Molecular Characterization and the Antimicrobial Resistance Profile of Salmonella spp. Isolated from Ready-to-Eat Foods in Ouagadougou, Burkina Faso

The emergence of antimicrobial-resistantfood-borne bacteria is a great challenge to public health. This study was conducted to characterize and determine the resistance profile of Salmonella strains isolated from foods including sesames, ready-to-eat (RTE) salads, mango juices, and lettuce in Burkina Faso. One hundred and forty-eight biochemically identified Salmonella isolates were characterized by molecular amplification of Salmonella marker invA and spiC, misL, orfL, and pipD virulence genes. After that, all confirmed strains were examined for susceptibility to sixteen antimicrobials, and PCR amplifications were used to identify the following resistance genes: blaTEM, temA, temB, StrA, aadA, sul1, sul2, tet(A), and tet(B). One hundred and eight isolates were genetically confirmed as Salmonella spp. Virulence genes were observed in 57.4%, 55.6%, 49.1%, and 38% isolates for pipD, SpiC, misL, and orfL, respectively. Isolates have shown moderate resistance to gentamycin (26.8%), ampicillin (22.2%), cefoxitin (19.4%), and nalidixic acid (18.5%). All isolates were sensitive to six antibiotics, including cefotaxime, ceftazidime, aztreonam, imipenem, meropenem, and ciprofloxacin. Among the 66 isolates resistant to at least one antibiotic, 11 (16.7%) were multidrug resistant. The Multiple Antimicrobial Resistance (MAR) index of Salmonella serovars ranged from 0.06 to 0.53. PCR detected 7 resistance genes (tet(A), tet(B), blaTEM, temB, sul1, sul2, and aadA) in drug-resistant isolates. These findings raise serious concerns because ready-to-eat food in Burkina Faso could serve as a reservoir for spreading antimicrobial resistance genes worldwide.


Introduction
Food-borne diseases present a signifcant public health concern around the world. According to a recent report, 1 in 10 people become ill from consuming contaminated food, with 420,000 deaths yearly [1]. Among the diverse number of biological contaminants in existence, Salmonella is the most common pathogen responsible for thousands of deaths worldwide [2]. Salmonella is a Gram-negative, nonsporeforming bacillus, facultative anaerobe, and a member of the Enterobacteriaceae family. Based on diferences in their 16S rRNA sequence analysis, the genus Salmonella is divided into two taxonomical species, including Salmonella enterica and Salmonella bongori [3]. Most isolates that cause diseases in humans and animals belong to S. enterica. Tis species is further divided into more than 2,600 serovars capable of causing illnesses in humans and animals [4]. Teir reservoir is the gastrointestinal tract of a wide range of domestic and wild animals, with the ability to grow and multiply under various environmental conditions outside the living host. Salmonellosis occurs after the consumption of food or water that has been contaminated by Salmonella [5].
In humans, S. enterica serovars Typhi and Paratyphi cause severe systemic typhoid fever [6]. In contrast, nontyphoidal Salmonella (NTS), such as Salmonella ser. Typhimurium and Salmonella ser. Enteritidis, are commonly associated with gastroenteritis, the most common clinical presentation of Salmonella infections [6,7]. Te worldwide incidence of NTS is considerable, with an estimated 1.3 billion acute cases of gastroenteritis annually leading to 3 million deaths [8]. Africa has a high mortality rate, accounting for 4,100 deaths per year at a rate of 320 per 100,000 people [9]. Te severity and outcome of the disease are associated with diferences in host susceptibility, virulence factors, and serovar ftness [10]. Te pathogenicity is mediated by virulence factors such as Salmonella pathogenicity islands (SPIs), plasmids, pili, and enterotoxins [10,11]. SPIs, which are acquired through horizontal gene transfer events, carry the majority of the virulence genes of Salmonella [12].
In general, many cases of salmonellosis result in a selflimiting process in humans [6]. However, severe invasive disease or prolonged illness may require antimicrobial therapy, especially in children and immunocompromised [6]. Hence, antimicrobial resistance (AMR) in food-borne pathogens such as Salmonella severely threatens global public health. Currently, the annual death toll from AMR is estimated to be 700,000; by 2050, this situation is expected to rise to 10 million, with an economic loss of $100 trillion [13]. AMR may emerge in bacteria during the evolutionary process as cells accumulate spontaneous genetic mutations (resistance-mediating) in preexisting genes and then transfer them through vertical gene transfer [14]. However, the selective pressure created by antimicrobial agent abuse and overuse in humans and animals has accelerated the phenomenon [15]. Of particular concern is the widespread emergence of multidrug resistance (MDR) in Salmonella to clinically critical antimicrobial agents such as fuoroquinolones and β-lactams that could present a severe problem in human salmonellosis treatment [16,17]. Contamination of food with MDR Salmonella is a signifcant public health problem, as food can be a vehicle for spreading and distributing antimicrobial-resistant strains to humans either by direct contact or indirectly by consuming contaminated foods [18]. Indeed, by consumption of food containing antibiotic-resistant bacteria, exchange between bacteria from food and human intestinal microorganisms could occur, leading to the accumulation of antibioticresistant genes in humans, which may afect the efcacy of antibiotics [19]. In developing countries such as Burkina Faso, the role of food chains in the emergence, selection, dissemination, and ultimately transmission of AMR has received much less attention. As a result, little scientifc data on the antimicrobial resistance profles of Salmonella serotypes in food matrices are available. Te main objective of this study was to primarily characterize and determine the antibiotic resistance and virulence profles of Salmonella isolated from foods received at the Laboratoire National de Santé Publique (LNSP) of Burkina Faso. Te results could lay the foundation for further research on public health security and food safety problems caused by Salmonella.

