Tracking *Salmonella* Enteritidis in the Genomics Era: Clade Definition Using a SNP-PCR Assay and Implications for Population Structure

*Dele Ogunremi, Ruimin Gao, Rosemarie Slowey, Shu Chen, Olga Andrievskaia, Sadjia Bekal, Lawrence Goodridge and Roger C. Levesque*

### **Abstract**

*Salmonella enterica* serovar Enteritidis (or *Salmonella* Enteritidis, SE) is one of the oldest members of the genus *Salmonella*, based on the date of first description and has only gained prominence as a significant bacterial contaminant of food over the last three or four decades. Currently, SE is the most common *Salmonella* serovar causing foodborne illnesses. Control measures to alleviate human infections require that food isolates be characterized and this was until recently carried out using Pulsed-Field Gel Electrophoresis (PFGE) and phage typing as the main laboratory subtyping tools for use in demonstrating relatedness of isolates recovered from infected humans and the food source. The results provided by these analytical tools were presented with easy-to-understand and comprehensible nomenclature, however, the techniques were inherently poorly discriminatory, which is attributable to the clonality of SE. The tools have now given way to whole genome sequencing which provides a full and comprehensive genetic attributes of an organism and a very attractive and superior tool for defining an isolate and for inferring genetic relatedness among isolates. A comparative phylogenomic analysis of isolates of choice provides both a visual appreciation of relatedness as well as quantifiable estimates of genetic distance. Despite the considerable information provided by whole genome analysis and development of a phylogenetic tree, the approach does not lend itself to generating a useful nomenclature-based description of SE subtypes. To this end, a highly discriminatory, cost-effective, high throughput, validated single nucleotide based genotypic polymerase chain reaction assay (SNP-PCR) was developed focussing on 60 polymorphic loci. The procedure was used to identify 25 circulating clades of SE, the largest number so far described for this organism. The new subtyping test, which exploited whole genome sequencing data, displays the attributes of an ideal subtyping test: high discrimination, low cost, rapid, highly reproducible and epidemiological concordance. The procedure is useful for identifying the subtype designation of an isolate, for defining the population structure of the organism as well as for surveillance and outbreak detection.

**Keywords:** *Salmonella* Enteritidis, clades, WGS, SNP-PCR, PFGE, phage typing, nomenclature, population structure

### **1. Introduction**

The genus *Salmonella* contains a large number of Gram-negative bacteria primarily found in the gastrointenstinal tract of vertebrate organisms including humans, cattle, pigs, horses, companion animals, avian, reptiles and fish [1]. There are two species of *Salmonella*, namely *Salmonella enterica* and *S. bongori* [2]. *Salmonella enterica* is the species of relevance in food safety, and consists of five subspecies of varying importance in human health. *Salmonella enterica* subspecies *enterica* has received the greatest attention because of its large number of constituent organisms, now estimated at about 2,600, each defined as a serovar based on the Kauffman-White classification [1]. *Salmonella enterica* serovar Enteritidis (commonly written as *Salmonella* Enteritidis or SE) is the most prominent. The organism was originally described as a distinct species and named as *Salmonella enterica* alongside two other species namely *Salmonella choleraesuis* and *Salmonella typhi*. Since those early days, the taxonomy of *Salmonella* has changed to reflect two species and hundreds of serovars. Curiously, a limited number of *S. enterica* serovars is associated with foodborne illnesses of which SE has emerged over the last few decades as the most prevalent cause of foodborne salmonellosis in humans worldwide [3]. However, this has not always been the case and prior to the 1970s there was only the occasional report of foodborne salmonellosis attributable to SE.

