Virulence and Antimicrobial-Resistant Gene Profiles of Salmonella spp. Isolates from Chicken Carcasses Markets in Ibague City, Colombia

Salmonella spp. is one of the leading causes of foodborne bacterial infections, with major impacts on public health and healthcare system. Salmonella is commonly transmitted via the fecal-to-oral route, and food contaminated with the bacteria (e.g., poultry products) is considered a common source of infection, being a potential risk for public health. The study aims to characterize the antimicrobial resistance- and virulence-associated genes in Salmonella isolates recovered from chicken marketed carcasses (n = 20). The presence of 14 antimicrobial and 23 virulence genes was evaluated using end-point PCR. The antimicrobial genes were detected in the following proportion among the isolates: blaTEM 100%, dfrA1 and blaCMY2 90% (n = 18), aadA1 75% (n = 15), sul1 and sul2 50% (n = 10), floR 45% (n = 9), qnrD 20% (n = 4), and aadA2 15% (n = 3). catA, sul3, qnrS, and aac(6′)-Ib genes were absent in all isolates. Regarding virulence-associated genes, all Salmonella strains contain invA, fimA, avrA, msgA, sopB, and sopE. The cdtB gene was present in 95% (n = 19) of isolates, whereas spvC and spvB were present in 55% (n = 11). Other virulence genes such as spiC, lpfC, lpfA, and csgA were present in 90% (n = 18) of strains. The presence of antimicrobial and virulence genes in several Salmonella strains in chicken meat suggests the potential pathogenicity of the strains, which is relevant given the possibility of cross-contamination which represents a significant threat to public health.


Introduction
Foodborne diseases caused by microorganisms are a significant global public health concern [1].According to the Centers for Disease Control and Prevention (CDC), foodborne pathogens with the highest annual burden of disease and overall impact on public health are Campylobacter, Listeria monocytogenes, Salmonella, STEC, Shigella, Vibrio, and Yersinia [2,3].Salmonella spp.infections are among the leading causes of foodborne bacterial infections, with major impacts on human health and the economy [4][5][6].Salmonella remains one of the most burdensome foodborne pathogens globally, with an estimated 200 million to over 1 billion infections, 93 million gastroenteritis cases, and 155,000 fatalities [7].
Currently, more than 2600 diferent serotypes of Salmonella have been identifed, but only a small number of these are commonly associated with foodborne illnesses in humans [8].In clinical human medicine, Salmonella serotypes can be grouped into typhoidal serotypes (Salmonella Typhi and Salmonella Paratyphi A, B, C) and non-typhoidal Salmonella serotypes (NTS) (caused by other Salmonella strains) [9,10].Te typhoidal Salmonellas are invasive and cause systematic infection [11,12].In contrast, NTS serotype infections cause diarrhea, nausea, vomiting, abdominal pain, myalgia, and arthralgia and are usually self-limiting, although immunocompromised patients can develop a sepsis [13,14].
Te route of Salmonella spp.transmission is commonly through a fecal-to-oral route with the consumption of contaminated food or water [15,16].Many foods containing Salmonella are derived from animals (such as eggs, poultry, seafood, and meat), and the transfer of bacteria to these products occurs as a result of cross-contamination during food processing [17,18].Epidemiological studies have identifed poultry meat as the main vehicle for Salmonella infection [17,19].Poultry meat serves as a good environment for the growth of pathogenic Salmonella spp.due to its high nutrient content, pH of 5.5 to 6.5, and water [7,20].However, the prevalence of Salmonella in poultry products depends on the type of poultry store (supermarkets and fresh food markets) [21].
Currently, several studies have identifed that Salmonella isolates from chicken farms carry mobile genetic elements (MGEs) that contribute to increased pathogenicity and antimicrobial resistance [22][23][24][25].MGEs include plasmids, integrons, and transposons, which play a crucial role in the dissemination of antibiotic resistance genes (ARGs) and the development of bioflm-mediated antibiotic resistance [26].Salmonella pathogenicity is mediated by virulence genes, which facilitate the survival, colonization, and damage of the host [27].Consequently, identifcation and assessment of pathogenic potential and antimicrobial resistance of Salmonella serovars in chicken and poultry products is crucial for disease assessment and epidemiological surveillance [21].Tus, this study aimed to characterize the antimicrobial resistance-and virulence-associated genes in Salmonella isolates recovered from chicken carcasses marketed in Ibague, Colombia.
In Colombia, a study on 1.003 retail broiler chicken carcasses reported that 27% were contaminated with Salmonella, indicating a risk of infection from poultry products in the country [28].However, the prevalence of Salmonella spp. in chicken carcasses varied among Colombian cities, ranging from 0% to 57% [28].For example, in Ibague (central region of Colombia), Salmonella Enteritidis was identifed as a signifcant source of salmonellosis, particularly linked to contaminated eggs and raw chicken meat, emphasizing the genetic relationship between isolates from poultry and human gastroenteritis cases [29].Additionally, studies on raw chicken meat sold in Ibague revealed a high prevalence of Salmonella, with specifc serotypes such as S. Paratyphi B, S. Hvittingfoss, and S. Muenster being predominant and posing a potential threat to human health due to multidrug resistance patterns [30].Tus, this study aimed to characterize the antimicrobial resistance-and virulenceassociated genes in Salmonella isolates recovered from chicken carcasses marketed in Ibague, Colombia.