Bacterial Collection.
Te study was conducted at the Direction du Contrôle des Aliments et de la Nutrition Appliquée of LNSP, the reference center for food quality control and applied nutrition in Burkina Faso. Te studied strains were obtained through routine food surveillance activities from 2018 to 2020. Te studied strains were obtained through routine food surveillance activities from January 2018 to December 2019. Four diferent food categories (sesames, n � 480; mango juices n � 156, lettuces, n � 92, and RTE salads, n � 324) comprising 1052 samples were analyzed during this period (Table 1).
Te routine isolation and identifcation of Salmonella in this laboratory are performed according to the International Organization for Standardization procedure [20]. In brief, food samples (25 g or 25 mL) were preenriched in 225 mL of bufered peptone water (Lioflchem, Italy) and incubated at 37°C for 24 hours. One milliliter aliquots of the preenriched cultures were transferred to 10 mL of selenite cystine (Lioflchem, Italy). After incubation at 42°C for 24 hours, one loopful of each broth was streaked onto selective agar plates; xylose lysine deoxycholate agar (XLD, Lioflchem, Italy). Te XLD plates were incubated at 37°C for about 24 hours. After incubation, the plates were examined for morphologically typical Salmonella colonies. Suspected Salmonella colonies were picked from each plate and subjected to biochemical tests using API20E Biochemical Test Strip (BioMérieux, France). Strains were isolated from sesames, ready-to-eat (RTE) salads, mango juices, and lettuce. All isolates were kept at −80°C in the brain heart infusion or BHI (Lioflchem, Italy) medium with 30% (v/v) glycerol until further testing.

DNA Extraction.
All Salmonella isolates previously preserved were cultured on nutrient agar (micro master, India) at 37°C for 18-24 hours. Te boiling method was utilized for each strain to extract the total DNA as defned earlier [21]. In brief, one loop full of Salmonella from the nutrient agar plates was subcultured overnight in 1000 µL of BHI. Te following day, each culture broth was centrifuged at 8, 000g for 5 minutes at 4°C, and the cell pellets obtained were resuspended in 500 µL of sterile distilled water and boiled at 100°C for 10 minutes in a water bath. Te cell suspension was frozen at −20°C for 10 minutes and centrifuged at 8, 000g for 5 minutes at 4°C. Te supernatant was transferred into a fresh Eppendorf tube and stored at −20°C until use. DNA concentration and quality were assessed using a Jenway 737501 Genova Nano Micro-Spectrophotometer (Jenway, United Kingdom).

Molecular Confrmation of Salmonella.
Biochemically identifed Salmonella isolates were subjected to confrmation by polymerase chain reaction (PCR) using the invA gene, which contains unique sequences specifc to the genus Salmonella and has been proven to be an essential target for their detection [22]. Furthermore, iroB gene amplifcation was used to detect S. enterica isolates. PCR reactions were performed in a reaction mixture comprising 4 µL FIREPol ® Master Mix (Solis BioDyne, Estonia), 0.4 µL of each primer, 4 µL of DNA template, and nuclease-free water to make a volume up to 20 µL. Te primers, the size in base pairs of the respective amplifcation products, and the references used for the detection of targeted genes are presented in Table 1. Aliquots (5 μL) of amplifcation products were analyzed on 1.5% agarose gel by electrophoresis in 0.5X TBE bufer (0.1 M Tris, 0.1 M boric acid, and 0.002 M NaEDTA) and stained with ethidium bromide. A 100-bp DNA ladder (Termo Scientifc, USA) was used as a molecular size standard and visualized by UV light illumination. A negative control without template DNA was included in all experiments.