The earliest reports of foodborne illnesses caused by *Salmonella* were attributed to duck egg sources as summarized by Scott [4]. Subsequently, the organisms was found in live chicks, ducks and ducklings [5, 6]. Although these early reports came from different countries, SE did not become a common cause of foodborne illnesses until the 1980s [7]. By 1994, SE was the most commonly reported *Salmonella* serotype, with an incidence of 110 laboratory-confirmed infections per 100,000 population in the Northeast of US, and shell eggs from hens were identified as the major vehicle for SE infection in humans [8], in contrast to the earlier reports incriminating duck eggs. A 2010 outbreak of egg-related SE infections in the US resulted in an estimated 1,939 illnesses and a recall of over 500 million eggs, which ranked as the largest egg recall in history and one of the most expensive food recalls ever [9]. Similar events occurred in other parts of the world and were severe enough to warrant a warning of a new pandemic [7]. Together with two other serovars namely, Typhimurium and Heidelberg, the three most common serovars alone account for 59% of *Salmonella* outbreaks in humans in Canada, while the 10 most commonly observed *Salmonella* serovars account for about 76% of the total *Salmonella* infections reported. Establishing epidemiological linkages between contaminated products and human disease for *Salmonella* serovars has been particularly difficult for a number of reasons. One of the historically important reasons has been the clonal nature of many of the dominant serovars, especially Enteritidis which makes discrimination of strains difficult and an attribution of a particular strain linked with illness to a food source particularly challenging.

One resource that has been used by researchers to study SE is the strain P125109 phage type 4 (PT4) which was isolated from an outbreak of human food poisoning in the United Kingdom, and traced back to a poultry farm. The strain is highly virulent in newly hatched chickens and is also invasive in laying hens, resulting in egg contamination [10, 11]. The complete genome sequences of the host-promiscuous SE PT4 isolate P125109 was determined by Thomson *et al.* in 2008 [12].

*Tracking* Salmonella *Enteritidis in the Genomics Era: Clade Definition Using a SNP-PCR Assay… DOI: http://dx.doi.org/10.5772/intechopen.98309*

Next generation sequencing (NGS) and especially whole genome sequencing (WGS) has emerged in recent years and has made it possible to sequence bacterial genomes within hours, a remarkable feat that is revolutionizing the field of microbiology. With the advent of microbial WGS, new light is shed on the nature of pathogens and our understanding of the biology of *Salmonella* is steadily increasing as *Salmonella* genomes are generated increasingly at a rapid rate and are deposited in public databases. Further understanding of genome diversity and variation of bacterial pathogens has the potential to improve quantitative risk assessment and assess the evolution of *Salmonella*, relationship among strains and serovars, emergence of new strains and the role of mobile genetic elements especially plasmids and bacteriophages in *Salmonella* [13]. The recent development of the *Salmonella* SystOmics database (SalFoS https://salfos.ibis.ulaval.ca/), a rich collection of over 3000 *Salmonella* genomes and their metadata represents a milestone and an important resource for future approaches to mitigate the burden of foodborne salmonellosis [14].

Food safety which is significantly impacted by *Salmonella* has gained from the advent of microbial genomics. Subspecies characterization including serovar identification and strain differentiation can now be done using genomics approach. As will soon be evident to the reader, there is much work yet to be done as the new capacity is yet to translate to tangible benefits to the consumer. Outbreaks caused by SE have remained at a high level or even increasing and there is a need to evaluate the efficacy of procedures used to detect the organism in food as well as approaches used in tracking the organism through the entire spectrum of the food chain, from farm to fork.

## **2. Laboratory culture and identification of organism**

#### **2.1 Culture procedures for** *Salmonella*

Culture-based methods are commonly employed to detect pathogens in food, and in clinical and environmental samples. The Compendium of Analytical Methods (https://www.canada.ca/en/health-canada/services/food-nutrition/ research-programs-analytical-methods/analytical-methods/compendium-methods. html) and the Bacteriological Analytical Manual (https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam) are compilations of laboratory procedures developed by the food safety regulatory agencies in Canada and the United States, respectively and each contains a catalog of official and recommended methods for isolating and detecting *Salmonella*. Briefly, *Salmonella* detection in food relies on a series of culture steps in broth formulations optimized to resuscitate *Salmonella* following injury caused by food handling, processing and storage and to reduce the abundance of competing bacteria [15]. In many enrichment protocols, broth and culture plates have been described for the isolation of *Salmonella* in different types of samples and matrices [16–18]. Typically, the first step is to culture a suspect food sample in a non-selective pre-enrichment broth, examples of which are lactose broth, buffered peptone water, trypticase soy, brilliant green water, powdered milk with brilliant green and universal pre-enrichment [16]. Following an overnight incubation commonly performed at 37**°**C, the culture material is subsequently transferred into a selective enrichment broth which suppresses and inhibits the growth of non-salmonellae while expanding the *Salmonella* population, facilitating isolation by plating on the appropriate media plates [19, 20]. Tetrathionate (TT) and Rappaport-Vassiliadis (RV) broths and RV semi-solid medium are the most commonly used selective culture conditions, performed at 37° or 42**°**C overnight for several days [15, 19].

