Molecular Detection of Virulence Factors in Salmonella serovars Isolated from Poultry and Human Samples

Salmonellosis is a common infectious disease in humans caused by Salmonella spp., which in recent years has shown an increase in its incidence, with products of avian origin being a common source of transmission. To present a successful infective cycle, there are molecular mechanisms such as virulence factors that provide characteristics that facilitate survival, colonization, and damage to the host. According to this, the study aims to characterize the virulence factors of Salmonella spp. strains isolated from broilers (n = 39) and humans (n = 10). The presence of 24 virulence genes was evaluated using end-point PCR. All the strains of Salmonella spp. isolated from broiler chickens revealed presence of 7/24 (29, 16%) virulence genes (lpfA, csgA, sitC, sipB, sopB, sopE, and sivH). Regarding the strains isolated from cases of gastroenteritis in humans, all strains contained (14/24, 58, 33%) virulence genes (lpfA, csgA, pagC, msgA, spiA, sitC, iroN, sipB, orgA, hilA, sopB, sifA, avrA, and sivH). In summary, the presence of virulence genes in different strains of Salmonella isolated from broilers and humans could be described as bacteria with potential pathogenicity due to the type and number of virulence genes detected. These findings are beneficial for the pathogenic monitoring of Salmonella in Colombia.


Introduction
Salmonellosis is a foodborne disease with the greatest impact worldwide on both humans and animals [1,2]. Tis disease is caused by the Salmonella in which more than 2,700 serotypes have been reported so far [3]. In humans, the consumption of chicken meat and eggs that were contaminated is conduced to develop the disease because they are considered the main reservoir and vehicle of Salmonella [1,4]. Moreover, food contamination could occur in various stages of the food chain such as production, distribution, and sale [5]. Te serotype, infective dose, virulence factors, and host immunity will infuence the disease's clinical presentation [6].
Salmonellosis in humans is characterized by symptoms such as acute fever, abdominal pain, diarrhea, nausea, and vomiting; however, immunocompromised people and children under 5 years of age and older adults can present severe symptoms [7,8]. In 2018, the EU member states reported 5146 foodborne outbreaks where 33% correspond to illnesses caused by Salmonella [7].
Host-pathogen interactions in bacteria can modulate the expression of some genes to adapt to the environment, infuencing their ability to cause illness [9]. Terefore, the virulence genes facilitate the survival, colonization, and damage of the host [10]. Expression of virulence genes will initiate when Salmonella spp. faces the hostile environment of the hosts' gastrointestinal tract compound of a wide variety of conditions such as osmolarity, oxygen tension, and pH which favor interaction with the target cell during pathogenesis [2]. Te molecular mechanisms of pathogenicity used by Salmonella involve genes, grouped in regions called pathogenicity islands that provide new characteristics that allow it to undergo a successful infective cycle [11]. Tese genetic segments linked to virulence functions are known as Salmonella pathogenicity-island (SPI) and Salmonella has 24 identifed [12]. In addition, SPI could be transmitted between bacteria by horizontal gene transfer and is related to virulence mechanisms such as host colonization, capsules, toxins, invasiveness, bioflm, fmbriae, fagella, serotype conversion, and secretion systems [12][13][14].
Overall, bacterial virulence factors are critical elements for systemic infections [11]. As a result, the pathogenicity of Salmonella has been associated with the number and type of virulence genes present in the chromosomal SPIs [15]. For example, genes such as SopB/SigD and SopE2 allow a rapid internalization of the bacteria playing an important role in Salmonella virulence [16]. Moreover, genes involved in the intracellular survival of Salmonella play a signifcant role in systemic disease in humans [17]. Meanwhile, adherence factors like fmbrial operons mediate the attachment of Salmonella serovars to epithelial cell lines [18]. Besides, Salmonella virulence plasmid plays a crucial role in enhancing the ability of particular serovars to multiply in tissues outside the intestinal tract [19]. Other genes, such as cdtB, code for the CdtB subunit considered as a toxin with a possibly important role in the unusually lengthy, persistent, and development of systemic diseases [20,21].
Despite being a public health concern, there are insufcient studies on virulence factors in Salmonella spp. isolates from broilers and humans in Colombia; also, without specifc information, it is difcult to predict the success of Salmonella control schemes. Tus, it is important to know the genomic particularities in each of the serotypes belonging to this genus; this allows to clarify the bacterial dynamics in the diferent animal hosts and prevent outbreaks in humans and animals [11]. Tus, the aim of this study was to evaluate the potential virulence of Salmonella isolates from poultry and human by detecting the presence of 24 genes involved in virulence and pathogenicity using the polymerase chain reaction (PCR). Accordingly, the results of this study could lay the foundation for further research on public health security and food safety problems caused by Salmonella infections in Colombia.

