sales, usage and regulation of anti-

*Inappropriate prescribing and dispensing*: Lack of access to update information makes prescribers to prescribe less rationally [55]. Economic incentive and enticement from pharmaceutical companies further pressure the prescriber to prescribe unnecessarily or inappropriately [56]. Moreover, it is a common practice in many developing countries for antibiotics to be dispensed without a prescription. Also, over the counter sales of antibiotics is common. These practices had been attributed to weak enforcement of laws in such countries with resultant increase in acquisition of drug resistance in *Salmonella* species and high morbidity and mortal-

*Patients*: Patient attitude contributes to the emergences of resistance through poor compliance to the prescribed course of treatment [1], especially if their symptoms are mild and resolved quickly [57]. The attitudes of self-medication in most patients has seriously contributed to the emergence of MDR in *S*. typhi to most first line drugs such as chloramphenicol, cephalosporin, streptomycin, tetracycline, ampicillin, etc. [10]. More also, poor hygiene practices of handling raw animal product and food in general with inadequate heat treatment has greatly contributed to the spread of antibiotic resistant strains from animal products and food to human [58, 59]. Furthermore, improper cooking methods; re-heating of food by food handlers in restaurants and canteens have been identified as also a major factor responsible for the spread of multidrug resistant *Salmonella* species since most of developing countries (e.g. Nigeria) population live below the average level of \$1 per day meal, hence they resort to patronizing restaurants and canteens of questionable cooking

**2.5. Some identified factors for the emergence and spread of resistance in** *Salmonella*

*Salmonella* species, which can be grouped into two, namely:

practices and poor hygiene practices.

biotics. These are elucidated as follows:

ity of *Salmonella*-associated diseases [10, 56].

genic organisms [51, 52].

104 Current Topics in Salmonella and Salmonellosis

**species**

grons, etc.

*2.5.1. Behavioural factors*

standards [60, 61].


It should also be noted, that lack of information about prevalence of resistance problems or poor supply chain management or long-term facilities (poor diagnostic facilities) contribute to poor diagnosis and effective treatment of disease that are of public health concern, which result in emergence of multi-drug resistance strains that eventually result to high cost of treatment of these diseases [1, 10, 63]. Furthermore, non-availability of *S*. typhi vaccines in developing countries like Nigeria is also one of the contributing factors to the spread of resistance in *Salmonella* species [64].

*Contribution of non-human uses of antimicrobials*: The worldwide increase in the use of antibiotics in poultry, fishery and livestock production industries to treat and prevent infections, or as growth promoter, has greatly contributed to the increased antibiotics resistance in potential food-borne pathogens (*Salmonella, Shigella, Campylobacter*, etc.) in the past years [58, 64]. The increased use of antibiotics in agriculture has played a significant role in the emergence and spread of antibiotic resistant food-borne pathogens in human as a result of the consumption of poultry and dairy products [59]. The summary of the behavioural factors and their contribution to antibiotic resistance is shown in **Figure 1**.

#### *2.5.2. The genetic factors*

The major mechanism in the spread (dissemination of resistance genes between or among bacteria of the same species or different species is through genetic mechanism. Since resistance genes for instance in *Salmonella* have often been located within plasmid, integrons sometimes

**Figure 1.** A flow chart showing the spread of antibiotic resistance in *Salmonella* species in both man and animal populations.

associated with transposons and also insertion sequence [1, 56, 65]. The major factors involved in the genetic mechanism of spread are the integrons, transposomes and plasmids [13].

*Integrons* are mobile genetic elements of specific structure that consist of two conserved segment capable of flanking through a central region in which resistance gene cassettes are inserted [36]. Also, on the 51 -conserved segment is an *int* gene that encodes a site-specific recombinase, capable of capturing DNA, including resistance genes [66].

*Transposons*: These are mobile genetic elements that contain insertion sequence (0.2–6.0kb), which can move (transpose) from one site to the other site within the same or different chromosome or plasmid and thus replicate along with it [56].

*Plasmid* (4–400kb) are self-replicating, extra chromosomal DNA that contain genes either for resistance, virulence and other functions and are dispensable under certain conditions. Incompatibility group of plasmid DNA (Inc) HI1 are important vectors of antibiotic resistance in *S*. typhi. It was first detected from a large outbreak in 1972 in Mexico [64]. However, some larger plasmids are conjugative (R-plasmid) and are transferable between organisms, spreading along resistance genes in *S*. typhi [38]. It is should be noted that, as resistance genes move to other plasmids or chromosomes, they sometimes link with other resistance genes in resistance clusters, whose transfer can then result in spontaneous acquisition of resistance to several unrelated drugs, which eventually result to multi-drug resistance by recombination process [56].

#### **2.6. Consequences of resistance in** *Salmonella* **species to public health**

There are several clinical and public health consequences associated with antimicrobial drugs resistance in *Salmonella* species. These include:


of poultry and dairy products [59]. The summary of the behavioural factors and their contri-

The major mechanism in the spread (dissemination of resistance genes between or among bacteria of the same species or different species is through genetic mechanism. Since resistance genes for instance in *Salmonella* have often been located within plasmid, integrons sometimes

**Figure 1.** A flow chart showing the spread of antibiotic resistance in *Salmonella* species in both man and animal

bution to antibiotic resistance is shown in **Figure 1**.

*2.5.2. The genetic factors*

106 Current Topics in Salmonella and Salmonellosis

populations.


#### **2.7. Strategies to combat resistance problems posed by** *Salmonella* **species**

Several efforts have been adopted by several organizations, government and researchers to combat antimicrobial resistance imposed by some pathogenic organism that are of public health significant (*Salmonella* spp.) [68]. The 2006 IFT report led to the publication of the World Health Organization (WHO) list of critically important antimicrobials for human medicine and veterinary importance. This propelled the U.S Food and Drug Administration (FDA) to categorize various classes of anti-microbials as important, highly important and critically important and has since issued rules that prohibit most extra labelled use of some critically important antimicrobials such as fluoroquinolones and cephalosporin in food animal species [69].

Other efforts to address threats posed by antimicrobial resistance include: monitoring programmes for antimicrobial resistance microbes that integrates human, animals and food sampling scheme. Examples include: National Antimicrobial Resistance Monitoring System (NARMS) in the United States and the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP) in Denmark. These programmes in collaboration with CDC, WHO and FDA had really helped to trace the incidence of resistance particularly in foodborne pathogens (*Salmonella* and *Campylobacter*) globally, thereby embarking several strategies such as giving updated information, rules/laws, social and financial help, etc. to combat resistance threats.

Also, effort to combat resistance posed by *Salmonella* is the modification of drugs that led to the production of third – and fourth generation cephalosporins, and the use of medicinal plants also known as herbal medicine had been proven to have bactericidal effect on *S*. typhi; the causative agent of typhoid fever, also with the use of combined therapy, that is, the use of two or more different classes of antibiotics for the treatments of a particular disease e.g. Salmonellosis [1, 57].

Furthermore, the following are also needed to be done to track-down the current rise in the spread of resistant *S. enterica*: Intensive surveillance of vended foods in developing countries to reduce microbial risk associated with their consumption [60]. Public enlightenment to discourage the patronage of vended foods should be intensified as vended foods especially in Lagos is a potential vector responsible for the spread of resistant *Salmonella* species, or high level of hygiene practice should be maintained by food vendors under strict supervision and monitoring by food regulatory authorities if at all vended foods will be patronized [60].

## **3. Conclusion**

Several factors such as misuse of antibiotics, use of antibiotics in agriculture, poor hygiene practices by hospitals and individuals, unregulated sales of antibiotics and genetic factors, such as plasmids, integron, transposons, etc., contribute to selective pressure on antibiotics and resistance gene transfer, respectively, in *Salmonella* species. This has led to the emergence and spread of resistance in this microorganism and resultant therapeutic failures. Several strategies have been adopted by governmental organizations and pharmaceutical companies in the areas of resistance monitoring, restriction in the use of antibiotics in agriculture, production of modified drugs, the use of combined therapy, future plans on the use of bioactive compounds from medicinal plants against MDR bacterial strains. There is a need to enforce regulatory laws governing procurement and sales of antibiotics in developing countries. Also, good sanitation and hygiene practices as well as sensitization of people about the danger associated with indiscriminate purchase and use of antibiotics are essential to steam the growing trends of antibiotic resistance in bacterial pathogens especially *Salmonella* species.

## **Author details**

health significant (*Salmonella* spp.) [68]. The 2006 IFT report led to the publication of the World Health Organization (WHO) list of critically important antimicrobials for human medicine and veterinary importance. This propelled the U.S Food and Drug Administration (FDA) to categorize various classes of anti-microbials as important, highly important and critically important and has since issued rules that prohibit most extra labelled use of some critically important antimicrobials such as fluoroquinolones and cephalosporin in food animal species [69].

Other efforts to address threats posed by antimicrobial resistance include: monitoring programmes for antimicrobial resistance microbes that integrates human, animals and food sampling scheme. Examples include: National Antimicrobial Resistance Monitoring System (NARMS) in the United States and the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP) in Denmark. These programmes in collaboration with CDC, WHO and FDA had really helped to trace the incidence of resistance particularly in foodborne pathogens (*Salmonella* and *Campylobacter*) globally, thereby embarking several strategies such as giving updated information, rules/laws, social and financial help, etc. to combat resistance threats. Also, effort to combat resistance posed by *Salmonella* is the modification of drugs that led to the production of third – and fourth generation cephalosporins, and the use of medicinal plants also known as herbal medicine had been proven to have bactericidal effect on *S*. typhi; the causative agent of typhoid fever, also with the use of combined therapy, that is, the use of two or more different classes of antibiotics for the treatments of a particular disease e.g. Salmonellosis [1, 57]. Furthermore, the following are also needed to be done to track-down the current rise in the spread of resistant *S. enterica*: Intensive surveillance of vended foods in developing countries to reduce microbial risk associated with their consumption [60]. Public enlightenment to discourage the patronage of vended foods should be intensified as vended foods especially in Lagos is a potential vector responsible for the spread of resistant *Salmonella* species, or high level of hygiene practice should be maintained by food vendors under strict supervision and monitoring by food regulatory authorities if at all vended foods will be

Several factors such as misuse of antibiotics, use of antibiotics in agriculture, poor hygiene practices by hospitals and individuals, unregulated sales of antibiotics and genetic factors, such as plasmids, integron, transposons, etc., contribute to selective pressure on antibiotics and resistance gene transfer, respectively, in *Salmonella* species. This has led to the emergence and spread of resistance in this microorganism and resultant therapeutic failures. Several strategies have been adopted by governmental organizations and pharmaceutical companies in the areas of resistance monitoring, restriction in the use of antibiotics in agriculture, production of modified drugs, the use of combined therapy, future plans on the use of bioactive compounds from medicinal plants against MDR bacterial strains. There is a need to enforce regulatory laws governing procurement and sales of antibiotics in developing countries.

patronized [60].