Detection of Virulence Genes.
All confrmed Salmonella isolates were screened for the presence of virulent genes (spiC, misL, orfL,and pipD). PCR conditions, primers, the size in base pairs of the respective amplifcation products, and the references used for the detection of targeted genes are presented in Table 2. . Tey were selected based on their clinical or epidemiological signifcance to human and animal health. Escherichia coli (ATCC 25922) was used as a reference strain for quality control. After testing, the interpretation of the zones of inhibition was in accordance with the CLSI guidelines. Resistance patterns were determined by taking into account any nonsusceptible (R + I) phenotype. A strain was classifed as MRD if it is resistant to at least one antimicrobial in 3 diferent antimicrobial classes. Te Multiple Antimicrobial Resistance (MAR) index was determined according to the method of Krumperman [27] using the formula: a/b, where "a" is the number of antibiotics to which the particular isolate was resistant, and "b" is the number of antibiotics to which the particular isolate was exposed.

Antimicrobial Resistance Gene Screening.
Genes with reported contributions to antimicrobial resistance were tested in this study. Tus, genes encoding resistance to ampicillin, bla TEM , temA, et, temB), tetracycline (tet(A), et, tet(B)), sulfonamides (sul1 and sul2), and gentamycin (StrA and aadA), selected according to the resistance phenotypes, were screened by PCR. Te primers, the size in base pairs of the respective amplifcation products, and the references used for detection of targeted genes are presented in Table 2.

Statistical Analysis.
Te data were statistically analyzed using IBM SPSS 25.0 software. Chi-square (χ 2 ) was used to determine if there was any signifcant diference between the prevalence of each virulence gene in Salmonella from mango juice, sesame, RTE salad, and lettuce. P values ˂0.05 were considered statistically signifcant.

Results and Discussion
Antimicrobial-resistant Salmonella in RTE foods is a big concern. As a result, regular monitoring of the prevalence and antibiotic resistance characteristics of Salmonella strains isolated from foods can provide epidemiological information about food-borne infection in a specifc area. We characterized and then determined the antibiotic resistance profles and resistance genes of Salmonella spp. strains isolated from RTE foods received at the LNSP of Burkina Faso.

Presumptive Salmonella Isolates and Molecular
Confrmations. From the 1052 samples analyzed during the study period, 148 were found as presumptive Salmonella isolates after cultural, biochemical identifcations (Table 1). Presumptive Salmonella strains were isolated from sesame, RTE salad, lettuce, and mango juice samples. In the present study, we conducted PCR amplifcation using invA gene to confrm presumptive Salmonella strains isolated from sesame, RTE salad, lettuce, and mango juice samples. Out of the 148 biochemically identifed isolates obtained from LNSP,   International Journal of Microbiology only 108 were genetically confrmed as Salmonella through PCR amplifcation of the invA marker. Tis marker was amplifed in 67, 21, 11, and 9 isolates from sesames, lettuce, mango juices, and RTE salads, respectively ( Figure 1). Rahn et al. [22] demonstrated that the invA gene contains Salmonella-specifc sequences and could be used as a PCR target with potential diagnostic applications. Furthermore, Salmonella serovars' invA gene enables bacteria to penetrate the host and induce infection, enhancing the pathogenicity of isolates [28]. Interestingly, we have noticed that the number of Salmonella is decreased after molecular confrmation demonstrating the sensitivity and reliability of the molecular amplifcation tests than traditional cultural methods. In addition to invA gene detection, we used the PCR assay with primers targeting the iroB gene. Te PCR detection assay targeting iroB distinguishes S. enterica strains from other bacterial species, including S. bongori [29]. Out of 108 Salmonella isolates, 66 (61.1%) were identifed as S. enterica. Most isolates that cause diseases in humans and animals belong to S. enterica. In developing countries such as Burkina Faso, numerous studies have previously reported the presence of Salmonella spp. in RTE foods [30][31][32]. Te potential hygiene breakdowns at various stages of the food processing and distribution chain due to the lack of food safety regulations and food handler educations are responsible for food contamination in developing countries [33]. As the presence of pathogens such as Salmonella could cause illnesses with substantial health risks, including the death of consumers, routine monitoring is necessary to be undertaken.