Salmonella Strains.
In this study, 49 Salmonella enterica strains from the Bacterial Strain Collection of the Laboratory of Immunology and Molecular Biology were included, and Salmonella enteritidis (ATCC ® 13076 ™ ) were used as a positive control. Te strains were previously serotyped using the Kaufmann−White scheme and correspond to the serotypes, namely, S. enteritidis (n � 4), S. typhimurium (n � 2), S. braenderup (n � 1), S. newport (n � 1), S. grupensis (n � 1), and S. uganda (n � 1) isolated from cases of gastroenteritis in humans [22] and S. paratyphi B (n � 24) and S. heidelberg (n � 15) isolated from poultry farms located in the region of Tolima [23] and Santander [24].

DNA Extraction.
Fresh bacterial colonies were used for Genomic DNA (gDNA) extraction using the Invisorb Spin Universal Kit (Stratec Molecular, Berlin, Germany) following the protocol suggested by the fabricant and were stored at −20°C until further use. Molecular confrmation of Salmonella isolates was done by amplifcation of a fragment of invA gene (accession number M90846.1) by endpoint PCR.

Virulence
Genes. Te molecular characterization of 24 genes involved in virulence and pathogenicity was conducted using the gDNA of Salmonella spp. (Table 1). A single PCR assay was used to detect each one of the 24 virulence genes. Primers and annealing temperature used for PCR are listed in Table 1. Te reactions were carried out following the manufacturer's recommendation for the GoTaq ® Flexi DNA Taq polymerase (Promega, Madison, WI, United States), 1 μL of DNA, and 1 μL of each primer (10 pmol/μL). Te ProFlex ™ 3 × 32-well PCR System (Applied Biosystems, Carlsbad, CA, United States) was used to perform the amplifcation using an initial denaturation for 3 minutes at 95°C, 35 cycles of denaturation for 30 seconds at 95°C, 30 seconds of annealing (Table 1), extension at 72°C, and fnal extension for 5 minutes at 72°C. Te PCR products were detected by electrophoresis in agarose gel using HydraGreen (ACTGene, Piscataway, NJ, United States) as an intercalant agent, and the visualization of the gel was conducted in the gel documentation equipment ENDURO GDS (Labnet International, Edison, NJ, United States).

Confrmation of Salmonella.
All Salmonella strains amplifed the expected DNA fragment of the invA gene that was used to confrm the Salmonella genus ( Figure 1).
Te pattern III was the most predominant that was detected in 10 isolates (Heidelberg 3, Paratyphi B 4, Enteritidis 2). In poultry farm isolates, pattern II with 21 virulence genes was detected in 8 isolates (Heidelberg 4, and Paratyphi B 4). Te pattern I was detected in 6 isolates (Heidelberg 3, Paratyphi B 3). Patterns X, XI, XII, XIII, XIV, XV, XVII, XVIII, and XIX were detected only in serotypes of Paratyphi B. Also, patterns II, IV, VII, and IX were only observed in Heidelberg isolates. Patterns I, III, V, and VIII were observed in poultry isolates.