108 Current Topics in Salmonella and Salmonellosis

**3. Conclusion**

Kabiru Olusegun Akinyemi\* and Samuel Oluwasegun Ajoseh

\*Address all correspondence to: akinyemiko@yahoo.com

Department of Microbiology, Faculty of Science, Lagos State University, Ojo, Lagos, Nigeria

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**Provisional chapter**

## **Quinolone Resistance in Non-typhoidal** *Salmonella* **Quinolone Resistance in Non-typhoidal** *Salmonella*

Siriporn Kongsoi, Chie Nakajima and Yasuhiko Suzuki Siriporn Kongsoi, Chie Nakajima and Yasuhiko Suzuki

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67154

#### **Abstract**

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**3**: 155–160.

114 Current Topics in Salmonella and Salmonellosis

Non‐typhoidal *Salmonella* is the primary foodborne zoonotic agent of salmonellosis in many countries. Non‐typhoidal *Salmonella* infections are transmitted to humans primar‐ ily through consumption of contaminated foods from animal origin, whereas *S.* Typhi and Paratyphi infections are spread directly or indirectly by contact with an infected person. Quinolones exhibit potent antibacterial activity against *Salmonella* and are usu‐ ally the first choice of treatment for life‐threatening salmonellosis due to multidrug‐ resistant strains. However, by the early 1990s, quinolones have been approved for use in food‐producing animals. The increased use of this group of antimicrobials in animal has led to the concomitant emergence of quinolone‐resistant non‐typhoidal *Salmonella* strains. However, in some countries, there are no legal provisions, which apply to vet‐ erinary drugs. This situation provides favorable conditions for spread and persistence of quinolone‐resistant bacteria in food‐producing animals. The objective of this chapter is to review the current regulatory controls for the use of quinolones in food‐producing animals, its effect on development of quinolone resistance, and the potential impact on human and animal health. Moreover, this chapter reviews the current knowledge of qui‐ nolone resistance mechanisms and the future directions of research with particular atten‐ tion to the strategies to control the emergence of quinolone‐resistant *Salmonella*.

**Keywords:** non‐typhoidal *Salmonella*, quinolones, resistance

#### **1. Introduction**

Non‐typhoidal *Salmonella* refers to a group of bacteria that cause diarrheal illness in humans and domestic animals. More than 2500 different serovars of non‐typhoidal *Salmonella* have been described: all serovars of *Salmonella* except for Typhi, Paratyphi A, Paratyphi B (tartrate negative), and Paratyphi C. Non‐typhoidal *Salmonella* are important causes of foodborne infection because *Salmonella* have a broad host range and are strongly associated with animal

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and plant products. Humans are infected by consumption of food or water contaminated with *Salmonella* and direct contact transmission between infected animals and humans in a variety of ways or contaminated environment and directly between humans. The recent outbreaks show that fresh fruits and vegetables can be contaminated with non‐typhoidal *Salmonella* especially sprouts, tomatoes, fruits, peanuts, and spinach [1–5]. Non‐typhoidal *Salmonella* is commonly found in food products derived from the animal species such as poultry, eggs, dairy products, and contaminated pets such as cats, dogs, rodents, reptiles, or amphibians [6–9].

Non‐typhoidal *Salmonella* is a leading cause of bacterial diarrhea worldwide, in contrast to typhoid fever, which remains endemic in developing countries. There are an estimated 93.8 million cases of non‐typhoidal *Salmonella* gastroenteritis, resulting in approximately 155,000 deaths globally each year [10]. Gastroenteritis is the most frequent clinical symptom of non‐typhoidal *Salmonella* infection. The incubation period of non‐typhoidal *Salmonella* gas‐ troenteritis is 6–72 h, usually 12–36 h after initial exposure. The classic presentation in non‐ typhoidal *Salmonella* gastroenteritis has self‐limiting, acute gastroenteritis, watery diarrhea, abdominal pain, fever, nausea, and sometimes vomiting [11]. The gastroenteritis usually lasts 4–7 days, and most people recover with little or no treatment [12]. Non‐typhoidal salmonel‐ losis clinical presentations differ significantly by serovars such as *S*. Typhimurium and *S*. Enteritidis, have a broad host range, and can cause gastrointestinal infections with less sever‐ ity than typhoidal enteric fever which affects both humans and a wide variety of animal hosts. An infection with *S*. Choleraesuis is primarily responsible for the severe systemic illness of salmonellosis in human and swine. Some serotypes such as *S.* Dublin are responsible for the systemic salmonellosis in humans and also cause death in young calves, occasionally death in mature cattle and results in decreased milk production, diarrhea, and abortion in cattle. Rates of invasive systemic salmonellosis and death are generally higher among persons with high‐risk conditions, infants aged <3 months, elderly aged ≥60 years, the debilitated, immu‐ nosuppressive conditions, and malignant neoplasms.

Antimicrobial therapy can prolong the duration of excretion of non‐typhoidal *Salmonella* and, therefore, is only considered for gastroenteritis patients caused by *Salmonella* species with moderate‐to‐severe diarrhea, high fever, or systemic infection and for gastroenteritis in peo‐ ple at increased risk of invasive disease (persons with high‐risk conditions). Current recom‐ mendations are that fluoroquinolones (FQs) be reserved for patients with moderate‐to‐severe diarrhea by non‐typhoidal *Salmonella* infection. Resistance among non‐typhoidal *Salmonella* serovars to the first‐line antibiotics such as chloramphenicol, ampicillin, trimethoprim‐sulfa‐ methoxazole, and cotrimoxazole has been present for many years, and resistance to FQs has also increased over the last decade.

The emergence of quinolone‐resistant non‐typhoidal *Salmonella* varies by serotype and geo‐ graphic location. Therefore, the control of quinolone‐resistant non‐typhoidal *Salmonella* infec‐ tion is difficult. There is a high need to understand the quinolone resistance mechanisms for preventing the further quinolone resistance development through the better interventional strategies that prevent spread of quinolone‐resistant *Salmonella* between humans and animal reservoirs along the food chain.

## **2. Quinolone use in food‐producing animals**

and plant products. Humans are infected by consumption of food or water contaminated with *Salmonella* and direct contact transmission between infected animals and humans in a variety of ways or contaminated environment and directly between humans. The recent outbreaks show that fresh fruits and vegetables can be contaminated with non‐typhoidal *Salmonella* especially sprouts, tomatoes, fruits, peanuts, and spinach [1–5]. Non‐typhoidal *Salmonella* is commonly found in food products derived from the animal species such as poultry, eggs, dairy products, and contaminated pets such as cats, dogs, rodents, reptiles, or

Non‐typhoidal *Salmonella* is a leading cause of bacterial diarrhea worldwide, in contrast to typhoid fever, which remains endemic in developing countries. There are an estimated 93.8 million cases of non‐typhoidal *Salmonella* gastroenteritis, resulting in approximately 155,000 deaths globally each year [10]. Gastroenteritis is the most frequent clinical symptom of non‐typhoidal *Salmonella* infection. The incubation period of non‐typhoidal *Salmonella* gas‐ troenteritis is 6–72 h, usually 12–36 h after initial exposure. The classic presentation in non‐ typhoidal *Salmonella* gastroenteritis has self‐limiting, acute gastroenteritis, watery diarrhea, abdominal pain, fever, nausea, and sometimes vomiting [11]. The gastroenteritis usually lasts 4–7 days, and most people recover with little or no treatment [12]. Non‐typhoidal salmonel‐ losis clinical presentations differ significantly by serovars such as *S*. Typhimurium and *S*. Enteritidis, have a broad host range, and can cause gastrointestinal infections with less sever‐ ity than typhoidal enteric fever which affects both humans and a wide variety of animal hosts. An infection with *S*. Choleraesuis is primarily responsible for the severe systemic illness of salmonellosis in human and swine. Some serotypes such as *S.* Dublin are responsible for the systemic salmonellosis in humans and also cause death in young calves, occasionally death in mature cattle and results in decreased milk production, diarrhea, and abortion in cattle. Rates of invasive systemic salmonellosis and death are generally higher among persons with high‐risk conditions, infants aged <3 months, elderly aged ≥60 years, the debilitated, immu‐

Antimicrobial therapy can prolong the duration of excretion of non‐typhoidal *Salmonella* and, therefore, is only considered for gastroenteritis patients caused by *Salmonella* species with moderate‐to‐severe diarrhea, high fever, or systemic infection and for gastroenteritis in peo‐ ple at increased risk of invasive disease (persons with high‐risk conditions). Current recom‐ mendations are that fluoroquinolones (FQs) be reserved for patients with moderate‐to‐severe diarrhea by non‐typhoidal *Salmonella* infection. Resistance among non‐typhoidal *Salmonella* serovars to the first‐line antibiotics such as chloramphenicol, ampicillin, trimethoprim‐sulfa‐ methoxazole, and cotrimoxazole has been present for many years, and resistance to FQs has

The emergence of quinolone‐resistant non‐typhoidal *Salmonella* varies by serotype and geo‐ graphic location. Therefore, the control of quinolone‐resistant non‐typhoidal *Salmonella* infec‐ tion is difficult. There is a high need to understand the quinolone resistance mechanisms for preventing the further quinolone resistance development through the better interventional strategies that prevent spread of quinolone‐resistant *Salmonella* between humans and animal

amphibians [6–9].

116 Current Topics in Salmonella and Salmonellosis

nosuppressive conditions, and malignant neoplasms.

also increased over the last decade.

reservoirs along the food chain.

The first quinolone was generated in the early 1960s. The first member of the quinolones is nalidixic acid (NAL), a 1,8‐naphthyridine as shown in **Figure 1**, which had a good activity against Gram‐negative pathogens and was used to treat urinary tract infections. However, the use of NAL was decreased due to the increasing resistance of this drug and because of the synthesis of new, broad‐spectrum, and safer antimicrobials. The molecular modifications of the core quinolone structure significantly affect their antimicrobial activity, allowing the synthesis of various compounds of this drug class.

FQs (fluorinated derivatives of quinolones) were first developed since the 1980s. The presence of fluorine in position 6 of the core quinolone structure provides broad and potent antimicrobial activity against Gram‐positive and Gram‐negative bacteria because it significantly enhances the antibiotics' penetration into the bacterial cell membrane. Norfloxacin (NOR), launched in 1980, is a first broad‐spectrum FQ which consisted of a piperazinyl ring that replaces the methyl group at position 7 (**Figure 1**) results in enhancing activity against Gram‐negative bac‐ teria [13]. Ciprofloxacin (CIP) has similar structure to NOR except the ethyl group at N‐1 of CIP is replaced by a cyclopropyl group (**Figure 1**) that increasing the spectrum of action which not only active against Gram‐negative bacteria but also against Gram‐positive bacteria [14]. The structure of enrofloxacin (ENR) is similar to CIP but with an additional ethyl group on the piperazinyl ring (**Figure 1**).