Materials and Methods
2.1.Ethical Approval.Ethical approval was not required for this study.Salmonella spp.strains were obtained from the Bacterial Strain Collection at the Laboratory of Immunology and Molecular Biology (Universidad del Tolima).

Genomic DNA Extraction and Molecular Confrmation of
Salmonella.Genomic DNA (gDNA) was extracted from fresh bacterial colonies using Wizard ® Genomic DNA Purifcation Kit (Promega, USA) according to the manufacturer's conditions.DNA samples were stored at −20 °C prior to use.Molecular confrmation of Salmonella isolates was performed by amplifcation of a 284 bp fragment of the invA gene (accession number: M90846.1)by endpoint polymerase chain reaction (PCR), using Salmonella Enteritidis (ATCC 13076 ® ) as positive control and E. coli (ATCC 25922 ® ) as negative control [31].

Molecular Detection of Antibiotic Resistance Genes and
Virulence-Associated Genes.Fourteen antibiotic resistance genes (ARGs) were examined in all isolates by endpoint PCR using specifc primers described in Table 1.Te ProFlex ™ PCR System (Applied Biosystems, Waltham, MA, USA) was utilized for conducting the reactions, using a fnal volume of 25 μL made up of 14,875 µL deionized distilled water, 5 μL of Flexi Bufer 5× Colorless GoTaq ® (Promega, Madison, WI, USA), 1 µL of dNTPs (Invitrogen, Carlsbad, CA, USA), 1 µL of each primer (forward and reverse) (10 pmol/Μl) (Macrogen, Seoul, Korea), 1 μL of MgCl 2 (25 mM) (Promega, Madison, WI, USA), 0.125 μL of GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA), and as template 1 µL of gDNA.PCR amplifcation was performed with the following conditions: initial denaturation at 95 °C for 3 minutes, 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 30 seconds, and fnal extension at 72 °C for 8 minutes.For all experiments, Salmonella spp.strains previously characterized in the Laboratory of Immunology and Molecular Biology were used as reference strains.Te annealing temperature and extension time were defned based on the primer melting temperatures and the expected amplicon size (Table 1).

Molecular Detection of Virulence-Associated Genes and
Integrons.Te determination of twenty-three Salmonella virulence genes was performed by PCR using the conditions described previously.Primers sequences, annealing temperature, amplicon size, and corresponding references are listed in Table 2.For integron detection, gDNA from isolates was used as a template for the reaction, using gene-specifc primer sets (Table 2).PCR conditions were as described above, and the annealing temperature is listed in Table 2.

Statistical Analysis.
All data were analyzed using Microsoft Excel.Prevalence as the ratio of positive animals to the total number of samples is expressed as a percentage [39].

Distribution of Antibiotic Resistance Genes among Salmonella
Serovars.Among the β-lactamase-encoding genes (bla TEM and bla CMY2 ), only bla CMY2 , which confers resistance to ampicillin, was detected in all the strains (100%), and bla CMY2 , which confers resistance to ceftriaxone, was found at a high frequency (90%; n � 18) (Table 3).Among the genes specifc for chloramphenicol, such as catA and foR, only foR, which confers resistance to forfenicol, was detected in 45% (n � 9) of Salmonella strains.Regarding the genes that encode aminoglycoside resistance, aadA1 and aadA2 were found to be in 75% (n � 15) and 15% (n � 3) of strains, respectively (Table 3).Gene cassettes encoding resistance to trimethoprim, such as dfrA1 and dfrA12, were present in 95% (n � 15) and 20% (n � 4) of strains, respectively.Regarding sulfonamide resistance, only sul1 and sul2 genes were detected in 50% (n � 10) of Salmonella strains.Moreover, among the quinolone and fuoroquinolone resistance genes evaluated, only qnrD, a plasmid-mediated quinolone resistance gene, was detected among Salmonella strains (20%; n � 4).

