**2. Mechanisms and propagation of resistance**

In microorganisms, drug resistance arises in order to contend against a harmful stimulus that threatens their survival. In bacteria, the mechanisms that confer the resistance against antibiotics could be classified as intrinsic (mutations originating in the organism itself) or acquired by transfer of genetics elements during the replication of DNA (vertical transfer) or from different species or genera (horizontal transfer) [6] (**Figure 1**).

#### **2.1 Intrinsic mechanisms or natural resistance**

The intrinsic mechanisms can be found in the cell in a natural manner. They are conditions that are universally found in bacterial species and that are independent of antibiotic selectivity [7]. Some of the intrinsic mechanisms are described below.

#### *2.1.1 Permeability or impermeability of the outer membrane or cell wall*

Gram-positive bacteria are more susceptible to various antibiotics since they have a thick outer layer of peptidoglycan with polymers of teichoic acid and covalently bound proteins, which allows the easy penetration of small molecules up to 30–57 kDa [8, 9]. In contrast, Gram-negative bacteria have an outer membrane that surrounds them with a relatively thin peptidoglycan layer. The composition of the outer membrane is based on lipid molecules covalently linked to polysaccharide units [10].

The lipid molecule has a large chain of fatty acids that contribute to reduce the fluidity of the lipopolysaccharide (LPS) membrane [10]. The central region of the LPS plays an important role providing a barrier to hydrophobic antibiotics and other compounds. It has been reported that strains that express full-length LPS have an intrinsic resistance to hydrophobic antibiotic class such as macrolides and aminoglycosides. Another modification observed is the alteration of the anionic nature

**171**

**Figure 1.**

*Schematic representation of the mechanisms of multidrug-resistance acquisition in bacteria.*

*Multidrug-Resistant Bacterial Foodborne Pathogens: Impact on Human Health and Economy*

of the LPS; the most common LPS modifications are the cationic substitution of phosphate groups with 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), which decreases the net negative load of lipid A from minus 1.5 to minus 1 or from minus 1.5 to 0, respectively [11]. The net positive charge resulting from the LPS modification reduces the binding of some cationic antibiotics such as polymyxins, leading the resistance of the bacteria such as *Escherichia coli*,

*DOI: http://dx.doi.org/10.5772/intechopen.88789*

*Klebsiella pneumoniae*, and *Salmonella enterica* [12].

*Multidrug-Resistant Bacterial Foodborne Pathogens: Impact on Human Health and Economy DOI: http://dx.doi.org/10.5772/intechopen.88789*

of the LPS; the most common LPS modifications are the cationic substitution of phosphate groups with 4-amino-4-deoxy-L-arabinose (L-Ara4N) or phosphoethanolamine (PEtN), which decreases the net negative load of lipid A from minus 1.5 to minus 1 or from minus 1.5 to 0, respectively [11]. The net positive charge resulting from the LPS modification reduces the binding of some cationic antibiotics such as polymyxins, leading the resistance of the bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, and *Salmonella enterica* [12].

**Figure 1.**

*Schematic representation of the mechanisms of multidrug-resistance acquisition in bacteria.*

*Pathogenic Bacteria*

the ecology of microorganism. In order to contend with the residues present in their environment, microorganisms may acquire genetic elements that allow them to cope with these compounds and survive. In some cases, the use of poor quality drug, counterfeit products, incorrect product, and modified dosage can accelerate the development of microbial resistance. Another relevant factor for development of AMR is the inadequate disposal of waste generated in the agricultural production and pharmaceutical and wastewater treatment plants as they can be spread through the environment [3]. One of the recently described phenomena observed is the association between the emergence of multiresistant microorganisms (MMR) and the increase in the isolates that show the production of extended-spectrum betalactamase enzymes (ESBL). Currently, more than 200 varieties of BLEE enzymes are recognized with different substrates, and the frequency of isolates producing

these enzymes varies from country to country (from 20 to 48%) [3].

antimicrobials in the production of animals and crops.

**2. Mechanisms and propagation of resistance**

**2.1 Intrinsic mechanisms or natural resistance**

transfer) [6] (**Figure 1**).

Although there are many factors that favor the spread of antibiotic resistance, it affects different sectors, such as human health, animal health, agriculture, environment, and commercial trade [4]. It is estimated that 700,000 people die each year from infections caused by microorganisms resistant to antimicrobials and a large number of sick animals that do not respond to treatments [5]. Within the agricultural and food industry, resistant microorganisms represent a risk for production that threatens the global economy. For this reason, it is important to implement supervised agricultural regulations and practices that ensure the responsible use of

In microorganisms, drug resistance arises in order to contend against a harmful stimulus that threatens their survival. In bacteria, the mechanisms that confer the resistance against antibiotics could be classified as intrinsic (mutations originating in the organism itself) or acquired by transfer of genetics elements during the replication of DNA (vertical transfer) or from different species or genera (horizontal

The intrinsic mechanisms can be found in the cell in a natural manner. They are conditions that are universally found in bacterial species and that are independent of antibiotic selectivity [7]. Some of the intrinsic mechanisms are described below.