**Figure 1.** The structural features of four different quinolones.

All these structural modifications in the molecular molecule of quinolones improved a spec‐ trum of drug activity, tissue penetration, long half‐life in the body, lower toxicity, and greater capacity to cross bacterial cell membranes and consequently better activity against Gram‐ negative bacteria and Gram‐positive species. Their treatment indications developed from uri‐ nary infection to applications against many other systemic diseases. The last generations of quinolones provide the activity against anaerobic bacteria.

FQs have been licensed for use in food animals at the beginning of the 1990s, and subse‐ quently, a new FQs extensively have been authorized, and a large number of different veterinary pharmaceutical products have been launched in the market [15]. ENR exhibits good activity against most Gram‐negative bacteria, including *Escherichia coli*, *Campylobacter*, *Enterobacter*, *Serratia*, *Chlamydia,* and *Mycobacterium,* and has a variable effect on *Pseudomonas*, *Enterococcus*, *Clostridium*, *Staphylococcus*, and *Streptococcus*. The efficacies of ENR treatment in food‐producing animals have been reported in turkeys against *Pasteurella multocida* infections and in chickens against *E. coli* infections. Danofloxacin (DFX) and ENR are licensed for use in food‐producing animals in the United States. ENR and DFX are currently approved to be good choices for therapy of bovine respiratory disease (BRD) in high‐risk cattle. ENR is also currently approved for treatment of swine respiratory disease (SRD). DFX and ENR are only available as a sterile injectable solution for animal usage and should be administered under a prescription from a veterinarian. ENR is FQ antimicrobial agent frequently used in poultry production, sold by the Bayer Corporation under the trade name Baytril; however, it is also sold under the various generic names. ENR is a FQ antibiotic that is very similar to the human drug CIP. Under current legislation, if a small number of chickens present the clinical signs and symptoms, ENR can be used to treat the whole flock by adding the drug into the drinking water, even when most of the chickens are not sick. FQs can also be used to treat infections in breeding flocks, and the transmission of drug‐resistant organisms may occur among chicks.

Finland and Denmark ban all the uses of FQs in poultry; however, they are used in other species of farm livestock. Australia has never approved the use of FQs in poultry and any farm animals, and consequently, resistance to FQs in zoonotic bacteria such as *Campylobacter* and *Salmonella* has a low prevalence in farm animals. The prevalence in human infected with resistant bacteria is also much lower than in many other countries. Resistant *Campylobacter* infections were low just 0% in 2003 and 2.6% in 2006; however, nearly all of these cases were returning travelers [16]. Human infections with resistant *E. coli* were also low in preva‐ lence at 4–5% [16]. Finland does not approve the use of FQs in poultry result in no resis‐ tant *Campylobacter* from poultry productions in 2007, and the resistance in *Campylobacter* was found only 1% in 2008 and 2009. Resistant *Campylobacter* infections of Finnish patients who had not traveled abroad were found 2–3% and 61% were investigated from the patients who have traveled abroad within 2 weeks [17].

In September 2005, the U.S. Food and Drug Administration (FDA) banned the use of FQs for treating bacterial infections in U.S. poultry result from concerns about increasing in FQ resis‐ tance among *Campylobacter* isolates of poultry and humans. Although the FQs were banned in the US in 2005, the impact of the ban on resistance in human *C. jejuni* is not clear because the resistant isolates in 2013 remained at the same level as in 2005 (22*%*). In retail chicken, CIP resistance in *C. coli* has decreased to 13.5*%* in 2010 from 29% in 2005; however, resistance in *C. jejuni* significantly increased from 15.2 to 22.5*%* from 2002 to 2010. It may be caused by the illegal use of FQs in the U.S. poultry industry.

## **3. A contribution of veterinary usage of quinolones to resistance in human non‐typhoidal** *Salmonella* **isolates**

All these structural modifications in the molecular molecule of quinolones improved a spec‐ trum of drug activity, tissue penetration, long half‐life in the body, lower toxicity, and greater capacity to cross bacterial cell membranes and consequently better activity against Gram‐ negative bacteria and Gram‐positive species. Their treatment indications developed from uri‐ nary infection to applications against many other systemic diseases. The last generations of

FQs have been licensed for use in food animals at the beginning of the 1990s, and subse‐ quently, a new FQs extensively have been authorized, and a large number of different veterinary pharmaceutical products have been launched in the market [15]. ENR exhibits good activity against most Gram‐negative bacteria, including *Escherichia coli*, *Campylobacter*, *Enterobacter*, *Serratia*, *Chlamydia,* and *Mycobacterium,* and has a variable effect on *Pseudomonas*, *Enterococcus*, *Clostridium*, *Staphylococcus*, and *Streptococcus*. The efficacies of ENR treatment in food‐producing animals have been reported in turkeys against *Pasteurella multocida* infections and in chickens against *E. coli* infections. Danofloxacin (DFX) and ENR are licensed for use in food‐producing animals in the United States. ENR and DFX are currently approved to be good choices for therapy of bovine respiratory disease (BRD) in high‐risk cattle. ENR is also currently approved for treatment of swine respiratory disease (SRD). DFX and ENR are only available as a sterile injectable solution for animal usage and should be administered under a prescription from a veterinarian. ENR is FQ antimicrobial agent frequently used in poultry production, sold by the Bayer Corporation under the trade name Baytril; however, it is also sold under the various generic names. ENR is a FQ antibiotic that is very similar to the human drug CIP. Under current legislation, if a small number of chickens present the clinical signs and symptoms, ENR can be used to treat the whole flock by adding the drug into the drinking water, even when most of the chickens are not sick. FQs can also be used to treat infections in breeding flocks, and the transmission of drug‐resistant organisms may occur among chicks.

Finland and Denmark ban all the uses of FQs in poultry; however, they are used in other species of farm livestock. Australia has never approved the use of FQs in poultry and any farm animals, and consequently, resistance to FQs in zoonotic bacteria such as *Campylobacter* and *Salmonella* has a low prevalence in farm animals. The prevalence in human infected with resistant bacteria is also much lower than in many other countries. Resistant *Campylobacter* infections were low just 0% in 2003 and 2.6% in 2006; however, nearly all of these cases were returning travelers [16]. Human infections with resistant *E. coli* were also low in preva‐ lence at 4–5% [16]. Finland does not approve the use of FQs in poultry result in no resis‐ tant *Campylobacter* from poultry productions in 2007, and the resistance in *Campylobacter* was found only 1% in 2008 and 2009. Resistant *Campylobacter* infections of Finnish patients who had not traveled abroad were found 2–3% and 61% were investigated from the patients who

In September 2005, the U.S. Food and Drug Administration (FDA) banned the use of FQs for treating bacterial infections in U.S. poultry result from concerns about increasing in FQ resis‐ tance among *Campylobacter* isolates of poultry and humans. Although the FQs were banned in the US in 2005, the impact of the ban on resistance in human *C. jejuni* is not clear because the resistant isolates in 2013 remained at the same level as in 2005 (22*%*). In retail chicken,

quinolones provide the activity against anaerobic bacteria.

118 Current Topics in Salmonella and Salmonellosis

have traveled abroad within 2 weeks [17].

Multidrug resistance in non‐typhoidal *Salmonella* is a global problem, and these strains are linked to more severe disease outcome. Serovars Typhimurium and Newport, two of most common serotypes, are more resistant to multiple antimicrobial agents than the other sero‐ types [18]. Multidrug‐resistant *S.* Typhimurium definitive type (DT) 104, was first detected in 1980s, emerged as a public health concern because of its global distribution in diseases among animal species such as poultry, pigs, and sheep and humans [19, 20]. The emergence and worldwide spread of multidrug‐resistant *S.* Typhimurium DT104 isolates are associated with the intake of contaminated meat and meat products. Many strains of *S.* Typhimurium DT104 are generally resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracycline [21]. Moreover, new resistant strains of non‐typhoidal *Salmonella* are constantly rising worldwide and resistant against ampicillin, chloramphenicol, kanamycin, streptomy‐ cin, trimethoprim, and cotrimoxazole [22–24], for example, a multidrug‐resistant strains of serovars Virchow [25], Heidelberg [26], and Infantis [27, 28]*.*

Quinolones were introduced for veterinary use in various countries, and subsequent use has been followed by the development of quinolone resistance in bacteria of food‐producing animals and consequently transmits the resistant zoonotic bacteria to humans [29]. In many countries, FQs are drug of first choice for prescription in acute gastrointestinal symptoms caused by *Salmonella* infection, and resistance to this drug group has often been described, particularly to NAL [15]. In a study performed between 1996 and 2003, *Salmonella* isolates were investigated for quinolone susceptibility; the results revealed that NAL and CIP resis‐ tances were 1.6 and 7%, respectively. A significant upward trend in resistance was observed for NAL from 0.4% in 1996 to 2.3% in 2003 [30]. In Germany, an increase in the frequency of NAL‐resistant *Salmonella* strains was discovered after the approval and use of ENR [31]. Concurrent increase in resistance was observed in France among *Salmonella* isolates from animals and humans, and the same clones were determined among the different hosts [32]. In the United Kingdom, also in Spain, the incidence of NAL‐resistant *Salmonella* illnesses in humans was increased followed the introduction for veterinary use of FQs in 1993 [33, 34]. A study from Denmark and Taiwan described the emergence of salmonellosis caused by multidrug and quinolone‐resistant *S*. Typhimurium DT104 linked to a swine herd and the subsequent spread of those isolates to humans [35–37]. In European countries, similar associa‐ tions between FQ resistance development in *Salmonella* infecting humans and retail poultry products have been described. Therefore, the FQ‐resistant *Salmonella* in poultry has reached alarming proportions in some countries [38]. In the United States, there was an increase in the proportion of FQ resistance development in *Salmonella* infections following the first approved use of FQs in food‐producing animals in 1995 [39].

The data indicate that it would be reasonable to assume that the veterinary usage of FQs will have made a remarkable contribution to FQ resistance in human *Salmonella* infections.