Virulence Genes among Salmonella Isolates.
Virulence determinants infuence bacterial pathogenicity, and their presence in Salmonella can result in infections. During contamination, the host infection outcomes are shaped by a range of factors, including age, environment, and pathogenicity of the pathogen [26]. Te present study established the prevalence of four virulence genes, including spiC, pipD, misL, and orfL, located on SPI. Te SPIs contain a number of functionally related genes necessary for a specifc virulence phenotype of Salmonella [34]. In this study, 57.4% of Salmonella isolates harbored the pipD gene, while 55.6%, 49.1%, and 38% were positive for spiC, misL, and orfL, respectively. High prevalence rates of misL and pipD (91% each) among Salmonella isolates from human diarrhea, environment, and lettuce samples in Burkina Faso have been reported [35]. In addition, Zishiri et al. [26] have found high detection (85%) of the spiC gene, followed by pipD (80%), misL (75%), and orfL (20%) among Salmonella isolated from human clinical samples in South Africa. Furthermore, Dione et al. [36] reported high prevalence rates of pipD (95%) and spiC (78%) in Salmonella isolated from humans and livestock samples. In contrast, low prevalence rates of 47% for SpiC and 35% for pipD in Salmonella spp. isolated from chickens in South Africa were reported [26]. Te frequency of virulence genes in Salmonella isolates from lettuce, mango juice, RTE salad, and sesame are shown in Figure 2. After statistical analysis, there was no signifcant diference between the prevalence of each virulence gene in Salmonella from mango juice, sesame, RTE salad, and lettuce. Because Salmonella pathogenicity is determined by the presence or absence of virulence genes, the detection of diferent virulence genes in the Salmonella isolates in this study poses a potential public health risk.