Discussion
Salmonella species are ubiquitous pathogens that are considered the major agents of foodborne disease worldwide [27,28]. In Salmonella spp., physiological and environmental stimuli drive the expression of virulence genes, which are responsible for the main pathogenic mechanisms in this microorganism [10,29]. Virulence factors can maximize the ftness of pathogens via host exploitation [30]. Virulence factors are encoded by a number of genes and may be located on Salmonella pathogenicity islands (SPI), virulence plasmids (pSLT), bacteriophages, or at another location on the chromosome [25,27,31].
A few virulence factors are related with the cellular structure of the bacteria, such as fmbriae [32]. Fimbrial virulence genes represent a major player in pathogenesis by allowing bacteria to interact with host cells [33,34]. In the present study, all the isolates carried the lpfA and csgA genes. Similarly, previous studies have found high detection of the csgA gene among Salmonella serotypes [25,35]. Te csgA gene is related to bioflm production and the maintenance of the bacteria in the environment, including inert surfaces [36]. Likewise, the presence of the csgA gene is relevant to public health because the csg genes in Salmonella are related to the ability to produce bioflms, leading to increased drug resistance [37]. In this way, the presence of the csgA gene in all the strains could suggest that the Salmonella strains could be kept on inert surfaces such as those used in food production, which is relevant to public health. In poultry isolates, the detection rate of fmbria-associated genes such as sefA and pefA were 51.3% and 74.4%, respectively, lower than the other virulence genes evaluated. Additionally, the detection rate of pefA was 60% in isolates from cases of gastroenteritis in humans. Te presence of the sefA gene in Salmonella isolates is relevant because this gene is a promotor of the sef operon, and this operon is a mechanism by which Salmonella serotypes can adapt to an increasing number of hosts [25,38]. Previous studies of virulence gene detection in Salmonella Heidelberg isolated from chicken carcasses did not report isolates with the sefA gene [25]. For this reason, it is possible that the presence of the sefA gene in Salmonella Heidelberg isolates could indicate a major virulence of the strains. Furthermore, sefA gene has been associated with the serotypes Enteritidis, Moscow, but the  Veterinary Medicine International horizontal transfer methods allowed other serotypes to obtain diferent genes than that in the case of this study serotypes such as Paratyphi B and Braenderup, and Typhimurium carried the gene [39]. Te absence of the pefA gene in some Salmonella serotypes is related to the location of the gene, which is plasmidial [40].
Te type III secretion system (TTSS) encoded by Salmonella mediates, in a contact-dependent manner, the translocation of efector proteins from the bacterial cytoplasm into the host cell [41]. Some genes of TTSS are related to structure, efector protein, or regulatory protein of these systems [42]. Te sipB, invA, orgA, prgH, and spaN genes are Table 2: Patterns of virulence genes of Salmonella isolates obtained from poultry farms and cases of gastroenteritis in humans. * For PCR-based patterns, black area represents a positive result and white area represents negative result for the presence of a virulence gene. Virulencerelated function is a Fimbriae gene function; b is type three secretion system gene function, b1 structure, b2 is efector protein, b3 is regulatory protein; c is survival inside cells gene function; d is plasmid gene function, e is iron metabolism gene function, and f is toxins gene function.
Veterinary Medicine International associated to the structure of TTSS, which allows Salmonella to invade phagocytic and nonphagocytic cells [40,43]. Te sipB and invA genes were found in 100% of the isolates that were assessed (n � 49/49). Te sipB gene may play a vital role in Salmonella pathogenesis [44]. In the case where detection rates of the invA gene were expected, this gene is recognized as a rapid detection agent for the genus Salmonella, and this gene also indicates that all the strains are able to produce gastroenteritis and invade the cells [45,46]. A high prevalence of orgA, prgH, and spaN genes in poultry and human isolates were observed in this study (71-100%). In the same way, previous research has detected sipB, orgA, prgH, and spaN genes in Salmonella isolates from poultry-related sources [47].