## **4. The potential impact on human health**

FQ resistance in *Salmonella* is clearly associated with FQ use in food‐producing animals, and foodborne infections caused by such resistant bacteria are well investigated in human. FQ resistance in *S.* Typhimurium DT104 has been associated with increased hospitalization, more frequent and longer illness, treatment failures, and a higher risk of death [40]. Many studies also investigated that infections with multidrug‐resistant *Salmonella* were associated with longer hospitalization and a higher death rate than infections with susceptible isolates [41–43]. Previous study has found a 3.15 times increased mortality when patients infected with NAL‐resistant *S.* Typhimurium compared to patients infected with susceptible isolates [44]. For treatment of the infections with FQ‐resistant *Salmonella,* alternative antimicrobials are the third or fourth generation cephalosporin. Nevertheless, it should be considered con‐ traindications for treatment of uncomplicated non‐typhoidal *Salmonella* infection because FQ treatment can induce prolonged excretion of *Salmonella* and increased frequency of relapses [45]. However, for patients at risk such as immunocompromised, severely infected and elderly, FQs are considered first choice drugs and effective in reducing the disease length if the treatment starts early in the infection.

## **5. The potential impact on animal health**

FQs are highly potent antimicrobial agents rapidly absorbed after oral administration and have a long half‐life and widespread distribution to most body tissues, which made them suitable for using in herd treatment of food‐producing animals. FQs are effective for serious infections in food‐producing animals such as systematic gastroenteritis and severe respira‐ tory diseases and are also used to treat urinary tract, skin, and soft‐tissue infections caused by Gram‐negative or some Gram‐positive aerobic bacteria. Moreover, they also have potential for treatment of infections caused by *Mycoplasma*, *Mycobacterium*, *Chlamydia*, *Ehrlichia*, and *Rickettsia*. However, documentation about authorized dosages and the effectiveness of FQs to treat all these infections in animals have not been determined on the base of the pharmaco‐ kinetic and pharmacodynamics properties. Sufficient knowledge about the selecting optimal dose and duration of FQs could help to develop appropriate dosing regimens to maximize the clinical efficacy, avoid therapeutic failure, and decrease the selection of resistance which would ensure for the benefit of animals and their future use.

However, the potential clinical disadvantage associated with FQ use was a rapid selection for resistance. Several pathogenic bacteria of food‐producing animals have been investigated the increasing of resistance to FQs following the introduction of ENR [46]. If FQ resistance emerges in animal pathogenic bacteria, this may result in treatment failure and increased mortality. This is a risk for poor animal welfare conditions and will result in economical losses. Consequently, for some animal infectious diseases, antimicrobial therapeutic use will be complicated if FQs lose their efficacy. As described in a previous study, multidrug‐resis‐ tant *S.* Typhimurium infections in veal calves were resistant to most conventionally used antimicrobials and also resistant to ENR resulted in a mortality exceeding 90%. FQs are also considered effective in other infections such as pneumonia, neonatal diarrhea, and mastitis caused by Gram‐negative organisms in piglets and calves. However, there were insufficient data to support the animal health or welfare problems when diseases cannot be treated result from FQ resistance during treatment.

The data indicate that it would be reasonable to assume that the veterinary usage of FQs will

FQ resistance in *Salmonella* is clearly associated with FQ use in food‐producing animals, and foodborne infections caused by such resistant bacteria are well investigated in human. FQ resistance in *S.* Typhimurium DT104 has been associated with increased hospitalization, more frequent and longer illness, treatment failures, and a higher risk of death [40]. Many studies also investigated that infections with multidrug‐resistant *Salmonella* were associated with longer hospitalization and a higher death rate than infections with susceptible isolates [41–43]. Previous study has found a 3.15 times increased mortality when patients infected with NAL‐resistant *S.* Typhimurium compared to patients infected with susceptible isolates [44]. For treatment of the infections with FQ‐resistant *Salmonella,* alternative antimicrobials are the third or fourth generation cephalosporin. Nevertheless, it should be considered con‐ traindications for treatment of uncomplicated non‐typhoidal *Salmonella* infection because FQ treatment can induce prolonged excretion of *Salmonella* and increased frequency of relapses [45]. However, for patients at risk such as immunocompromised, severely infected and elderly, FQs are considered first choice drugs and effective in reducing the disease length if

FQs are highly potent antimicrobial agents rapidly absorbed after oral administration and have a long half‐life and widespread distribution to most body tissues, which made them suitable for using in herd treatment of food‐producing animals. FQs are effective for serious infections in food‐producing animals such as systematic gastroenteritis and severe respira‐ tory diseases and are also used to treat urinary tract, skin, and soft‐tissue infections caused by Gram‐negative or some Gram‐positive aerobic bacteria. Moreover, they also have potential for treatment of infections caused by *Mycoplasma*, *Mycobacterium*, *Chlamydia*, *Ehrlichia*, and *Rickettsia*. However, documentation about authorized dosages and the effectiveness of FQs to treat all these infections in animals have not been determined on the base of the pharmaco‐ kinetic and pharmacodynamics properties. Sufficient knowledge about the selecting optimal dose and duration of FQs could help to develop appropriate dosing regimens to maximize the clinical efficacy, avoid therapeutic failure, and decrease the selection of resistance which

However, the potential clinical disadvantage associated with FQ use was a rapid selection for resistance. Several pathogenic bacteria of food‐producing animals have been investigated the increasing of resistance to FQs following the introduction of ENR [46]. If FQ resistance emerges in animal pathogenic bacteria, this may result in treatment failure and increased mortality. This is a risk for poor animal welfare conditions and will result in economical

have made a remarkable contribution to FQ resistance in human *Salmonella* infections.

**4. The potential impact on human health**

120 Current Topics in Salmonella and Salmonellosis

the treatment starts early in the infection.

**5. The potential impact on animal health**

would ensure for the benefit of animals and their future use.

## **6. The current state of knowledge of quinolone resistance mechanisms**

FQs are strong inhibitors of bacterial enzymes, which are necessary enzymes associated in major biological processes including DNA replication [47–49]. In prokaryotes, DNA is known as a double helix because there are two strands that intertwine around each other. However, additional complexity comes from the further twisting (supercoiling) of the double‐strand structure to put the double helix under torsion stress [50]. This supercoiling process that enables the long strands of DNA is condensed into compact supercoils permitting large amounts of DNA to be packed into the cell [51].

Topoisomerase I and topoisomerase II enzymes are enzymes that regulate the overwinding or underwinding of DNA and control the level of twisting within DNA. Topoisomerase I removes the number of negative supercoils, in contrast to topoisomerase II, which intro‐ duces negative supercoils that facilitate the unwinding of the over‐twisted DNA and can further change the DNA topology into an under‐twisted DNA [50]. DNA gyrase and DNA topoisomerase IV are type II topoisomerase comprising 2 A subunits and 2 B subunits enco‐ ded by the *gyrA* and *gyrB* genes or 2 C subunits and 2 E subunits encoded by the *parC* and *parE* genes, respectively [52]. DNA gyrase and topoisomerase IV have distinct roles although both enzymes have homologous action to relax positively supercoiled DNA. DNA gyrase decatenates replicating DNA by introducing negative supercoils into relaxed DNA while topoisomerase IV unlinks the newly replicated daughter chromosomes during cell division [52–54].

FQs are direct inhibitors of bacterial DNA synthesis by inhibiting two enzymes, DNA gyrase and topoisomerase IV, which have important roles in DNA replication. The quinolones bind to these enzymes with DNA to form drug‐enzyme‐DNA complexes (known as a ternary complex) subsequently induces double‐strand DNA breaks and blocks replication, therefore, results in damage to bacterial DNA and bacterial cell death [55–58]. However, the primary target enzyme, either DNA gyrase or topoisomerase IV, of FQs varies depending on the bacte‐ rial species. The preferential target of FQs in Gram‐negative bacteria is DNA gyrase, whereas in Gram‐positive microorganisms, topoisomerase IV is the primary target [58].

Resistance to quinolones occurs by different ways. The major mechanisms of bacterial resis‐ tance to FQs are altered target enzymes, expression of an active efflux, and altered membrane permeability.

#### **6.1. Target‐site mutation**

The main mechanism of FQ resistance is due to mutation in target genes (*gyrA*, *gyrB*, *parC*, and *parE*) that encode the primary and secondary target enzymes of these drugs. The mutations in quinolone resistance‐determining region (QRDR) of target genes alter the target enzyme conformation by amino acid substitutions and subsequently decrease in the drug binding affinity of the target enzyme, leading to FQ resistance [59–62].

In *Salmonella*, quinolone resistance was firstly investigated in the *gyrA* gene coding for the A subunit of gyrase. Mutations associated with FQ resistance in GyrA have been clustered between amino acids 67 and 106, termed the QRDR region. Amino acid substitutions of GyrA at Ser83 (to Phe, Tyr, or Ala) or at Asp87 (to Gly, Asn, or Tyr) are most usually identified in NAL‐resistant *Salmonella* strains. Previous studies have observed that single point mutation in QRDR of *gyrA* led to reduced sensitivity to CIP in *Salmonella* isolates [63]. Similar decreasing in CIP susceptibility was also found in three amino acid mutations of *parC* at Ser67 (to Cys), Arg76 (to Cys), and Cys80 (to Arg) in *S.* Enteritidis [64, 65]. Nevertheless, less frequently, the previous study detected novel mutations inside QRDR of GyrA at codon Asp72, Asp82, and Ala119 and also outside the QRDR [66]. Moreover, in another studies, the authors found double mutations in GyrA at both Ser83 and Asp87 in *S.* Typhimurium DT204 [67] and a single mutation at Asp87 (to Tyr) in all *Salmonella* strains [68] showing high‐level resistance to FQs. A *gyrB* gene mutation has also been observed in a quinolone‐resistant *S*. Typhimurium at Ser463 (to Tyr) [69].

These target‐site mutations show that different mutations of FQ‐resistant *Salmonella* isolates can result in very different resistance levels of quinolones, and this is not the same for all strains and all resistance mutations. Therefore, amino acid substitutions in topoisomerases are inadequate to clarify the level of resistance to quinolones in *S. enterica*. Nevertheless, it remains to be investigated what the specific role of these mutations on quinolone resistance in *Salmonella*.

#### **6.2. Transmissible quinolone‐resistance mechanisms**

Plasmid‐mediated quinolone resistance (PMQR) genes on mobile genetic elements are able to reduce susceptibility of quinolone or FQ antimicrobials. The PMQR gene, *qnr*, encodes a pen‐ tapeptide repeat motif protein (Qnr) that protects the target enzyme DNA gyrase and topoi‐ somerase IV by blocking the quinolone inhibition [70]. Recently, several Qnr proteins were investigated in Enterobacteriaceae (QnrA, QnrB, QnrC, QnrD, QnrS) [71, 72]. A recent study reported six variants of *qnrB* genes in *Salmonella* and *E. coli* isolates of human and animal isolates [73]. Nonetheless, the prevalence of *qnrS* genes is higher than the other *qnr* genes in *Salmonella*. A study from different European countries investigated a *qnrS* gene in 10% of the *Salmonella* isolates [73]. Moreover, *qnrS* gene has been identified in non‐typhoidal *Salmonella* clinical isolate from the USA [74]. The *qnrD* gene also has been investigated in eight different *Salmonella* serovars from 13 European countries [73].