Gram-positive bacteria are more susceptible to various antibiotics since they have a thick outer layer of peptidoglycan with polymers of teichoic acid and covalently bound proteins, which allows the easy penetration of small molecules up to 30–57 kDa [8, 9]. In contrast, Gram-negative bacteria have an outer membrane that surrounds them with a relatively thin peptidoglycan layer. The composition of the outer membrane is based on lipid molecules covalently linked to polysaccharide units [10].

The lipid molecule has a large chain of fatty acids that contribute to reduce the fluidity of the lipopolysaccharide (LPS) membrane [10]. The central region of the LPS plays an important role providing a barrier to hydrophobic antibiotics and other compounds. It has been reported that strains that express full-length LPS have an intrinsic resistance to hydrophobic antibiotic class such as macrolides and aminoglycosides. Another modification observed is the alteration of the anionic nature

*2.1.1 Permeability or impermeability of the outer membrane or cell wall*

**170**

On the other hand, embedded in the outer layer membrane of Gram-negative bacteria, there are proteins called porins that function as a channel through which the molecules can diffuse. Porins could restrict the influx of numerous antibiotics and contribute to the resistance against them [13]. The mechanism of resistance promoted by the porins consists in changing the hydrophilic composition of some antibiotics such as beta-lactam, chloramphenicol, fluoroquinolones, and tetracyclines. Likewise, the alteration of the amount or modification of the structure of the porins promotes resistance to antibiotics [11].

#### *2.1.2 Expulsion of the antibiotic by active mechanisms*

In general those mechanisms are mediated by bacterial flow pumps that actively transport many toxic molecules out of the cell [14]. The outflow pumps can interact with only one molecule (enzyme substrate specific), or they can have a broader spectrum and export distinct classes of molecules. Antibiotic resistance mediated by active outflow pumps may be incidental, since the pumps exhibit a broad-range substrate [9]. However, efflux pumps associated with antibiotic resistance have been described in Gram-positive and Gram-negative bacterial pathogens. The energy of some flow pumps depends of the antibiotic agents in order to extract it from the periplasm to the outside. The overexpression of one or more of these flow pumps prevents the intracellular accumulation of antibiotics at the thresholds necessary to exert their inhibitory activity [15].

#### *2.1.3 Modification of the target site*

Most of the antibiotics bind specifically to their targets with high affinity. Changes in target structure prevent an effective binding to antibiotics but still allow the target to carry out its normal function. The target modification could be originated by a mutation in the gene that encodes for the antibiotic target [14]. An example of this mutation mechanism is the linezolid antibiotic, a member of the oxazolidinone class, which inhibits the initiation of bacterial translation by altering multiple copies of the V domain of the 23S rRNA in Gram-positive bacteria. The mutation in one of these copies of the V domain can confer antibiotic resistance [16].

Another mechanism relies in avoiding or releasing the binding of the antibiotic to their target site [17]. One of the most representative examples of this mechanism and of current importance is that used by quinolones, which exert their function by inhibiting important enzymes of bacterial DNA replication such as gyrase and topoisomerases II and IV. The mechanism of evasion of the antibiotic function is by the expression of repeating pentapeptides (PRP), encoded by *qrn* genes, which bind and promote the release of the quinolone from the target enzymes, allowing the normal activity of the topoisomerases [18].

#### *2.1.4 Enzymatic inactivation or modification of antibiotics*

In this case, the mechanism of action could be by enzymatic hydrolysis [19] or modification of chemical groups by transfer or addition of different chemical compounds [14]. The classic example of a hydrolytic enzyme is the beta-lactamase, which hydrolyzes the beta-lactam ring, a common structural element in penicillins, cephalosporins, carbapenems, and monobactams [20]. Four classes of beta-lactamases have been described: the classes A, C, and D have a serine hydrolase activity; in contrast, class B has a metalloenzyme activity [21]. Another example of this type of resistance is provided by the enzymes erythromycin esterases EreA and EreB. These enzymes hydrolyze the macrolactone rings of macrolides such as erythromycin. It should be

**173**

chromosome [29].