Discussion
Poultry meat is one of the most economical and consumable sources of animal protein [40,41].However, poultry meat production involves several stages before the meat reaches consumers [42].Among the stages of poultry production, food safety is an important consideration because of the bacterial pathogens present in broilers, such as Salmonella [43].Contaminated poultry products are the major source of human Salmonella infection [44].Contamination can occur at various stages during production on the farm (preharvest stage), during transportation to the processing plant, at slaughter and evisceration (processing stage), postevisceration processing, cutting and boning, packaging and storage, distribution and retail, and transportation and handling [45,46].Also, Salmonella strains isolated from poultry products exhibit high levels of resistance to antibiotics, making disease control difcult and posing serious risks to global public health [47].To minimize the impact of Salmonella-related outbreaks, determining the susceptibility of the bacteria to antimicrobial agents is essential [48].
Among the 20 Salmonella strains, antimicrobial resistance-and virulence-associated genes were determined to explore the molecular mechanisms underlying multidrug resistance (MDR) and pathogenicity.Te most common resistance genes found were those encoding for β-lactams (bla TEM and bla CMY−2 ), tetracycline (dfrA1), streptomycin (aadA1), and sulfonamide (sul1 and sul2).Te family of β-lactam antibiotics is the most prescribed family of antibiotics prescribed to treat bacterial infections [49].Nevertheless, their use has been limited by the emergence of bacteria with resistance mechanisms, such as β-lactamases [50].Some mechanisms by which bacteria acquire resistance to β-lactams include the production of β-lactamases, efux pumps, and alterations in penicillin-binding proteins [51].Te β-lactamase-encoding genes bla TEM and bla CMY−2 were predominant among the antimicrobial resistance genes detected.Tese fndings are similar to previous studies that have reported bla TEM and bla CMY−2 as the main genes involved in the mechanisms of resistance to β-lactam antibiotics and cephalosporins, respectively [52,53].Tis difers with the prevalence rates reported in a previous study conducted in China on Salmonella isolated from chickens, where the rates of bla TEM and bla CMY−2 β-lactamase genes were 61.11%, and 63.89%, respectively.
Regarding sulfamethoxazole resistance (sul) genes, high positivity rates for sul1 (50%), and sul2 (50%) were observed, which is in accordance with previous reports that indicate sul1 and sul2 genes are the most frequently detected Sul genes in Salmonella spp.[54,55].Tis is relevant because sulfonamide resistance usually arises from the acquisition of sul1 and sul2 genes, which encode forms of dihydropteroate synthase that are not inhibited by the drug [56].Also, in Brazil, 68% of Salmonella isolated from chicken meat samples contained sul2 [57].Te detection of sul genes in Salmonella strains isolated from carcasses is relevant because of the potential transfer of these genes from commensal bacteria into more virulent bacteria via integrons, transposons, or plasmids [58,59].Concerning chloramphenicol resistance genes, the foR gene was found in 45% of the Salmonella strains.In addition, in previous studies from China, foR was identifed in 35.1% of Salmonella strains isolated from chickens, ducks, and pigs [60].Te frequency of this gene could be related to the long-term use of forfenicol in veterinary medicine, leading to the appearance of these resistance genes [61].For example, foR is particularly prevalent in multidrug-resistant strains of Salmonella isolated from poultry [52].
Dihydrofolate reductase gene (dfrA1), which conferred resistance to trimethoprim (integron-encoded dihydrofolate reductase), was commonly detected between Salmonella strains (95%).Also, high positivity rates for dfrA1 gene were reported on isolates recovered from clinical and environmental samples [62].A comparative study conducted in Iraq reported 77.6% of isolates resistant to dfrA1 gene [63].Furthermore, 15 (75%) Salmonella strains contained the aminoglycoside resistance gene aadA1, which is relevant because of the association between aadA1 and streptomycin resistance in Salmonella strains isolated from chicken meat [64].Likewise, its frequency is also relevant because this gene is encoded by a conjugal plasmid, which can be transferred to E. coli, with or without selective pressure from antibiotics [65].Regarding plasmid-mediated quinolone resistance (PMQR) genes, this study identifed high frequency of qnrD (20%) compared to a previous report from Colombia [33].Genetic elements, such as integrons, are able to recognize and capture cassettes carrying the antibiotic resistance genes, leading to the spread of multidrug resistance (MDR) [66].Class 2 integrons are one of the most common integrons in pathogenic bacteria [67].Also, the presence of integrons in Salmonella has been associated with antimicrobial resistance [60].In this study, class 2 integron was detected in 95% of Salmonella isolates, which was higher than previous studies in South Africa (33%) and Iran (9.2%) [68,69].