Another plasmid‐encoded quinolone resistance determinant is a variant of an aminogly‐ coside acetyl transferase gene, *aac(6*'*)‐Ib‐cr*, which is able to acetylate the amino nitrogen on the piperazinyl substituent in aminoglycoside, and FQ drug classes lead to decreased susceptibility of these drugs [75–77]. However, the variant enzyme is not able to acetylate moxifloxacin and levofloxacin due to the absence of a piperazinyl substituent at position C‐7. Recently, this *aac(6*'*)‐Ib‐cr* gene has been reported in *Salmonella* isolated from chickens in China [78]. Plasmid‐mediated quinolone resistance determinants in *Salmonella* isolated from food‐producing animals are serious public health concern. Continuous surveillance of quinolone resistance determinants at national and international levels needs for limiting the dissemination of quinolone‐resistant *Salmonella* strains.

#### **6.3. Membrane permeability**

**6.1. Target‐site mutation**

122 Current Topics in Salmonella and Salmonellosis

in *Salmonella*.

The main mechanism of FQ resistance is due to mutation in target genes (*gyrA*, *gyrB*, *parC*, and *parE*) that encode the primary and secondary target enzymes of these drugs. The mutations in quinolone resistance‐determining region (QRDR) of target genes alter the target enzyme conformation by amino acid substitutions and subsequently decrease in the drug binding

In *Salmonella*, quinolone resistance was firstly investigated in the *gyrA* gene coding for the A subunit of gyrase. Mutations associated with FQ resistance in GyrA have been clustered between amino acids 67 and 106, termed the QRDR region. Amino acid substitutions of GyrA at Ser83 (to Phe, Tyr, or Ala) or at Asp87 (to Gly, Asn, or Tyr) are most usually identified in NAL‐resistant *Salmonella* strains. Previous studies have observed that single point mutation in QRDR of *gyrA* led to reduced sensitivity to CIP in *Salmonella* isolates [63]. Similar decreasing in CIP susceptibility was also found in three amino acid mutations of *parC* at Ser67 (to Cys), Arg76 (to Cys), and Cys80 (to Arg) in *S.* Enteritidis [64, 65]. Nevertheless, less frequently, the previous study detected novel mutations inside QRDR of GyrA at codon Asp72, Asp82, and Ala119 and also outside the QRDR [66]. Moreover, in another studies, the authors found double mutations in GyrA at both Ser83 and Asp87 in *S.* Typhimurium DT204 [67] and a single mutation at Asp87 (to Tyr) in all *Salmonella* strains [68] showing high‐level resistance to FQs. A *gyrB* gene mutation

has also been observed in a quinolone‐resistant *S*. Typhimurium at Ser463 (to Tyr) [69].

These target‐site mutations show that different mutations of FQ‐resistant *Salmonella* isolates can result in very different resistance levels of quinolones, and this is not the same for all strains and all resistance mutations. Therefore, amino acid substitutions in topoisomerases are inadequate to clarify the level of resistance to quinolones in *S. enterica*. Nevertheless, it remains to be investigated what the specific role of these mutations on quinolone resistance

Plasmid‐mediated quinolone resistance (PMQR) genes on mobile genetic elements are able to reduce susceptibility of quinolone or FQ antimicrobials. The PMQR gene, *qnr*, encodes a pen‐ tapeptide repeat motif protein (Qnr) that protects the target enzyme DNA gyrase and topoi‐ somerase IV by blocking the quinolone inhibition [70]. Recently, several Qnr proteins were investigated in Enterobacteriaceae (QnrA, QnrB, QnrC, QnrD, QnrS) [71, 72]. A recent study reported six variants of *qnrB* genes in *Salmonella* and *E. coli* isolates of human and animal isolates [73]. Nonetheless, the prevalence of *qnrS* genes is higher than the other *qnr* genes in *Salmonella*. A study from different European countries investigated a *qnrS* gene in 10% of the *Salmonella* isolates [73]. Moreover, *qnrS* gene has been identified in non‐typhoidal *Salmonella* clinical isolate from the USA [74]. The *qnrD* gene also has been investigated in eight different

Another plasmid‐encoded quinolone resistance determinant is a variant of an aminogly‐ coside acetyl transferase gene, *aac(6*'*)‐Ib‐cr*, which is able to acetylate the amino nitrogen on the piperazinyl substituent in aminoglycoside, and FQ drug classes lead to decreased

affinity of the target enzyme, leading to FQ resistance [59–62].

**6.2. Transmissible quinolone‐resistance mechanisms**

*Salmonella* serovars from 13 European countries [73].

The membrane permeability and the ability of FQs to enter the bacterial cells are an important determinant of the potency of these drugs that have intracellular targets [79]. The outer‐ membrane proteins (OMPs) of Gram‐negative bacteria consist of pore‐forming outer‐mem‐ brane proteins which serve as a particular barrier for the entry of hydrophilic molecules into the cell. It has been shown that CIP (hydrophilic quinolones) preferentially entry into the cells via porin pathway [80]. Down‐regulation of OMPs results in reduced FQ susceptibility in FQ‐resistant isolates of different species [81–84]. Very few researches have investigated on alterations of OMP expression or the role of lipopolysaccharide composition in quinolone‐ resistant *Salmonella* isolates [68, 85–89]. The lengthening of the O chains has been studied in quinolone‐resistant *Salmonella* that could also lead to a lower level in the permeability of the outer membrane [85]. The previous studies have found the lack of OmpF porin expression result from SoxS up‐regulates micF transcription in quinolone‐resistant *Salmonella* strains [86–88, 90]. However, it remains unclear whether such alterations contributed to signifi‐ cant reduction of outer‐membrane permeability and reduced susceptibility of quinolones in *Salmonella* isolates.

#### **6.4. Efflux**

Chromosomal multidrug efflux pumps are capable of actively removing FQs and a broad range of antimicrobial agents from the bacterial cell and are mostly encoded by chromo‐ somal genes. These efflux systems consist of different classes of transporters such as the resistance nodulation division (RND) family of tripartite transporters of Gram‐negative pathogens [91, 92]. These systems are mainly responsible for the intrinsic pattern of reduced susceptibility to FQs and other antimicrobial agents but are also responsible for increased resistance resulting from derepression of the transporter. Previous studies showed the evi‐ dence for the participation of active efflux in quinolone‐resistant *Salmonella* isolates [85, 93]. It was concluded that the overproduction of the AcrAB‐TolC efflux pump appeared prior to *gyrA* mutations in *in vitro* selected quinolone‐resistant *Salmonella* mutants [85]; therefore, the AcrAB‐TolC efflux system is the major mechanism that involved in quinolone resis‐ tance in *S*. Typhimurium DT104 strains. However, both target gene mutations and active efflux mediated by AcrAB*‐*TolC are necessary to obtain high‐level FQ resistance for *S*. Typhimurium DT204 strains [94]. Nevertheless, there is no direct evidence to demonstrate the role of the AcrAB‐TolC efflux system in quinolone‐resistant *Salmonella*; therefore, sub‐ stantial work remains to be done in order to understand the role of efflux and its regulation in *Salmonella*.

#### **6.5. The fitness costs**

Mechanisms associated with high‐level FQ resistance are multiple mutations in the type II topoisomerase‐encoding genes and the over‐expression of multidrug resistance efflux pumps. The presence of mutations in these structural or regulatory genes not only increases resistance to quinolones but also affects fitness costs such as reduced growth rates and virulence of the bacterial cell in a lack of antibiotic selective pressure [95–99]. However, maintenance of resis‐ tance can arise through the development of second‐site compensatory mutations that restore fitness and virulence without loss of resistance [100].

The fitness cost of the genes responsible for quinolone resistance traits has not been fully elucidated in high‐level FQ‐resistant *Salmonella*. Nevertheless, results from previous stud‐ ies suggest that high‐level CIP resistance mechanisms in *Salmonella* lead to restrictive condi‐ tions of fitness costs and minimizing the emergence and spread of highly resistant clones in the absence of drug selection pressure [101, 102]. As demonstrated in previous study [103], high‐level CIP‐resistant *S*. Enteritidis *in vitro* derived mutants in the absence of antibiotic selective pressure result in compensatory evolution favoring a reversion back to a more sensi‐ tive phenotype associated with lesser fitness costs, rather than the compensatory mutations that would restore resistance. However, under *in vivo* conditions, a previous study has found that chromosomal mutations of *S*. Typhimurium that confer resistance to NAL, streptomy‐ cin, or rifampicin decrease growth rate and ability to colonize in mice rather than a rever‐ sion to the susceptible phenotype and restore virulence [104]. In contrast to the high‐level FQ resistance, an intermediate level of resistance to CIP of *S*. Typhimurium mutants apparently favored a partial reversion to a susceptible level and a normal growth rate with successfully colonized the gut of chickens, rather than the acquisition of resistance to FQs [101].

Quinolone resistance of non‐typhoidal *Salmonella* is complicated. The understanding of the various mechanisms of quinolone resistance, the fitness costs of each *Salmonella* strain, and the interplay between different quinolone resistance mechanisms has increased in recent years. Increased resistance to quinolones could be selected under a wide range of selective conditions even in the absence of quinolone selective pressure. Therefore, minimizing the emergence and spread of quinolone resistance will not be as simple as limiting the use of these drugs.

## **7. To decrease the emergence and spread of quinolone resistance**

FQs are intensively used in animal production and have allowed better treatment of several animal infectious diseases. The risks of the overuse and misuse of FQs in food‐animal produc‐ tion can contribute to higher levels of resistance in human *Salmonella* infections. Therefore, the FQ resistance of *Salmonella* should be taken into account and prevented as resistant bacteria or resistance genes may be transferred to humans through the food chain. Given the importance of FQ resistance as a global health concern, many researchers have reviewed the existing sci‐ entific literatures and developed guidelines to limit all compounds of FQ use, including use in food‐producing animals. FQs should be banned for all preventive use and mass medication, but only used as life‐saving therapeutic treatment of individual sick animals.

Priority setting of agendas for research on minimizing the emergence of FQ resistance in *Salmonella* is needed to identify missing scientific data and to specify research designs and methods to address these resistance problems in food‐producing animals and human medi‐ cine. The priorities identified by the research agenda must include contributions by different experts in basic genetics and microbiology sciences, veterinary medicine, human medicine, public health organization, social sciences, economics sciences, and public policy.

