Occurrence, Antimicrobial Resistance, and Virulence Profiles of Salmonella Serovars Isolated from Wild Reptiles in South Africa

Reptiles are carriers of an array of microorganisms, including significant zoonotic bacteria of the genus Salmonella, which cause a disease referred to as salmonellosis that affects both animals and humans. This study investigated the occurrence of Salmonella serovars in wild reptiles at Timbavati Private Game Reserve in Limpopo Province, South Africa, and examined their virulence and antimicrobial resistance gene profiles. A total of 19 wild reptiles were sampled, which resulted in 30 presumptive Salmonella isolates. The isolates were identified using polymerase chain reaction (PCR) by amplifying the invA gene and were further confirmed by 16S rRNA gene sequencing. Salmonella serovars were detected in chameleons (36.8%), lizards (31.6%), snakes (15.8%), and tortoises (15.8%). The use of 16S rRNA gene sequencing revealed that Salmonella enterica subsp. enterica serovar Salamae (30%), S. enterica subsp. enterica (16.7%), S. enterica subsp. enterica serovar Typhimurium (13.3%), and S. enterica subsp. enterica serovar Indiana (13.3%) were the four most common subspecies among the investigated 30 isolates. Detected virulence genes included pagN (100%), hilA (96.7%), ssrB (96.7%), prgH (86.7%), and marT (86.7%). The isolates exhibited resistance to nalidixic acid (43.3%) and kanamycin (43.3%), followed by streptomycin (16.7%) and ciprofloxacin (3.3%). Antibiotic-resistant genes were detected as follows: strA, strB, qnrA, qnrS, parC, aadA, aac(6′)-Ib, and aac(6′)-Ib-cr at 33.3%, 6.7%, 16.7, 13.3%, 10%, 23.3%, 6.7%, and 10%, respectively. The findings highlight the necessity of educational initiatives aimed at reducing reptile-related infections. Effective antibiotic treatment appears promising for infection, given the minimal drug resistance observed in reptile Salmonella serovars in the current study.


Introduction
Salmonella is a genus that belongs to the family Enterobacteriaceae, a Gram-negative facultative anaerobic bacterium, and is regarded as one of the most concerning zoonotic bacteria in the world [1,2].Salmonella is naturally present in the gastrointestinal tracts of many species of animals, including humans, birds, reptiles, and livestock [3,4].Te species S. enterica is comprised of six subspecies: indica, salamae, enterica, houtenae, arizonae, and diarizonae.It is estimated to have more than 2659 serovars, which are divided into 60 serogroups [5,6].
According to the current nomenclature, Salmonella spp. is taxonomically classifed into two species: S. bongori and S. enterica [7].Salmonella is generally considered a normal constituent of the reptilian intestinal microbiota with a subclinical presentation [1].Nevertheless, some reptiles harbor and shed Salmonella spp.asymptomatically in their faeces, and up to 90% of them are considered reservoirs for the bacteria [8].In South Africa, Salmonella serovars have previously been documented in farmed crocodiles and a few other, mostly captive reptiles [9].However, the association of reptile-associated Salmonella in South Africa is largely unknown.
Tere have even been several outbreaks of human salmonellosis associated with reptiles from various countries [8,10,11].Assessing the risk of humans being infected through direct contact with reptiles becomes challenging due to the lack of a robust understanding of the natural occurrence of Salmonella spp.circulating in reptiles and their propensity to switch hosts [1].Te risk of zoonotic disease is higher with the transmission of multidrugresistant Salmonella spp.strains.Te presence of plasmids, transposons, integrons, and insertion sequences can contribute to the development of antibiotic resistance [12,13].Tere have been numerous studies on antibiotic resistance genes identifed in Salmonella spp.[12][13][14].Most virulence and resistance genes have been transferred between species by horizontal gene transfer (HGT) [15].Virulence plasmids, pili, and enterotoxins are among the reported Salmonella pathogenicity islands (SPIs) [16].Virulence mechanisms are required to defeat host defense systems, and the development of antimicrobial resistance is required to allow pathogenic bacteria to overcome antimicrobial therapy and adapt to and thrive in competitive and demanding environments [15,17,18].Te virulence genes contribute to pathogenesis through host cell attachment and overcoming host defense mechanisms [14].Infection and virulence are often associated with antibiotic resistance, as seen in bioflm-producing bacteria or intracellular infections [15,16].Terefore, the aim of this study was to determine the prevalence of Salmonella spp. in various wild reptile species and to evaluate their antimicrobial resistance and virulence gene profles.Te remarkable array of reptile diversity in this region acts as a catalyst for the exploration of antibiotic resistance, with ultimate benefts for reptile conservation.

Field Site. Te Timbavati Private Game
Reserve is situated between 24 °24′S and 31 °21′E.It covers an area of 550 km 2 and is located on the central west border of Kruger National Park.Te reserve comprises Combretum apiculatum, Acacia nigrescens, and Colophospermum mopane as the dominant vegetation types, with mostly granite or basalt as the principal soil types [19].