When used to detect the presence of a microorganism in a food sample, laboratory culture procedures are slow and time consuming, requiring the sequential use of non-selective and selective enrichment broths and could take a week or longer. Another disadvantage is the documented inherent bias in the performance of selective broths which results in the preferential recovery of certain *Salmonella* serovars and not others [17, 21, 22]. For instance, different *Salmonella* serotypes are recovered by culture procedures performed on non-clinical, non-human sources when compared to samples tested in hospitals and other clinical settings from patients experiencing symptoms. Experimental results show that members of some *Salmonella* serogroups are unable to effectively compete with other serovars leading to a reduced efficiency of recovery of some *Salmonella* organisms including SE, from contaminated food [21]. The use of culture-independent procedures that can lead to rapid and sensitive detection of *Salmonella* [23] may in time eclipse the routine use of culture methods for detection. Nevertheless, the recovery of *Salmonella* in food is currently required to establish risk to the consumer and in support of a regulatory action. For this reason, and for the purpose of building inventories of microbial organisms for clinical and regulatory food microbiology, culture procedures are expected to remain in use. A wide variety of selective plating media are available for the isolation of *Salmonella* and a number of them will now be examined.

#### *2.1.1 Xylose lysine desoxycholate (XLD) agar*

XLD agar is a selective growth medium originally shown to facilitate the isolation of *Shigella* but was demonstrably useful for *Salmonella* isolation and has been further modified since its first description [24, 25]. At pH 7.4, the XLD agar appears bright pink or red as a result of the phenol red indicator. *Salmonella* ferments xylose, a sugar molecule, to produce acid and the bacterial colony turns yellow. In time, xylose is consumed and lysine is in turn utilized which upon decarboxylation produces an acidic environment and colonies turn back to red. In contrast, *Shigella* cannot ferment xylose and the colony remains red. *Salmonella* is able to metabolize thiosulfate to produce hydrogen sulphide, leading to the formation of colonies with black centres, which is an important feature in differentiating *Salmonella* colonies from *Shigella*. XLD agar is capable of supporting other members of *Enterobacteriaceae* such as *Escherichia coli* however the colonies and media turns yellow because of the fermentation of lactose which is also present in the agar. *Pseudomonas aeruginosa* is also able to grow on XLD plates as pink, flat, rough colonies but will not metabolize thiosulfate nor turn black. *Proteus* organisms can grow on XLD to give rose colored colonies and can sometimes metabolize thiosulfate to render the colonies black which will be readily confused with *Salmonella*. In addition, *Salmonella* strains have been described that do not metabolize thiosulfate and will grow as pink colonies which will be readily confused with *Shigella.* Thus, XLD agar is a moderately selective medium for isolating *Salmonella* and for differentiating it from other organisms.

#### *2.1.2 Xylose lysine Tergitol-4 (XLT-4) agar*

Similar to XLD agar, XLT-4 agar is also a selective culture medium which is used to isolate and identify *Salmonella* in food and environmental samples. Compared to XLD agar, XLT-4 is supplemented with a surfactant, 7-ethyl-2-methyl-4-undecanol hydrogen sulfate commonly referred to as Tergitol 4 while lacking sodium chloride and sodium desoxycholate. The surfactant is responsible for the inhibition of *Proteus* spp. and other non-salmonellae. XLT-4 agar is

*Tracking* Salmonella *Enteritidis in the Genomics Era: Clade Definition Using a SNP-PCR Assay… DOI: http://dx.doi.org/10.5772/intechopen.98309*

clearly one of the most stringent of all selective culture plates used for isolating *Salmonella* with positive colonies growing up as red and eventually turning black starting from the centre as a result of hydrogen sulfide production. However, *Salmonella* strains that fail to produce hydrogen sulfide appear as yellow colonies on XLT-4 agar [26, 27].