Furthermore, sufficient research funding for minimizing the FQ resistance of *Salmonella* in human and food‐producing animals has likely contributed to the adequate scientific evidence which necessary for informing public health decisions. Given the scale of the FQ resistance problem and the demonstrated role of FQ uses in food‐producing animals in this public health crisis, adequate support for research specific to the role of food‐producing animal uses of FQs in the development of resistance must be a national priority.

Urgently address complex barriers that limit the quality of data on the use of FQs in food‐produc‐ ing animals and human medicine. Currently, such data from human and veterinary medicine are provided on a voluntary basis, and the methods used to collect, analyze, and report are not standardized because of political, economic, and social barriers. Effective surveillance of FQ use in food‐producing animals and humans is a key first step toward for estimating the full scope of FQ resistance in *Salmonella*. Despite increasingly widespread recognition that FQ use in food‐pro‐ ducing animals is a major factor of human infections with FQ‐resistant *Salmonella*, there remains a significant need for scientific evidence of the FQ use practices that affect the human health risk.

## **8. Conclusion**

**6.5. The fitness costs**

124 Current Topics in Salmonella and Salmonellosis

fitness and virulence without loss of resistance [100].

Mechanisms associated with high‐level FQ resistance are multiple mutations in the type II topoisomerase‐encoding genes and the over‐expression of multidrug resistance efflux pumps. The presence of mutations in these structural or regulatory genes not only increases resistance to quinolones but also affects fitness costs such as reduced growth rates and virulence of the bacterial cell in a lack of antibiotic selective pressure [95–99]. However, maintenance of resis‐ tance can arise through the development of second‐site compensatory mutations that restore

The fitness cost of the genes responsible for quinolone resistance traits has not been fully elucidated in high‐level FQ‐resistant *Salmonella*. Nevertheless, results from previous stud‐ ies suggest that high‐level CIP resistance mechanisms in *Salmonella* lead to restrictive condi‐ tions of fitness costs and minimizing the emergence and spread of highly resistant clones in the absence of drug selection pressure [101, 102]. As demonstrated in previous study [103], high‐level CIP‐resistant *S*. Enteritidis *in vitro* derived mutants in the absence of antibiotic selective pressure result in compensatory evolution favoring a reversion back to a more sensi‐ tive phenotype associated with lesser fitness costs, rather than the compensatory mutations that would restore resistance. However, under *in vivo* conditions, a previous study has found that chromosomal mutations of *S*. Typhimurium that confer resistance to NAL, streptomy‐ cin, or rifampicin decrease growth rate and ability to colonize in mice rather than a rever‐ sion to the susceptible phenotype and restore virulence [104]. In contrast to the high‐level FQ resistance, an intermediate level of resistance to CIP of *S*. Typhimurium mutants apparently favored a partial reversion to a susceptible level and a normal growth rate with successfully

colonized the gut of chickens, rather than the acquisition of resistance to FQs [101].

spread of quinolone resistance will not be as simple as limiting the use of these drugs.

**7. To decrease the emergence and spread of quinolone resistance**

but only used as life‐saving therapeutic treatment of individual sick animals.

Quinolone resistance of non‐typhoidal *Salmonella* is complicated. The understanding of the various mechanisms of quinolone resistance, the fitness costs of each *Salmonella* strain, and the interplay between different quinolone resistance mechanisms has increased in recent years. Increased resistance to quinolones could be selected under a wide range of selective conditions even in the absence of quinolone selective pressure. Therefore, minimizing the emergence and

FQs are intensively used in animal production and have allowed better treatment of several animal infectious diseases. The risks of the overuse and misuse of FQs in food‐animal produc‐ tion can contribute to higher levels of resistance in human *Salmonella* infections. Therefore, the FQ resistance of *Salmonella* should be taken into account and prevented as resistant bacteria or resistance genes may be transferred to humans through the food chain. Given the importance of FQ resistance as a global health concern, many researchers have reviewed the existing sci‐ entific literatures and developed guidelines to limit all compounds of FQ use, including use in food‐producing animals. FQs should be banned for all preventive use and mass medication, Infections in humans with quinolone‐resistant *Salmonella* resulted in increased risk of hospi‐ talization and mortality. FQs are efficient and valuable antimicrobials in some serious animal indications because FQs are the only alternative available. Therefore, if FQs lose their ability for the treatment of animal diseases, the therapeutic effect of some diseases will be compli‐ cated and may result in poor animal welfare and economical losses. Recently, it is now critical that food‐producing animal use of FQs be recognized as one of the major contributors to the development of resistant *Salmonella* strains that result in life‐threatening human infections and included as part of the strategy to control the public health crisis of FQ resistance.

## **Author details**

Siriporn Kongsoi<sup>1</sup> , Chie Nakajima2,3 and Yasuhiko Suzuki2,3\*

\*Address all correspondence to: suzuki@czc.hokudai.ac.jp


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**Provisional chapter**

## *Salmonella* **in Wastewater: Identification, Antibiotic Resistance and the Impact on the Marine Environment** *Salmonella* **in Wastewater: Identification, Antibiotic Resistance and the Impact on the Marine Environment**

Abdellah El Boulani, Rachida Mimouni, Hasna Mannas, Fatima Hamadi and Nouredine Chaouqy Hasna Mannas, Fatima Hamadi and Nouredine Chaouqy Additional information is available at the end of the chapter

Abdellah El Boulani, Rachida Mimouni,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67298

#### **Abstract**

Many coastal cities around the world discharge their wastewaters into the marine environment. These wastewaters contain a high variety of pathogenic microorganisms that would have a role in the contamination of this ecosystem and may have potential risks for public health and environment. Using an environmental approach, we inves‐ tigate the presence of *Salmonella* in wastewater treatment plants and its presence after the treatment in its receiving marine environment. In this environmental approach, we provide information about the inefficiency of wastewater treatment to remove *Salmonella*, especially that wastewater is considered as a good tank of high diversity of *Salmonella* serotypes. The identified *Salmonella* serotypes in the receiving marine environment almost coincide with those identified in wastewater. This characterization of *Salmonella* strains from wastewater and marine environment involves the direct impact of municipal wastewater discharges on this environment. Antibiotic susceptibility tests reveal gener‐ ally the presence of multiresistant *Salmonella* strains in wastewater, which usually end up in the marine environment and may have a significant risk on the public health.

**Keywords:** antibiotic susceptibility, marine environment, *Salmonella*, Wastewater, wastewater treatment

## **1. Introduction**

Many major coastal cities around the world discharge their wastewaters into the marine envi‐ ronment. Wastewater may consist of a combination of domestic, industrial and agricultural effluent. Generally, wastewater treatment plants (WWTPs) are mostly designed to effectively remove fecal bacteria, some chemical pollutants but are not provided to eliminate pathogenic

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

microorganisms [1, 2]. Furthermore, most of these treatment systems remain imperfect and continue to reject varied concentrations of enteric bacteria in the environment [1–5].

Pathogenic bacteria become normally more harmful to environment and humans when they acquire resistance to antibiotics. Moreover, many studies [5–7] confirmed the detection of antibiotic concentrations in wastewater that treatment systems generally fail to eliminate. Therefore, when there is a contact between bacteria and antibiotics traces, the wastewater could be an ideal environment to the exchange of genetic material between bacteria and consequently the acquisition of antibiotic resistance [5, 6, 8, 9].

The *Salmonella* genus is one of the pathogen bacteria that could be conveyed to the environmen‐ tal waters through sewage pollution. Therefore, the determination of the presence of *Salmonella*, its levels and its antibiotic resistance at the same time in wastewaters and environmental waters are crucial and are required to assess the related health risks.

Previous works [1, 9–13] have studied the presence of *Salmonella* in wastewater but they stay insufficient to provide enough information on the diversity and the antibiotic resistance pro‐ files of *Salmonella*. This makes transmission of *Salmonella* via wastewater, its impact on the environment and the origin of contamination poorly understood. In Morocco, *Salmonella* in wastewater is rarely discussed and its bibliography is very scarce. In this context, we are cur‐ rently leading the first study about *Salmonella* in the southern region of Morocco. This work is focusing on the identification, serotyping and antibiotic susceptibility of *Salmonella* strains isolated from Agadir wastewater treatment plant.

The aim of this chapter is first to provide qualitative information on the capacity of wastewater treatment process to eliminate *Salmonella* considered among the most pathogenic bacteria eas‐ ily transmissible in water. The second aim is to present the most *Salmonella* serotypes isolated from wastewater and marine environment and the possible impact of wastewater discharges on marine environment.

## **2. WWTPs removal efficiency of** *Salmonella*

Wastewater treatment plants know a growing pressure which leads to the evacuation of untreated or insufficiently treated effluents into the environment. Thus, the efficient removal of pathogenic bacteria in wastewater is a crucial task because wastewater discharges can significantly increase the contamination of surface water and lead to water and seafood‐ borne infections. Generally, an optimal wastewater treatment process can attain 90–99% of reduction of microbial load [2, 14, 15]. However, in some cases, the reduction level decreases are mostly due to a nonrespective operating mode or due to bad or inadequate plant maintenance.

Several studies have shown the presence of *Salmonella* in wastewater before and after treatment even if treatment processes are different [1, 9, 10, 13]. This could be explained by the fact that WWTPs are mostly designed just to reduce microbial load but are not conceived specifically to completely eliminate the pathogenic bacteria.

An activated sludge treatment process remains unable to totally remove *Salmonella* [9, 12]. Koivunen et al. have also shown that activated sludge process coupled with phosphorus pre‐ cipitation followed by a secondary settling or coupled with denitrification‐nitrification fails to remove *Salmonella* from municipal wastewater [1].

microorganisms [1, 2]. Furthermore, most of these treatment systems remain imperfect and

Pathogenic bacteria become normally more harmful to environment and humans when they acquire resistance to antibiotics. Moreover, many studies [5–7] confirmed the detection of antibiotic concentrations in wastewater that treatment systems generally fail to eliminate. Therefore, when there is a contact between bacteria and antibiotics traces, the wastewater could be an ideal environment to the exchange of genetic material between bacteria and

The *Salmonella* genus is one of the pathogen bacteria that could be conveyed to the environmen‐ tal waters through sewage pollution. Therefore, the determination of the presence of *Salmonella*, its levels and its antibiotic resistance at the same time in wastewaters and environmental waters

Previous works [1, 9–13] have studied the presence of *Salmonella* in wastewater but they stay insufficient to provide enough information on the diversity and the antibiotic resistance pro‐ files of *Salmonella*. This makes transmission of *Salmonella* via wastewater, its impact on the environment and the origin of contamination poorly understood. In Morocco, *Salmonella* in wastewater is rarely discussed and its bibliography is very scarce. In this context, we are cur‐ rently leading the first study about *Salmonella* in the southern region of Morocco. This work is focusing on the identification, serotyping and antibiotic susceptibility of *Salmonella* strains

The aim of this chapter is first to provide qualitative information on the capacity of wastewater treatment process to eliminate *Salmonella* considered among the most pathogenic bacteria eas‐ ily transmissible in water. The second aim is to present the most *Salmonella* serotypes isolated from wastewater and marine environment and the possible impact of wastewater discharges

Wastewater treatment plants know a growing pressure which leads to the evacuation of untreated or insufficiently treated effluents into the environment. Thus, the efficient removal of pathogenic bacteria in wastewater is a crucial task because wastewater discharges can significantly increase the contamination of surface water and lead to water and seafood‐ borne infections. Generally, an optimal wastewater treatment process can attain 90–99% of reduction of microbial load [2, 14, 15]. However, in some cases, the reduction level decreases are mostly due to a nonrespective operating mode or due to bad or inadequate plant

Several studies have shown the presence of *Salmonella* in wastewater before and after treatment even if treatment processes are different [1, 9, 10, 13]. This could be explained by the fact that WWTPs are mostly designed just to reduce microbial load but are not conceived specifically to

continue to reject varied concentrations of enteric bacteria in the environment [1–5].

consequently the acquisition of antibiotic resistance [5, 6, 8, 9].

are crucial and are required to assess the related health risks.

isolated from Agadir wastewater treatment plant.

