Virulence genes identification in Salmonella enterica isolates from humans, crocodiles, and poultry farms from two regions in Colombia

Background and Aim: Salmonella spp. is frequently found in the digestive tract of birds and reptiles and transmitted to humans through food. Salmonellosis is a public health problem because of pathogenicity variability in strains for virulence factors. This study aimed to identify the virulence genes in Salmonella isolates from humans, crocodiles, broiler cloacas, and broiler carcasses from two departments of Colombia. Materials and Methods: This study was conducted on 31 Salmonella enterica strains from humans with gastroenteritis (seven), crocodiles (seven), broiler cloacas (six), and broiler carcasses (12) from Tolima and Santander departments of Colombia, belonging to 21 serotypes. All samples were tested for Salmonella spp. using culture method on selective and non-selective mediums. Extraction of genomic DNA was performed from fresh colonies, DNA quality was verified by spectrophotometry and confirmed by amplification of InvA gene using conventional polymerase chain reaction (PCR). bapA, fimA, icmF, IroB, marT, mgtC, nlpI, oafA, pagN, siiD, spvC, spvR, spvB, Stn, and vexA genes were amplified by PCR. Results: The most prevalent gene was bapA (100%), followed by marT (96.77%), mgtC (93.55%), and fimA (83.87%). Likewise, IroB (70.97%), Stn (67.74%), spvR (61.29%), pagN (54.84%), icmF (54.8%), and SiiD (45.16%) were positive for more than 50% of the strains. Furthermore, none of the isolates tested positive for the vexA gene. Salmonella isolates presented 26 virulence profiles. Conclusion: This study reported 14 virulence genes in Salmonella spp. isolates from humans with gastroenteritis, crocodiles, and broiler cloacas and carcasses. The distribution of virulence genes differed among sources. This study could help in decision-making by health and sanitary authorities.


Introduction
Salmonella is a genus of Gram-negative bacteria from the Enterobacteriaceae family, classified into two species: Salmonella bongori and Salmonella enterica, commonly found in the digestive tract of mammals, birds, and reptiles.It represents a contagion source for humans through the consumption of foods such as beef, chicken meat, eggs, fish, pork, and vegetables [1][2][3][4].Salmonella enterica has 2700 serotypes and subspecies that cause 99% of infections, of which 20 serotypes are zoonotic, including Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Heidelberg, the most relevant serotypes in public health [4][5][6][7].
Salmonella spp.are the etiological agents of several diseases, such as gastroenteritis, typhoid, paratyphoid, septicemia, and meningitis [2,8,9].This zoonotic pathogen represents a public health problem leading per year to 93.8 million cases and 155,000 deaths worldwide; 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the USA result in an estimated $400 million in direct medical costs, 70%-80% of food poisoning incidents in China, and every 690 out of 100,000 Europe inhabitants have non-typhoidal salmonellosis [2,10,11].Moreover, a negative economic impact of $110 billion/per year has been estimated on the poultry industry [7,12].
Serotype, inoculum amount, host immunological status, and virulence factors that influence strain pathogenicity are the main problems associated with salmonellosis prevention [7].Moreover, genes on chromosomes, Salmonella pathogenicity islands (SPIs), mobile genetic elements (i.e., transposons, plasmids, and bacteriophages), and pili [12]; that code for adaptation to the host cell, resistance to antimicrobials, and the ability to overcome host defense mechanisms encoded virulence factors [7,12].Preliminary studies have established the presence and resistance to antibiotics of different serotypes of S. enterica in the poultry industry in Tolima and Santander departments, Available at www.veterinaryworld.org/Vol.16/October-2023/11.pdf as well as in various isolates from humans and crocodiles (Caiman crocodilus) in the department of Tolima [9,[13][14][15][16].To improve the understanding of the virulence profile and establish strategies that contribute to the control and prevention of salmonellosis, this study aimed to identify virulence genes in S. enterica isolates from patients with gastroenteritis, chicken carcasses and cloacal swabs, and crocodiles from two regions in Colombia.

Ethical approval
Ethical approval was not required for this study.Salmonella spp.strains were obtained from the bacterial strain collection of the Laboratory of Immunology and Molecular Biology (Universidad del Tolima).

Study period and location
Strains were collected and isolated from March 2018 to November of 2022 in the Department of Tolima and Santander.