#### *2.1.3 XA medium - modified XLD agar by adding D-arabinose*

XA medium is an improved selective and differential medium over XLD agar following its supplementation with arabinose, a sugar that is fermented by *Citrobacter* and *Proteus* but not by *Salmonella* [28]. The sensitivity of isolation of *Salmonella* using the XA and XLD media are equally high, however, the specificity of XA medium (92.0%) is superior to that of XLD (73.0%) [28]. Many *Salmonella* organisms appear as black colonies on XA agar whereas non-salmonellae will either not grow or appear as pink colonies. The use of arabinose to differentiate *Salmonella* from other closely related organisms represents a cost-effective approach, especially when compared to chromogenic plates (see Section 2.1.7).

#### *2.1.4 Hektoen enteric (HE) agar*

HE agar is a selective and differential medium for isolating and distinguishing members of the genera of *Salmonella* and *Shigella* from the other *Enterobacteriaceae*. HE agar has a blue appearance and contains indicators of lactose fermentation and hydrogen sulfide production while inhibiting the growth of Gram-positive bacteria. Species belonging to Enterobacteriaceae that are capable of fermenting one or more carbohydrates produces yellow or salmon-orange colored colonies, e.g., *Klebsiella pneumonia* which ferments lactose. Non-fermenters produce bluegreen colonies. Organisms that reduce sulfur to hydrogen sulfide such as *Salmonella* will produce black colonies or blue-green colonies with a black center. In contrast, colonies of *Shigella* remain green and do not turn black because of inability to metabolize sulfur.

#### *2.1.5 MacConkey agar*

MacConkey agar is used for the isolation of Gram-negative enteric bacteria which represents a large group of bacteria prominent among which includes *Salmonella, E. coli, Proteus*, *Citrobacter*, *Klebsiella*, *Pseudomonas*, *Shigella*, *Enterobacter* and *Yersinia*. These organisms grow on the agar because of the selective property conferred by crystal violet and bile salts to inhibit the growth of Grampositive bacteria. The indicator system is the neutral red dye which turns red at a pH below 6.8 but is colorless at higher pH. Thus, lactose fermenters such as *E. coli*, *Klebsiella* and *Enterobacter* which contain the *lac* operon form red or pink colonies on McConkey agar. In contrast, the other organisms including *Salmonella* which are generally non-lactose fermenters do not change color. Because *Salmonella* produce colonies similar to other non-lactose fermenters on MacConkey, the medium does not allow for identification of *Salmonella,* an objective that has to be achieved by employing other more selective agars. At the same time, lactose fermenting *Salmonella* have historically been shown to be causes of severe infections and outbreaks in humans [29] which is attributable to the presence of the *lac* operon carried in the chromosome or on plasmids [30] and leading to colonies that appear pink or reddish on MacConkey agar. Despite its limitations, the MacConkey agar can still be a very useful addition to the collection of media needed to comprehensively isolate and identify *Salmonella* in contaminated samples.

#### *2.1.6 Brilliant green sulfa (BGS) agar*

The selectivity of the BGS agar is due to the presence of brilliant green and sulfadiazine, two components that individually inhibits Gram-positive and most Gram-negative bacilli. Phenol red is the pH indicator that detects changes in pH due to the fermentation of sucrose and/or lactose. *Salmonella* colonies range from reddish or pink to nearly white in color with a red zone. Lactose or sucrose fermenters occasionally grow on this medium and appear as yellow-green colonies surrounded by a yellow-green zone. The presence of sulfadiazine in the media is effective in inhibiting the growth of *E. coli* and *Proteus* and to a large extent *Shigella* species [31]. In a latter modification of the BGS agar, the replacement of lactose with glucose and of sulfadiazine with novobiocin to create the novobiocin-brilliant green agar (NBG), led to a higher recovery of *Salmonella* but the medium could not differentiate it from hydrogen sulfide-positive *Citrobacter* organism [32].