**2. WWTPs removal efficiency of** *Salmonella*

completely eliminate the pathogenic bacteria.

on marine environment.

138 Current Topics in Salmonella and Salmonellosis

maintenance.

Until now, no study has been conducted on the elimination of *Salmonella* by infiltration percolation treatment process through sand. However, a current study conducted by our research team (unpublished data) showed the inefficiency of this treatment system to remove *Salmonella*. Thus, treated wastewater still contains *Salmonella* which continues to be detected at the treatment plant exit.

To get an efficient reduction or a total removal of *Salmonella*, a tertiary wastewater treatment is needed. Indeed, Koivunen et al. show that tertiary treatment by rapid sand contact filter allows a very good efficiency to remove *Salmonella.* Consequently, *Salmonella* was not found in wastewaters after tertiary filtration [1]. Furthermore, a current study conducted by our team showed that tertiary treatment by UV irradiation applied just on a portion of the secondary treated effluents (which are intended for the irrigation of golf courses) allows total elimination of *Salmonella* (unpublished data).

## **3. Microbiological risks associated with** *Salmonella* **in wastewater**

Wastewater is known to be a common vehicle for the transport and transmission of *Salmonella* serovars and is able to pollute environment and infect humans. Indeed, several outbreaks and contaminations have been related to *Salmonella* in wastewater through irrigation of crops [16–18], infiltration and transport in soil to groundwater [19–21] or to its discharge into the marine environment [22, 23]. In this last case, although some pathogens are natu‐ rally present in the aquatic environment, *Salmonella* may be introduced through animal or human fecal and sewage pollution [24].

The risk of contamination linked to *Salmonella* is enhanced by its ability to survive lon‐ ger in the environment. Indeed, contamination of groundwater, environmental water and soil by *Salmonella* is due to its ability to survive very long time in these environments [25]. Furthermore, contaminations related to *Salmonella* in seawater and seafood are increased by the capacity of *Salmonella* to survive in relatively high salt conditions [26]. Thus, the per‐ sistence of wastewater discharges in coastal waters may increase the load of *Salmonella* in marine environment, and therefore increase the higher incidence of seawater and seafood‐ borne infections.

## **4. Identification methods of** *Salmonella*

Various methods have been developed for the subtyping of *Salmonella*. Each of them has its advantages and drawbacks in terms of cost, speed, robustness, and sensitivity [27]. The choice of identification techniques of bacteria is generally done according to the objectives of identifying and the available means. Biochemical techniques are usually used to isolate and identify only species. For further characterization, determination of the origins and the relationships between different isolates, many other accurate methods are needed.

Conventional serotyping using somatic and flagellar *Salmonella* antisera is the most frequently used reference method for serotyping *Salmonella* isolated from wastewater [1, 9, 28, 29]. This method is based on the direct agglutination technique blade, involving *Salmonella* strains with different antisera to identify variants of the somatic O and flagellar H antigens. Serotyping is generally performed according to the antigenic formulae of Kauffmann‐White‐Le Minor scheme [30]. The main limitation of this technique is that not all O serotypes are included in Polyvalent O antisera which *Salmonella* species should agglutinate with [31]. A combina‐ tion between conventional serotyping and the ribosomal spacer‐heteroduplex polymorphism (RS‐HP) methods was also used to characterize *Salmonella* strains from wastewater [13]. This technique based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels [13].

PFGE allows a high discrimination and it is usually used for outbreak investigations and it is also widely used for characterizing epidemic *Salmonella* strains. The capacity of PFGE to differ‐ entiate strains of bacterial pathogens makes it a standard method used to assess the epidemic spread of infectious diseases and to trace *Salmonella* outbreaks. However, even if it is reproduc‐ ible and discriminatory, some strains of *Salmonella* cannot be typed by PFGE [27]. Recently, matrix‐assisted laser desorption/ionization time of light mass spectrometry (MALDI‐TOF MS) has been used for the identification of *Salmonella.* It is also utilized to discriminate *Salmonella Typhi* from other *Salmonella* serovars [32]. Despite its rapidity and simplicity, the preparation of MALDI‐TOF requires more modifications and improvements to available protocols before being adopted as an autonomous method [32]. Other molecular methods developed as alter‐ natives to conventional serotyping (MLST, MLVA, SNP, and molecular typing with composite microarrays) seem successful. However, these methods do not provide exactly similar results like those obtained by the current reference method which is agglutination serotyping [27].

## **5.** *Salmonella* **serotypes isolated from wastewater**

As reported, wastewater is an ideal tank of *Salmonella* strains. Furthermore, identified *Salmonella* showed generally high variability of serotypes. The major serotypes isolated in wastewater from different countries are presented in **Table 1**.

Data concerning *Salmonella* serotyping (**Table 1**) show high heterogeneity of serotypes iso‐ lated from wastewater. This high diversity can be explained by the variety of the origin of effluents carrying these *Salmonella* strains. The most frequent serotypes identified in France wastewater were Newport, Saintpaul, and Brandenburg [13]. Indeed, Serotypes Mbandaka, Virchow, Hadar, Indiana, Infantis, Saintpaul, and Senftenberg are commonly isolated from poultry farms. Also, Typhimurium and Indiana serotypes are generally isolated in human pathology and can derive from healthy carriers [13]. Espigares et al. have also explained this diversity by the variety of the origin of *Salmonella* which may be of human or animal origin [9]. *Salmonella* Agona, Saintpaul, Virchow, and Corvallis are the most frequent serotypes identi‐ fied in Finnish wastewater [1]. According to Koivunen et al., this diversity may be reported to the size of the population suggesting, therefore, that larger populations produce larger spec‐ tra of serovars [1]. However, despite these explanations, the origin of this diversity remains unknown. The large variability in serovars identified in USA wastewater indicates multiple sources of the isolates. This variability may be due to fecal shedding in clinical salmonel‐ losis or to animal agriculture [33]. Our current study (not shown data) concerning isolated *Salmonella* from wastewater in Agadir, a coastal city in Morocco, show high variability of serotypes. Thus, among 52 *Salmonella* strains we identified 18 different serotypes. *Salmonella Muenster* was the major serotype which showed a high incidence with 14 isolates followed by *Salmonella Infantis*, *Salmonella Senftenberg*, *Salmonella Montevideo*, and *Salmonella Kentucky* as the most frequent serotypes identified*.* This high diversity of serotypes in Agadir wastewater can be explained by its various origins including domestic and industrial effluents especially those coming from poultry slaughterhouse and fish processing plants very widespread in the city.


**Table 1.** *Salmonella* serotypes isolated from wastewater.

and the available means. Biochemical techniques are usually used to isolate and identify only species. For further characterization, determination of the origins and the relationships between

Conventional serotyping using somatic and flagellar *Salmonella* antisera is the most frequently used reference method for serotyping *Salmonella* isolated from wastewater [1, 9, 28, 29]. This method is based on the direct agglutination technique blade, involving *Salmonella* strains with different antisera to identify variants of the somatic O and flagellar H antigens. Serotyping is generally performed according to the antigenic formulae of Kauffmann‐White‐Le Minor scheme [30]. The main limitation of this technique is that not all O serotypes are included in Polyvalent O antisera which *Salmonella* species should agglutinate with [31]. A combina‐ tion between conventional serotyping and the ribosomal spacer‐heteroduplex polymorphism (RS‐HP) methods was also used to characterize *Salmonella* strains from wastewater [13]. This technique based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both

PFGE allows a high discrimination and it is usually used for outbreak investigations and it is also widely used for characterizing epidemic *Salmonella* strains. The capacity of PFGE to differ‐ entiate strains of bacterial pathogens makes it a standard method used to assess the epidemic spread of infectious diseases and to trace *Salmonella* outbreaks. However, even if it is reproduc‐ ible and discriminatory, some strains of *Salmonella* cannot be typed by PFGE [27]. Recently, matrix‐assisted laser desorption/ionization time of light mass spectrometry (MALDI‐TOF MS) has been used for the identification of *Salmonella.* It is also utilized to discriminate *Salmonella Typhi* from other *Salmonella* serovars [32]. Despite its rapidity and simplicity, the preparation of MALDI‐TOF requires more modifications and improvements to available protocols before being adopted as an autonomous method [32]. Other molecular methods developed as alter‐ natives to conventional serotyping (MLST, MLVA, SNP, and molecular typing with composite microarrays) seem successful. However, these methods do not provide exactly similar results like those obtained by the current reference method which is agglutination serotyping [27].

As reported, wastewater is an ideal tank of *Salmonella* strains. Furthermore, identified *Salmonella* showed generally high variability of serotypes. The major serotypes isolated in

Data concerning *Salmonella* serotyping (**Table 1**) show high heterogeneity of serotypes iso‐ lated from wastewater. This high diversity can be explained by the variety of the origin of effluents carrying these *Salmonella* strains. The most frequent serotypes identified in France wastewater were Newport, Saintpaul, and Brandenburg [13]. Indeed, Serotypes Mbandaka, Virchow, Hadar, Indiana, Infantis, Saintpaul, and Senftenberg are commonly isolated from poultry farms. Also, Typhimurium and Indiana serotypes are generally isolated in human pathology and can derive from healthy carriers [13]. Espigares et al. have also explained this

different isolates, many other accurate methods are needed.

**5.** *Salmonella* **serotypes isolated from wastewater**

wastewater from different countries are presented in **Table 1**.

serotype and intraserotype levels [13].