*2.2.1 Plasmids*

*Multidrug-Resistant Bacterial Foodborne Pathogens: Impact on Human Health and Economy*

noted that EreB enzyme confers resistance to almost all members of the macrolide class, with the exception of telithromycin, a semisynthetic erythromycin derivative, which belongs to a new class of antibiotics called ketolides [22]. In contrast, the EreA

The modification of antibiotics includes the modification of some element of their structure, which is essential in the union with the bacteria target diminishing its affinity. This mechanism involves the addition or transfer of groups such as N-acetyl, phosphoryl, O-nucleoside, O-ribosyl, and O-glycoside. Unlike hydrolysis, this modification does not destroy the essential structures of the antibiotic but obstructs the interaction of the antimicrobial with its target. An example of this mechanism occurs for polycationic antibiotics such as aminoglycosides, which act between the ionic bonds of the amino and hydroxyl groups of the antibiotics and the 16S rRNA region of the A site of the bacterial ribosome, deteriorating the translation mechanism. The enzymes responsible for the modification of the aminoglycosides are the aminoglycoside phosphotransferases (APH) and nucleotidyltransferases (ANT) that modify the hydroxyl groups and the aminoglycoside acetyltransferases (AAC) which modify the amino groups, changing

enzyme does not hydrolyze azithromycin and also telithromycin [23].

the size, structure, and electronic properties of the antibiotic [24].

**2.2 Genetic mobile elements transfer or acquired resistance mechanisms**

Once the bacterial cell acquires some degree of antibiotic resistance by an intrinsic mechanism that implies DNA modification, it can transfer the gene or genes encoding for the resistance marker to the offspring (vertical transfer) or to a different specie or genus (horizontal transfer) [25]. The gene resistance can be acquired by genetic mobile elements such as plasmids, transposons, or integrons [19].

The vertical transfer or vertical evolution occurs when a spontaneous mutation in the bacterial chromosome confers resistance to some members of the bacterial population. Once the resistance genes have arisen, they are transferred to the progeny of the bacteria during DNA replication [6]. When the bacterial genes that confer resistance to antibiotics are mobile, because they are contained within plasmids or are flanked by sequences recognized by some DNA transposition enzymes, they can be transferred between bacteria of a different taxonomic and ecological group. Some genetic mobile elements are plasmids, transposons, integrases, and genetic cassettes; in general this mechanism is called horizontal transfer gene [19, 26].

In bacterial cells, there are circular portions of extrachromosomal DNA that improve the survival characteristics of bacteria. This genetic information could be dispensable when it is no longer necessary to contend with the specific stress to which it imparts protection. Plasmids are self-replicating, given that they do so independently of chromosomal DNA replication. When plasmids contain antibiotic resistance genes, they are called plasmids R, and they can be transferred between bacteria of the same or different genera. Plasmids can be transferred to another bacterial cell by mechanisms called transformation or conjugation [19]. Transformation involves the acquisition of free DNA available in the medium. For this process the recipient bacteria must be in a competitive state; and the translocated DNA must be stabilized, either by integration into the host receptor genome or by recircularization (in the case of plasmid DNA) [27]. In contrast, conjugation involves the transfer of DNA through a multistep process that requires cell-tocell contact, via cell surface pili or adhesins [28]. The conjugative machinery is encoded by genes in plasmids or by integrative conjugative elements in the

*DOI: http://dx.doi.org/10.5772/intechopen.88789*

#### *Multidrug-Resistant Bacterial Foodborne Pathogens: Impact on Human Health and Economy DOI: http://dx.doi.org/10.5772/intechopen.88789*

noted that EreB enzyme confers resistance to almost all members of the macrolide class, with the exception of telithromycin, a semisynthetic erythromycin derivative, which belongs to a new class of antibiotics called ketolides [22]. In contrast, the EreA enzyme does not hydrolyze azithromycin and also telithromycin [23].

The modification of antibiotics includes the modification of some element of their structure, which is essential in the union with the bacteria target diminishing its affinity. This mechanism involves the addition or transfer of groups such as N-acetyl, phosphoryl, O-nucleoside, O-ribosyl, and O-glycoside. Unlike hydrolysis, this modification does not destroy the essential structures of the antibiotic but obstructs the interaction of the antimicrobial with its target. An example of this mechanism occurs for polycationic antibiotics such as aminoglycosides, which act between the ionic bonds of the amino and hydroxyl groups of the antibiotics and the 16S rRNA region of the A site of the bacterial ribosome, deteriorating the translation mechanism. The enzymes responsible for the modification of the aminoglycosides are the aminoglycoside phosphotransferases (APH) and nucleotidyltransferases (ANT) that modify the hydroxyl groups and the aminoglycoside acetyltransferases (AAC) which modify the amino groups, changing the size, structure, and electronic properties of the antibiotic [24].

#### **2.2 Genetic mobile elements transfer or acquired resistance mechanisms**

Once the bacterial cell acquires some degree of antibiotic resistance by an intrinsic mechanism that implies DNA modification, it can transfer the gene or genes encoding for the resistance marker to the offspring (vertical transfer) or to a different specie or genus (horizontal transfer) [25]. The gene resistance can be acquired by genetic mobile elements such as plasmids, transposons, or integrons [19].