Genomic DNA (gDNA) extraction
Frozen Salmonella colonies were thawed and seeded in Trypticase Soy Agar and Xylose Lysine Tergitol 4 medium (Oxoid, Germany).Genomic DNA was extracted from fresh colonies using the Wizard® gDNA Purification (Promega, USA) according to the manufacturer's conditions.
Polymerase chain reaction was performed in the ProFlex PCR System (Applied Biosystems, ThermoFisher, USA) following the parameters recommended by the manufacturer.The annealing temperature and extension time were defined based on the primer melting temperatures and the expected amplicon size.Products were detected by 2% agarose gel electrophoresis for 40 min at 100 V using PowerPac™ equipment (Bio-Rad, USA), HydraGreen™ as DNA dye (ACTGene, USA), and the ENDURO GDS gel documentation system (Labnet International, USA).

Results
All 31 S. enterica strains were tested by PCR for virulence genes presence.The amplification of gene operon invasion A (InvA) confirmed the presence of Salmonella in nearly all strains (96.77%), except for the Salmonella Kalina strain isolated from chicken carcasses as shown in  In this study, 12 virulence genes present in eight SPIs, one gene on chromosomal, and three genes of plasmids were evaluated.Regarding virulence genes frequencies, among different serovars denoting variable rates, the most prevalent gene was biofilm-associated The distribution of genes according to strain origin showed that 100% of the crocodiles, gastroenteritis, and cloacas presented marT and mgtC genes (Table-3).Furthermore, 100% of gastroenteritis in humans and cloacas in broiler isolates carried fimA and iroB gene (Table -3).Moreover, icmF, siiD, and spvR were present in 100% crocodile strains; and the nlpI gene present in 100% cloaca strains (Table-3).Neither crocodile nor cloaca chicken isolates had spvC (Table-3).In addition, no chicken cloaca strains presented icmF, spvB, and Stn genes, and oafA was not present in the crocodile strains (Table -3).
In addition, the virulence genes presence was classified into 26 profiles (P) (Table -3).Crocodile isolates had four genetic profiles (PI-PIV), six patrons in cases of human gastroenteritis (PV-PX), four profiles (PXI-PXIV) from broiler cloacas, and 12 profiles in broiler carcasses (PXV-PXXVI) (Table -3).PVII and PXVII were the genetic profiles with more virulence genes present (13 genes), followed by PI, PII, and PXXI with 12 genes, and PXXII had 2 genes being the patron with less virulence genes (Table -3).Most of the profiles were found once, except for PI present in the isolates of S. Braenderup, S. Infantis, and S. Soerenga from crocodiles, the PII found in S. Javiana and S. Saintpaul from crocodiles, and the PXIV detected in the three strains of S. Paratyphi B from broiler cloacas (Table-3).
Salmonella pathogenicity islands-1 and SPI-2 possess many virulence genes associated with extracellular pathogenesis and co-encode Type III secretion system [24].In this regard, the genes located in SPI-1 have been described and characterized in S. Typhimurium strains [26].However, S. Typhimurium isolates from broiler carcasses did not have fimA and iroB genes of SPI-1 (Table -3).Other strains did not present these genes, such as S. Powel, S. Othmarschen, S. Schwarzengrund, and S. Typhimurium (broilers carcasses) (Table -3).fimA gene is necessary for the aggregation of Type I fimbriae; in turn, the symbioses are essential for the colonization and biofilm formation of Salmonella spp.[27,28].Furthermore, the iroB gene encodes the glycosyltransferase that glycosylates enterobactin, preventing the host antimicrobial protein from sequestering the iroBCDEN siderophore [29][30][31].
Salmonella pathogenicity islands-2, SPI-3, and SPI-6-8 contain genes that allow Salmonella isolates to resist acidic environments, replicate intracellularly, and escape the host's immune system [24].According to the roles played by SPI-2 effector genes, the presence of oafA gene in humans with gastroenteritis and broiler cloacas could be due to the use of the acetylation reaction in cell infection, leading to the increased antimicrobial activity of macrophage and cell growth [4,24,32,33].
Regarding SPI-3, marT and mgtC genes were present in most strains (Table -3), which agrees with previous reports by Yue et al. [4].marT gene causes systemic infection because it plays a significant role in metabolism within the phagosome and may act as a general pathogenicity regulator by overexpression genes encoding main proteins in the fimbriae formation (e.g., fimA gene), biofilm regulators (e.g., nlpI gene), large surface proteins, antigenic surface proteins, and flagellar operons [34,35].The marT gene absence coincided with the fimA and nlpI genes lack in S. Othmarschen strain (Table-3).Besides, the mgtC gene is linked with independent flagellar growth and motility at low concentrations of Mg +2 [36].According to this, S. Othmarschen and S. Skansen of broiler carcasses were not lacked Mg +2 (Table -3).Moreover, the mgtC gene encodes the binding protein MgtC that plays a regulatory role in complex mgtCBR and mediates phosphate transport necessary for Salmonella spp.pathogenesis [37,38].