Antimicrobial Resistance Profles of Salmonella spp.
Isolates. Antimicrobial resistance in Salmonella is rapidly becoming a major concern for both animals and humans globally [37]. RTE foods have been recognized as AMR bacteria reservoirs, directly threatening human health [24]. As a result, determining Salmonella antimicrobial resistance is essential for the treatment regimens during epidemics in   International Journal of Microbiology a given location. In the present study, antimicrobial sensitivity testing using the disc difusion method was performed on 108 Salmonella isolates recovered from food samples with 16 antimicrobial agents. Globally, 66 (61.1%) isolates showed resistance to at least one antimicrobial. Te distribution of the susceptibility profle of Salmonella is presented in Table 3. Resistance to gentamycin (26.8%), ampicillin (22.2%), cefoxitin (19.4%), and nalidixic acid (18.5%) was the most common. Ampicillin, chloramphenicol, and trimethoprimsulphamethoxazole constitute the frst-line traditional antibiotics used for salmonellosis treatment [38]. High prevalence rates of resistance to these antibiotics have been described in Burkina Faso and elsewhere from other sources (e.g., humans and animals) [23,36,39]. Salmonella resistance to these afordable antimicrobials is a public health threat. Over the years, the misuse and abuse of these inexpensive and easily accessible over-the-counter drugs rendered them inefective [40]. Interestingly, no Salmonella isolate was resistant to the third-generation cephalosporins (cefepime, cefotaxime, and ceftazidime). Tis is important for public health since these antimicrobials may be essential in threatening drug-resistant Salmonella pathogens. By contrast, Karikari et al. [40] in Ghana have reported high resistance of Salmonella isolated from salad to thirdgeneration cephalosporins including cefotaxime (70%), ceftriaxone (85%), and ceftazidime (70%). In other countries, resistance against these antibiotics in Salmonella was highest, especially in poultry and pork [37,41]. Te high resistance to these antimicrobials results from the extensive use of these antibiotics for growth promotion and therapeutic and prophylactic purposes in livestock breeding in these areas [14]. Terefore, educating people about not to misuse these precious antimicrobials is essential to preserve their efcacy. In this study, Salmonella isolates showed moderate (18.5%) resistance to nalidixic acid (frstgeneration quinolones), which requires continuous monitoring since nalidixic acid resistance usually increases the risk of ciprofoxacin (second-generation quinolones) and other quinolone resistances [42]. Te antimicrobial resistance profle and MAR index of the Salmonella serovars are presented in Table 4. Te MAR index ranged from 0.06 (resistant to one antibiotic) to 0.53. A total of 10 (9.3%) of Salmonella were resistant to 2 antibiotics (AMP and FOX) which belonged to 2 diferent antimicrobial groups with a MAR index of 0.13. Furthermore, 1 (0.9%) Salmonella isolated from sesame was resistant to 8 antibiotics (AUG, AMP, FOX, TE, NA, CN, SXT, and C) with the MAR index of 0.53. Salmonella strains with a MAR index >0.2 originated from an environment where antimicrobials are overused and misused [27]. A total of 11 (10.2%) of Salmonella isolates were MDR bacteria (exhibited resistance to three or more classes of antimicrobial agents). Te number of MDR isolates found in the current study was low compared to other studies from diferent countries [43][44][45]. Nikiema et al. [46] also found a similar moderate percentage of MDR strains Salmonella strains isolated from sandwiches sampled at street food establishments in Ouagadougou, Burkina Faso. By contrast, a relatively high prevalence of MDR Salmonella (35.97%) from chicken and guinea fowl in Burkina Faso has been reported by Caroline Bouda et al. [47]. Te low number of MDR Salmonella isolates in this investigation can be attributed to the fact that the isolates were from samples that did not contain meat or fsh. MDR strains in foods directly threaten human health as they could provoke diseases challenging to cure. In addition, a genetic element such as integrons is often associated with multiresistant phenotypes among Salmonella isolates and plays an essential role in spreading antimicrobial resistance genes among Gram-negative bacteria [48]. Table 5 shows the distribution of resistance genes among phenotypically resistant Salmonella isolates. PCR detected 8 resistance genes (tet(A), tet(B), bla TEM , temB, sul1, sul2, and aadA) in drug-resistant isolates. Salmonella develops AMR by complex mechanisms, such as the production of enzymes that can alter the antibiotic target site by activating efux pumps and the production of β-lactamases, which can degrade the structure of antibiotic molecules [49]. Most AMR genes are on integrons, plasmids, or transposons, which may be transferred and mobilized to other or the same species of bacteria [48]. In the present study, 2 of the 5 tetracycline-resistant isolates harbored the tet(A) gene (40%), and only one isolate was positive for the tet(B) gene (20%). Te genes tet(A) and tet(B) encode energydependent efux proteins that help bacteria eliminate tetracycline from their cells [50]. Tese genes are widespread among Salmonella and have been located on transferable plasmids and code for an energy-dependent efux pump system [23]. In the present study, one of the 4 sulfamethoxazole-resistant isolates carried the sul1 gene (25%), 2 carried the sul2 gene (50%), and one carried both the sul1 and sul2 genes (25%). Tese genes encode dihydropteroate synthase and are often found in integronpositive isolates that carry other genes [51]. Te resistance of Gram-negative bacteria to cephalosporins and ampicillin is mainly mediated by β-lactamases [44]. Although TEM β-lactamase genes are nonextendedspectrumβ-lactamase lactamases, bla TEM is a major mediator of β-lactam resistance in Salmonella spp. worldwide [52]. In this study, 31 isolates resistant to ampicillin or cefoxitin were tested for three types of beta-lactamase genes. Among them, 15 (48.4%) gave positive amplicons for bla TEM , while 5 (20.8%) isolates exhibited the temB gene, and none of the temA genes were found. Similar to our fndings, high rates of bla TEM (44.11%) were found by Rajaei et al. [53] in Salmonella spp. isolates from raw kebab and hamburger in Iran and by Moawad et al. [54] in Egypt (73.3%) in Salmonella spp. isolates from chicken and beef meat. Aminoglycoside resistance occurs through several mechanisms that can coexist simultaneously in the same cell, the most common being chemically modifying aminoglycosides by aminoglycoside-modifying enzymes [55]. Enzymatic inactivation by acetylation, adenylylation, or phosphorylation at diferent locations of the aminoglycoside molecule is among the most clinically relevant strategies that bacteria use to resist the action of these   0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Ceftazidime 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Aztreonam 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Imipenem 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Meropenem 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Tetracycline 2 (3.0) 1 (9.0) 2 (9.5) 0 (6.7) 5 (4. antibiotics [56]. Aminoglycoside nucleotidyl-transferases can confer resistance to gentamicin, tobramycin, or streptomycin including aad and variant StrA/B among Gram-negative bacteria [57]. In our study, the aadA genes were found in high frequency (86.2%), while no isolate was found harboring StrA.

Conclusion
Te study provides additional information about the presence of Salmonella in foods, especially RTE foods in Burkina Faso. Moreover, these might act as reservoirs for antimicrobial-resistant Salmonella, which constitute a risk to consumers. Tus, our fndings highlight the urgent need for stringent sanitation and hygienic standards for food safety measures to minimize cross-contamination during the food chain process. In addition, it is crucial to implement strict guidelines for antimicrobial usage in hospitals and community settings to limit the potential spread of bacterial resistance.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon request.

Disclosure
Tis study was carried out as part of a PhD thesis.

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