Furthermore, TTSS is employed by Salmonella to inject diferent "efector proteins" into host cells [48]. Each efector protein activates or blocks a specifc host cell signaling pathway to establish symbioses or infectious diseases [49]. Some genes that encode the efector proteins are avrA, sopE, sopB, and sivH [50]. All the isolates carried the sivH, sopB, and sopE genes whilst the human isolates analyzed were 70%. Te SopB gene can regulate changes in phosphatidylinositol signaling that could generate chloride secretion by epithelial cells [51]. Tus, the signifcance of the presence of the sopB gene is because of the fact that strains with this gene can cause diarrhea, and this disease leads to the elimination of large numbers of bacteria in the host's environment [52]. Consequently, the possession of this gene could increase the spread of Salmonella. High frequency of sivH and sopE may be explained by the fact that these genes are associated with an island which is unique to Salmonella infecting warmblooded vertebrates [53,54]. Te detection rate of an efector protein gene like avrA gene was 97.4% in poultry and 100% in human isolates. AvrA protein plays a critical role in inhibiting infammation, regulating epithelial apoptosis, and enhancing proliferation during bacterial infections [55][56][57][58]. On the other hand, gene with regulatory protein functioning as hilA was found in 94.9% in poultry isolates. All the isolates from cases of gastroenteritis in humans carried the hilA gene.
Some virulence genes may contribute to survival within the macrophage or intracellular survival, for example, pagC, spiA, msgA, and tolC genes [59]. Te pagC gene is ubiquitously distributed among Salmonella serotypes [60]. As a result, prevalence found in pagC gene was 97.4% in poultry and 100% in human isolates. Te detection rate of the spiA, msgA, and tolC genes was higher than 92.3% in all the poultry isolates that were analyzed. On the other hand, all the isolates from cases of gastroenteritis in humans carried pagC, spiA, and msgA genes. Te high frequency of the spiA gene in poultry and human samples is considered critical due to the function of the gene that is related to the ability of the Salmonella serotypes to produce bioflms [25]. Bioflm is an important public health problem; it enhances resistance to physical forces, the host immune system, and antimicrobials [61,62]. In this way, Salmonella strains with the spiA gene would survive longer in poultry farms and could contaminate meat and eggs, where contaminated food is a vehicle in the transmission of Salmonella to humans. Te detection rate of tolC in human isolates was 60%. Te tolC gene plays a crucial role in the excretion of a wide range of molecules, including antibiotics [63].
Te detection rate of pSLT-mediated virulence genes such as spvB was 71.8% in poultry and 60% in human isolates, and the frequencies may be explained by the spvB gene that is located on virulence plasmids [64]. However, the spvB gene that is present in these isolates is relevant because spv genes are highly associated with strains that cause nontyphoid bacteremia and disseminated infection in humans [17,65]. In addition, genes related with iron metabolism such as iroN gene that are related to iron acquisition were 97.4% in poultry and 100% in human isolates that were analyzed [66]. Also, the sitC gene is another gene that is related to iron metabolism, and this gene encodes an important transporter of iron [67], and all the isolates carried the gene. Previously, the presence of the spaN gene was reported, but the iroN gene was not associated to bacteria isolated from poultry sources [68]. In the case of S. Heidelberg, the ffteen strains carried the two genes. Te signifcance of iroN gene cluster that is present on the Salmonella isolates is because of the fact that the iron gene that is present represents an adaptation to life at infamed mucosal surfaces [69]. On the other hand, Webber et al. reported that 88.9% (iroN; 112/126) and 79.4% (sitC; 100/ 126) of the Salmonella Heidelberg carried the gene [25]. Nevertheless, the presence of the virulence genes does not indicate that the bacteria is pathogenic, it necessarily combined the expression of multiple genes [70]. Finally, we suggest performing other methodologies to confrm the expression of genes or proteins related to virulence factors for a better characterization of each Salmonella strains.

Conclusions
An analysis of the virulence genes of Salmonella enterica was conducted to assess its pathogenic potential. In summary, this study provided a better insight into the epidemiology and pathogenicity of Salmonella serovars circulating in two Colombia regions. Also, the presence of virulence genes in diferent strains of Salmonella isolated from broilers and humans could describe it as bacteria with potential pathogenicity due to the type and number of virulence genes detected. In this way, we recommend active surveillance to have updated information on the pathogenicity of Salmonella enterica strains circulating and preventing outbreaks of Salmonella infection.

Data Availability
Te data were obtained from the study. Also, all the datasets generated or analyzed during this study are included in this manuscript.

Conflicts of Interest
Te authors declare that they have no conficts of interest.