#### *2.1.7* Salmonella *chromogenic agar*

Chromogenic plates have been developed for *Salmonella* as an improved alternative to procedures that rely on the ability of the organism to produce hydrogen sulfide or their inability to ferment lactose, attributes that are not fully diagnostic of *Salmonella*. This often result in *Citrobacter* and *Proteus* species being mistakenly identified as *Salmonella* while some atypical *Salmonella* are missed entirely, using agar plates described above*.* There are a number of commercially available chromogenic culture media which incorporate different chromogenic substrates and result in different colors of *Salmonella* colonies. Using the *Salmonella* chromogenic agar marketed by Oxoid (United Kingdom) as an example, the medium contains the substrate, Magenta-cap (5-bromo-6-chloro-3-indolylcaprylate) which is hydrolyzed by *Salmonella* species to give magenta colonies. The second substrate, X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside), is hydrolyzed by many non-*Salmonella* species including *Citrobacter* and *Proteus* to give blue colonies [33, 34]. The selection for *Salmonella* is further enhanced by the presence of bile salts which inhibit Grampositive bacteria, and of two antibiotics namely, novobiocin and cefsulodin which inhibit *Proteus* and *Pseudomonas*, respectively.

The isolation of *Salmonella* colonies in contaminated food demonstrates the presence of live organisms that can potentially cause harm. As indicated above, the procedure requires a combination of culture conditions, and takes time. Molecular procedures that can rapidly detect *Salmonella* are often used to accelerate the process, to improve on sensitivity of detection and also to confirm colonies as *Salmonella* because of the challenges with the isolation of the bacteria as outlined above. Many molecular techniques are now available for serotype-specific identification of SE.

#### **2.2 Identification of** *Salmonella* **Enteritidis**

Many laboratory diagnostic platforms have been applied to detect and identify *Salmonella* contamination in food and these include the PCR, enzyme-linked immunosorbent assay and the lateral flow assay [35–37]. Examples are available as commercial products. Currently, the most popular platform is the PCR and the most frequently used gene target is the *invA* gene. Nevertheless, many commercial offers do not disclose their target for proprietary reasons. PCR assays have also been developed with other gene targets present either in the chromosome, e.g., *flagellin* [38], *OriC* [39] *hilA* [40], *ttr* [41] or on plasmids, e.g., *SpvR*

*Tracking* Salmonella *Enteritidis in the Genomics Era: Clade Definition Using a SNP-PCR Assay… DOI: http://dx.doi.org/10.5772/intechopen.98309*

operon [42]. Multiplex PCR assays that are able to detect and distinguish among multiple serovars have also been developed by including serovar-specific gene targets such as STM4449 (Typhimurium [43]), STM 4497 (Typhimurium [44], *fliC* (Typhimurium [45]), *sdfI* (Enteritidis [46]) and *sefA* [29]. Recent work by Nadin-Davis and colleagues showed that many of the previously identified serovar specific markers were shared by other serovars especially *sefA* and *fliC* while highlighting the limitation with the use of a plasmid encoded target [47].

A multiplex PCR method which is capable of detecting all *Salmonella* spp., while identifying and distinguishing SE from the other two most prevalent serovars namely Typhimurium [48] and Heidelberg (Ogunremi et al., unpublished) is now available. The PCR was designed to amplify DNA fragments from four *Salmonella* genes, namely, *invA* gene (211-bp fragment), *iroB* gene (309-bp fragment), Typhimurium *STM 4497* (523-bp fragment), and Enteritidis *SE147228* (612-bp fragment) and has lately incorporated a 124-bp Heidelberg-specific fragment.

The identification of members of genus *Salmonella* to the subspecies level i.e., serovar is pivotal in tracking these pathogens along the food chain and the above molecular methods are very promising replacements to replace the traditional biochemical tests because of ease of application and high specificity for identifying SE and the other serotypes.