siiD gene of SPI-4 was found in S. Paratyphi B isolates from crocodiles and broiler cloacas but was absent in the strain from broiler carcasses, as described by Yue et al. [4].Likewise, it has a low occurrence in S. Enteritidis and S. Typhimurium strains [4], according to gene expression in one of the two S. Typhimurium strains and the absence in S. Enteritidis (Table -3).The fact that the strains have this gene denotes the union of the inner and outer membranes with the putative membrane fusion protein, a component of the Type I secretion system [39].
Salmonella pathogenicity islands-6 encodes the Type 6 secretion system that leads to survival within macrophages and successful establishment in the host intestine [40,41].pagN gene confers competitive advantages to the strains because it promotes hemagglutination, contributing to the adhesion of the pathogen to mammalian cells [42].Furthermore, the pagN gene is related to acidified environments, low Mg +2 concentrations, or the presence of antimicrobial peptides [43].In this way, it is possible to suggest that the S. Othmarschen and S. Skansen strains were in environments with a low concentration of Mg +2 because these isolates did not have the mgtC gene either (Table -3).
vexA gene is involved in the biosynthesis and export of capsule VI to the cell surface [44].This gene was not found in the serotypes from crocodiles, human cases of gastroenteritis, and poultry farms (Table -3), and other studies reported its absence in S. Typhimurium and Salmonella Dublin [24,45].On the other hand, nlpI gene is linked to biofilm formation and acclimation of S. Typhimurium [46,47], even though strain from human gastroenteritis lacked this gene.
bapA gene was present in all serotypes (Table -3), since codes for a large-secreted protein required for biofilm formation and host colonization [48].In the case of the icmF gene, it encodes for an inner membrane protein of Type 6 system secretion that contributes to the virulence of Salmonella spp.[24,49].
On the other hand, the stn gene chromosomal operon induces a loss of intestinal fluids, causing diarrhea and leading to severe acute gastroenteritis [50].According to this, the stn gene was present in 4/6 serotypes from human gastroenteritis cases (Table -3).Furthermore, the Stn gene may affect membrane integrity of Salmonella spp.through ompA localization regulation [51].
Salmonella virulence plasmid operon (spvRABCD) expression is induced by the host cells' intracellular environment, and operon genes are involved in survival and intracellular growth, and macrophage killing [24,52].The isolates that presented three plasmid genes (spvR, spvB, and spvC) include S. Budapest S. Enteritidis, S. Newport (gastroenteritis in humans), and S. Uganda.spvR and spvBC genes are required for the virulence phenotype of the spv operon [52].spvB gene was found in S. Braenderup, S. Enteritidis, S. Infantis, S. Javiana, S. Paratyphi B, S. Saintpaul, S. Soerenga, and S. Uganda, as well as in the S. Newport strains of cases of gastroenteritis and carcasses in broiler chickens.Nevertheless, Yue et al. [4] reported spvB gene in S. Typhimurium.
The presence of the spvC gene may be related to evading MAPK signaling, suppressing the inflammatory response, and spreading the bacteria in the late stages in specific serotypes [24,53,54].This agrees with its presence in two S. Typhimurium isolates from different sources (human gastroenteritis and broiler carcasses).Similarly, the prevalence of the spvC gene is higher in S. Typhimurium and S. Enteritidis [55], which is consistent with its finding in both serotypes.

Conclusion
This study reported 14 virulence genes in Salmonella spp.isolates from humans with gastroenteritis, crocodiles, and broiler cloacas, and broiler carcasses.The distribution of virulence genes differed among sources.Our results contribute to the characterization and monitoring of S. enterica isolates and their evolutionary process in the host from two departments of Colombia, and it could help in decision-making by health and sanitary authorities.
Copyright: Petano-Duque, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
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Table - 3
).Furthermore, none of the isolates tested positive for the vexA gene.Overall, 19 strains had more than nine virulence genes isolates from all gastroenteritis cases in humans, including Paratyphi B and Newport Salmonella strains (Table-3).