140 Current Topics in Salmonella and Salmonellosis

## **6.** *Salmonella* **serotypes isolated from a wastewater receiving marine environment**

In coastal cities, the marine environment is usually the final end of treated and untreated sewage. **Table 2** shows some studies throughout the world carried in this regard.


**Table 2.** *Salmonella* serotypes isolated from marine environment.

As reported in wastewater, data from marine environment also reveal a large diversity of *Salmonella* serotypes. However, the origin of these serovars remains little known. Following to data in **Tables 1** and **2**, it appears that the most serotypes found in wastewater are also isolated in the surrounding marine environment. Furthermore, studies from France [13] and Mexico [29] showed that Newport and Typhimurium were respectively the major *Salmonella* serotypes isolated in wastewater. These serotypes were also the same major serotypes isolated from marine environment [13, 29]. Serovar Typhimurium has been shown to be the most common serovar isolated from marine environment in different parts of the world [13, 29, 34–36]. It was suggested previously that this serotype shows an excellent adaptation to the marine water stress after passing through wastewater [37]. In Agadir, study conducted by our team [38] in marine environment has identified *S. Muenster* as the major serotype (13 strains among 46 isolates). This high occurrence of serotype Muenster coincides with its high inci‐ dence in Agadir wastewater. Therefore, these findings reveal that wastewater discharges into the marine environment could be the main source of contamination and the principal origin of *Salmonella* strains found in this environment.

## **7. Antimicrobial resistance of** *Salmonella* **isolated from wastewater**

Urban wastewater treatment plants (UWTPs) are suspected to be among the main anthropo‐ genic sources for antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bac‐ teria (ARB) spread into the environment [14, 39, 40]. The presence of antibiotics in effluents is generally due to incomplete metabolism in humans or because of the mismanagement of unused antibiotics [4, 41].

Generally, bacteria in wastewater are in permanent contact with various elements including antibiotics and other chemical products. Furthermore, antibiotic resistance was positively cor‐ related with the occurrence of antibiotic residues. In addition, a relationship between antibi‐ otic residues, bacterial community structure and antibiotic resistance was demonstrated [42]. Therefore, the elimination of these antibiotics from wastewater is a major concern. Sorption and hydrolysis could be a degradation pathway that may lead to the removal of antibiotics in wastewater [39]. However, in previous reports, it has been demonstrated that UV radiation is not effective to remove antibiotics [43]. This indicates that photolysis is not an important mechanism for degradation of these compounds in wastewater. A combination of physico‐ chemical and biological treatment and the optimization of their operating conditions might prove an effective removal increase of persistent antibiotic residues in wastewater [39].

According to previous studies [6, 44, 45], ARB have been detected widely in wastewater samples and comparatively to surface water, higher proportion of ARB was reported in raw and treated wastewater than in surface water [4, 40]. It has also been reported that *Salmonella* strains show a high proportion among other multidrug resistant bacteria [46]. According to these studies, conditions in wastewater treatment plants (WWTPs) seem to be favorable for the proliferation of ARB.

As reported in wastewater, data from marine environment also reveal a large diversity of *Salmonella* serotypes. However, the origin of these serovars remains little known. Following to data in **Tables 1** and **2**, it appears that the most serotypes found in wastewater are also isolated in the surrounding marine environment. Furthermore, studies from France [13] and Mexico [29] showed that Newport and Typhimurium were respectively the major *Salmonella* serotypes isolated in wastewater. These serotypes were also the same major serotypes isolated from marine environment [13, 29]. Serovar Typhimurium has been shown to be the most common serovar isolated from marine environment in different parts of the world [13, 29, 34–36]. It was suggested previously that this serotype shows an excellent adaptation to the marine water stress after passing through wastewater [37]. In Agadir, study conducted by our team [38] in marine environment has identified *S. Muenster* as the major serotype (13 strains among 46 isolates). This high occurrence of serotype Muenster coincides with its high inci‐ dence in Agadir wastewater. Therefore, these findings reveal that wastewater discharges into the marine environment could be the main source of contamination and the principal origin

**Country France Spain Mexico Morocco USA**

Serotypes Virchow Hadar Othmarschen Hadar Bardo

References [13] [34] [29] [35, 36, 38] [47]

Cerro Anatum Stanley Altona Adelaide Newport Enteritidis Galiema Anatum Agona Typhimurium Goldcoast Bulovka Corvallis Arizona

> London Tonev Senftenberg Hartford Newport Subaru Typhimurium Newport Typhimurium Typhimurium Kentucky Poona

> > Vejle London Reading Winnipeg Mbandaka Typhimurium Muenster Oakland Blockley Labadi

**7. Antimicrobial resistance of** *Salmonella* **isolated from wastewater**

Urban wastewater treatment plants (UWTPs) are suspected to be among the main anthropo‐ genic sources for antibiotics, antibiotic resistant genes (ARGs) and antibiotic resistant bac‐ teria (ARB) spread into the environment [14, 39, 40]. The presence of antibiotics in effluents

of *Salmonella* strains found in this environment.

**Table 2.** *Salmonella* serotypes isolated from marine environment.

142 Current Topics in Salmonella and Salmonellosis

Antibiotic resistance (ABR) of *Salmonella* isolated from wastewater has been discussed in some studies. A wide variety of antibiotic resistance patterns of *Salmonella* serotypes have been found in USA wastewater [33]. Indeed, 86% of 647 *Salmonella* isolates were susceptible, 4% were monoresistant and 10% were multiresistant. *Salmonella* serovars multiresistant to at least four antibiotics were *Salmonella Braenderup*, Derby, Edinburg, Hadar, Saintpaul, Typhimurium, Uganda and Virchow. A resistance to the third generation of cephalosporins considered as one of the first drugs of choice to treat human salmonellosis has also been reported [33]. From Finnish wastewater [1], among 197 isolates, 44% of the *Salmonella* strains were resistant to antimicrobial agents, almost 20% were multiresistant. The most multiresistant serotype was *Salmonella Saintpaul* (to 6 antibiotics). Moreover, 32% of strains were resistant to nalidixic acid which can indicates the reduction of sensitivity to ciprofloxacin considered as the selected drug in severe infections [1]. The most resistant serotype isolated in wastewater from Spain was *Salmonella Hadar* with a pattern of multiresistance to six antibiotics [9]. In Morocco, among 42 strains isolated from wastewater, 19 (45.2%) were resistant at least to two antibiotics. The most multiresistant serotype was *S. Typhimurium* followed by *S. Hadar* and *S. Senftenberg*. *S. Typhimurium* was resistant to ampicillin, chloramphenicol, streptomycin, sulfametoxazole/ trimetoprim, and tetracycline. Serovars Give, Newport and Anatum were susceptible to all used antibiotics [28]. Antibiotic susceptibility test (ATS) of *Salmonella* serotypes from Agadir wastewater treatment plant carried out by our team showed that among 52 isolates 23% of strains were multiresistant. ATS was carried out by the disk diffusion method, with a panel of 32 antimicrobial drugs (Bio‐Rad) and interpreted according to the EUCAST clinical guide‐ lines (http://www.eucast.org/clinical\_breakpoints/). Multiresistant serovars were Chester, Kentucky, and Typhimurium. The most multiresistant serotype identified in this study was Typhimurium with a pattern of multiresistance to 18 antibiotics.

All these data showed that wastewater is a tank of a wide variety of *Salmonella enterica* serovars and ABR patterns. Other data showed that multiresistant bacteria have been detected extensively in wastewater samples which pass through WWTPs and arrive to the receiving environment [14, 40, 44]. Consequently, this problem is of great concern for the wastewater management of coastal cities because multiresistant bacteria have become a significant public health problem. The caused infec‐ tion is much more difficult to be treated because the panel of effective antibiotics will be reduced.

## **8. Conclusions**


## **Acknowledgements**

We wish to thank the Institute Pasteur (IP) of Paris, France for help. We are grateful to the Laboratory of Microbiology, National Health Security Food Office (ONSSA) Agadir, Morocco for their excellent technical assistance. We thank also AGROTECH Agadir for their cooperation.

## **Author details**

Abdellah El Boulani1 , Rachida Mimouni1 \*, Hasna Mannas1 , Fatima Hamadi1 and Nouredine Chaouqy2

\*Address all correspondence to: r\_mimouni@yahoo.fr

1 Faculty of Sciences, Laboratory of Microbial Biotechnology & Plant Protection, University Ibn Zohr, Agadir, Morocco

2 Laboratory of Microbiology, National Health Security Food Office (ONSSA), Agadir, Morocco

## **References**

All these data showed that wastewater is a tank of a wide variety of *Salmonella enterica* serovars and ABR patterns. Other data showed that multiresistant bacteria have been detected extensively in wastewater samples which pass through WWTPs and arrive to the receiving environment [14, 40, 44]. Consequently, this problem is of great concern for the wastewater management of coastal cities because multiresistant bacteria have become a significant public health problem. The caused infec‐ tion is much more difficult to be treated because the panel of effective antibiotics will be reduced.

‐ Conventional wastewater treatment without efficient tertiary treatment, like filtration or dis‐ infection by UV, is generally insufficient and consequently constitutes a risk for public health. ‐ Wastewater is an ideal tank of high diversity of *Salmonella* serotypes and ABR patterns

‐ The presence of the same major *Salmonella* serovars simultaneously in sewage and in the marine environment confirms that the principal source of contamination of marine envi‐

‐ Improvement in the efficiency of treating antibiotics residues in WWTPs is the first line of defense against the potential ecological impacts of these chemicals in the environment. ‐ On the one hand, wastewater treatment must adopt effective methods for treatment, and on the other hand, there should be a rigorous approach for surveillance and monitoring effluents

We wish to thank the Institute Pasteur (IP) of Paris, France for help. We are grateful to the Laboratory of Microbiology, National Health Security Food Office (ONSSA) Agadir, Morocco for their excellent technical assistance. We thank also AGROTECH Agadir for their cooperation.

\*, Hasna Mannas1

1 Faculty of Sciences, Laboratory of Microbial Biotechnology & Plant Protection, University

2 Laboratory of Microbiology, National Health Security Food Office (ONSSA), Agadir,

, Fatima Hamadi1

and Nouredine

which usually end up in the marine environment in coastal cities.

before and after treatment and discharge into the environment.

, Rachida Mimouni1

\*Address all correspondence to: r\_mimouni@yahoo.fr

ronment by *Salmonella* is wastewater discharges.

**8. Conclusions**

144 Current Topics in Salmonella and Salmonellosis

**Acknowledgements**

**Author details**

Chaouqy2

Morocco

Abdellah El Boulani1

Ibn Zohr, Agadir, Morocco


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