The vertical transfer or vertical evolution occurs when a spontaneous mutation in the bacterial chromosome confers resistance to some members of the bacterial population. Once the resistance genes have arisen, they are transferred to the progeny of the bacteria during DNA replication [6]. When the bacterial genes that confer resistance to antibiotics are mobile, because they are contained within plasmids or are flanked by sequences recognized by some DNA transposition enzymes, they can be transferred between bacteria of a different taxonomic and ecological group. Some genetic mobile elements are plasmids, transposons, integrases, and genetic cassettes; in general this mechanism is called horizontal transfer gene [19, 26].

#### *2.2.1 Plasmids*

*Pathogenic Bacteria*

porins promotes resistance to antibiotics [11].

necessary to exert their inhibitory activity [15].

normal activity of the topoisomerases [18].

*2.1.4 Enzymatic inactivation or modification of antibiotics*

*2.1.3 Modification of the target site*

*2.1.2 Expulsion of the antibiotic by active mechanisms*

On the other hand, embedded in the outer layer membrane of Gram-negative bacteria, there are proteins called porins that function as a channel through which the molecules can diffuse. Porins could restrict the influx of numerous antibiotics and contribute to the resistance against them [13]. The mechanism of resistance promoted by the porins consists in changing the hydrophilic composition of some antibiotics such as beta-lactam, chloramphenicol, fluoroquinolones, and tetracyclines. Likewise, the alteration of the amount or modification of the structure of the

In general those mechanisms are mediated by bacterial flow pumps that actively transport many toxic molecules out of the cell [14]. The outflow pumps can interact with only one molecule (enzyme substrate specific), or they can have a broader spectrum and export distinct classes of molecules. Antibiotic resistance mediated by active outflow pumps may be incidental, since the pumps exhibit a broad-range substrate [9]. However, efflux pumps associated with antibiotic resistance have been described in Gram-positive and Gram-negative bacterial pathogens. The energy of some flow pumps depends of the antibiotic agents in order to extract it from the periplasm to the outside. The overexpression of one or more of these flow pumps prevents the intracellular accumulation of antibiotics at the thresholds

Most of the antibiotics bind specifically to their targets with high affinity. Changes in target structure prevent an effective binding to antibiotics but still allow the target to carry out its normal function. The target modification could be originated by a mutation in the gene that encodes for the antibiotic target [14]. An example of this mutation mechanism is the linezolid antibiotic, a member of the oxazolidinone class, which inhibits the initiation of bacterial translation by altering multiple copies of the V domain of the 23S rRNA in Gram-positive bacteria. The mutation in one of these copies of the V domain can confer antibiotic resistance [16]. Another mechanism relies in avoiding or releasing the binding of the antibiotic to their target site [17]. One of the most representative examples of this mechanism and of current importance is that used by quinolones, which exert their function by inhibiting important enzymes of bacterial DNA replication such as gyrase and topoisomerases II and IV. The mechanism of evasion of the antibiotic function is by the expression of repeating pentapeptides (PRP), encoded by *qrn* genes, which bind and promote the release of the quinolone from the target enzymes, allowing the

In this case, the mechanism of action could be by enzymatic hydrolysis [19] or modification of chemical groups by transfer or addition of different chemical compounds [14]. The classic example of a hydrolytic enzyme is the beta-lactamase, which hydrolyzes the beta-lactam ring, a common structural element in penicillins, cephalosporins, carbapenems, and monobactams [20]. Four classes of beta-lactamases have been described: the classes A, C, and D have a serine hydrolase activity; in contrast, class B has a metalloenzyme activity [21]. Another example of this type of resistance is provided by the enzymes erythromycin esterases EreA and EreB. These enzymes hydrolyze the macrolactone rings of macrolides such as erythromycin. It should be

**172**

In bacterial cells, there are circular portions of extrachromosomal DNA that improve the survival characteristics of bacteria. This genetic information could be dispensable when it is no longer necessary to contend with the specific stress to which it imparts protection. Plasmids are self-replicating, given that they do so independently of chromosomal DNA replication. When plasmids contain antibiotic resistance genes, they are called plasmids R, and they can be transferred between bacteria of the same or different genera. Plasmids can be transferred to another bacterial cell by mechanisms called transformation or conjugation [19]. Transformation involves the acquisition of free DNA available in the medium. For this process the recipient bacteria must be in a competitive state; and the translocated DNA must be stabilized, either by integration into the host receptor genome or by recircularization (in the case of plasmid DNA) [27]. In contrast, conjugation involves the transfer of DNA through a multistep process that requires cell-tocell contact, via cell surface pili or adhesins [28]. The conjugative machinery is encoded by genes in plasmids or by integrative conjugative elements in the chromosome [29].