Pathogenic Salmonella strains isolated in poultry contain various genes associated with Salmonella pathogenicity islands (SPIs), including pathogenicity islands 1 and 2 (SPI-1 and SPI-2) [44].SPI-1 allows the bacteria to penetrate nonphagocytic host cells, while SPI-2 is important for survival within the macrophage and for establishment of systemic infection [70,71].Te SPI-1 genes, such as fmA, invA, and avrA, were detected in 100% of the tested strains, followed by other genes such as spiC (90%), hilA (85%), sitC (80%), spaN (80%), and sipB (65%), with lower frequencies (Table 4).Te International Journal of Microbiology extensive presence of invA in Salmonella species has led to its use as a molecular marker for confrming Salmonella genus [72,73].Regarding Salmonella fmbriae virulence gene fmA, the prevalence rates were similar to a previous study in United States [74].Te fmA codes for the major fmbrial subunit in Salmonella, which helps to adhere to the cell International Journal of Microbiology surface and promotes colonization [75].Another relevant gene in Salmonella virulence is hilA, which stimulates the expression of invasive genes [76].In addition, similar results have been reported from commercial farms in Egypt, where the hilA gene was detected in 90% of Salmonella strains [77].
Likewise, the prevalence of avrA in strains is a concern because this gene can enhance bacteria proliferation during infections by inhibiting infammation and regulating epithelial apoptosis [78].Among SPI-2, various genes were detected (sifA (70%); spiA (90%)) (Table 4).Te spiA gene is associated with the ability of bioflm formation and virulence in Salmonella species [79].In this way, the presence of SPI-1 and SPI-2 genes among Salmonella strains isolated from poultry meat is signifcant, as it suggests the potential of these isolates to cause infections in humans [80].
Concerning SPI-3, msgA gene was present in all strains (Table 4), which agrees with previous reports on Salmonella isolated from poultry products in Iran [81].By contrast, siiD gene of SPI-4 was found only in 60% of strains, which is relevant because this gene encodes a protein that links the inner and outer membranes [82].Contributing to the virulence of Salmonella through the transport of specifc proteins that enhance the bacteria's ability to invade host cells, evade the host immune system, and establish infections [83].Another gene present among the strains was sopB of SPI-5, which is related to subverting host autophagy and inhibiting the fusion of Salmonella-containing vacuoles (SCVs) with lysosomes and autophagosomes [84].In contrast, a study conducted in South Africa found that 31.8% of the isolates had sopB [85].Terefore, the presence of sopB could facilitate the dissemination of bacteria in the host environment by inducing diarrhea during infection [86].Te detection rate of virulence plasmid-borne genes, such as spvB and spvC, was 55%, which was signifcantly higher than that in a previous report of Salmonella isolated from broiler chicken carcasses in the United States (5.5%) [87].Te spvB gene in Salmonella is associated with virulence and pathogenesis by promoting necroptosis of intestinal epithelial cells, leading to the destruction of the intestinal barrier and aggravation of infection [88].Te cdtB gene is another commonly detected gene.Tis gene encodes the cytolethal distending toxin B, which plays a signifcant role in the pathogenesis of Salmonella by causing cell cycle arrest, cytoplasmic distension, and nuclear enlargement in host cells [89].Fimbriae virulence genes, such as lpfC, lpfA, and pefA, mediate the adhesion of Salmonella serovar to host cells contributing to the pathogenesis [90].A high frequency of fmbriae virulence genes was observed among Salmonella strains, which is relevant because these genes contribute to infammation, intestinal colonization, and long-term carriage of Salmonella in vertebrate animals [90,91].
With respect to csgA gene, this gene encodes the major structural component of curli fmbriae, which stabilizes cellcell interactions during bioflm formation [92].Salmonella bioflm formation can enhance bacterial resistance to adverse conditions, such as environmental stresses, host defense mechanisms, and antibiotics, which is relevant because the detection rate of the csgA gene in Salmonella strains isolated from chicken meat was 90%, suggesting that Salmonella could be maintained on inert surfaces, such as those used in food production, being one of the main vehicles of foodborne salmonellosis outbreaks, which constitutes a public health problem [93].Finally, it is essential to recognize that the presence of virulence genes is not a conclusive indicator of a bacterium's pathogenic potential [94].It is the combined expression of multiple genes that is necessary for pathogenicity [95,96].Finally, we recommend using additional methods to confrm the expression of genes or proteins related to virulence factors.

Conclusions
Salmonella strains obtained from chicken meat containing antimicrobial and virulence genes raise concerns about their potential to cause disease and the health risk for humans.Te possibility of cross-contamination in retail chicken is especially alarming, as it represents a signifcant danger to public health, particularly for individuals undergoing antimicrobial therapy due to Salmonella-contaminated chicken infections.Tus, this research holds signifcance for the monitoring of salmonellosis.

Table 1 :
Primer sequences used for amplifcation of resistance genes.

Table 2 :
Primer sequences used for amplifcation of virulence genes.
* * For PCR-based patterns, black box indicates that the detection of the resistance gene was positive and white box indicates that the detection of the resistance gene was negative.Abbreviations: Ceft, Ceftriaxone; Amp, Ampicillin; Chlor, Chloramphenicol; Flor, Florfenicol.

Table 4 :
Distribution of virulence genes in Salmonella spp.isolates. *