Collection of Samples.
Samples were collected from wild reptiles (n � 19) in the Timbavati Private Game Reserve in the Limpopo Province.Collection consisted of active searching for wild reptiles and their subsequent release after sampling.Snakes were placed in transparent plastic tubes before sampling, while other reptiles were restrained by hand [20].Sterile cotton transport swabs (Transystem ™ ) were used to swab the cloaca of the reptiles and were stored at 4 °C during feld work [21].Te transport medium provides a nonnutritive environment that maintains the viability of microorganisms while restricting growth until samples can be processed.

Isolation, Identifcation, and Serotyping of Salmonella
Isolates.Te cloacal swabs were pre-enriched in bufered peptone water (BPW Oxoid, Biolab, South Africa) at 37 °C for 24 hours.A loopful of the bacterial cells in bufered peptone water was streaked onto xylose-lysine-deoxycholate agar (Merck, Wadeville, South Africa) and Brilliant Green agar (Scharlau Chemie S.A. Barcelona, Spain).Te streaked plates were then incubated at 37 °C for 24 hours.Te colonies were examined for their morphological appearance on the plate (colonies with or without black centers, colorless, or opaque-white colonies surrounded by pink or red zones on XLD).Te suspected Salmonella spp.colonies, those with glossy large black centers or almost black colonies, were examined for pure culture isolation on BGA.Between three and fve colonies were selected and purifed on nutrient agar (Merck, Wadeville, South Africa) and incubated at 37 °C for 18 to 24 hours.

DNA Extraction and Molecular Identifcation of Salmonella Serovars
Using invA Gene.Te bacterial genomic DNA was extracted using a genomic DNA extraction kit (Invitrogen, USA) from pure cultures.A NanoDrop spectrophotometer was used to measure the DNA concentrations.For the invA gene, PCR was carried out using the forward (GTG AAA TTA TCG CCA CGT TCG GGC AA) and reverse (TCA TCG CAC CGT CAA AGG AAC C) oligonucleotide primers with a reaction volume of 25 μL, containing: 8.5 μL nuclease-free water, 12.5 μL PCR Master Mix, 2 μL template DNA, and 1 μL of each primer utilizing an Engine T100 TermalTM cycler (BioRad, Singapore).Te thermal cycling conditions included an initial step of denaturation at 94 °C for 5 minutes, then 30 cycles of denaturation at 94 °C for 45 seconds, annealing at 58 °C for 45 seconds, and extension at 72 °C for 70 minutes, followed by a single, concluding extension step at 72 °C for 7 minutes [13].

Identifcation of Salmonella Species Using 16S rRNA.
All the positive samples for invA were subjected to 16S rRNA for sequencing.Te bacterial universal primers (27F: AGA GTT TGA TCM TGG CTC AG and 1492R: GGT TAC CTT GTT ACG ACT T) targeting the 16S rRNA gene segment were used for molecular identifcation using PCR.Te PCR conditions were as follows: initial denaturation step at 96 °C for 4 minutes, followed by 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 57 °C for 30 seconds, and extension at 72 °C for 1 minute, and fnally, a single and fnal extension step at 72 °C for 10 minutes [22].
2.6.Sequencing of PCR Amplicons.Te PCR products were sequenced at Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa.Te FintchTV [23] was used to edit the base pairs of the sequence chromatograms.Sequence identity was evaluated using the nucleotide Basic Local 2 International Journal of Microbiology Alignment Search Tool nucleotide (BLASTn) on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cg).Te generated 16S rRNA gene sequences were submitted to the GenBank database and assigned with the accession numbers as follows: OP683334-OP683363.

Discussion
Reptiles carry zoonotic pathogens that cause a variety of infectious diseases in both humans and other animals [6].
Tey are becoming increasingly appealing as pets and are popular attractions at wildlife education centers [20].Although the clinical relevance of Salmonella infections in wild and captive reptiles is poorly understood, it is believed that the majority of infections results in an asymptomatic carrier condition and do not cause disease in reptiles [6].S. enterica subsp.enterica serovar Houtenae has been associated with abdominal abscesses in a severely diseased captive African fat-tailed gecko [30].

International Journal of Microbiology
From a host-reservoir perspective, chameleons (Chamaeleo dilepis) were the most frequently infected with Salmonella serovars, i.e., S. enterica, S. Indiana, S. Salamae, S. Typhi, and S. Kentucky.Te prevalence rates of Salmonella serovars among chameleons, lizards, snakes, and tortoises were 36.8%,31.6%,15.8%, and 15.8%, respectively.Tese fndings difer in terms of the frequency of Salmonella spp.occurrence in various sectors of captive reptiles in Europe.Higher (76.9%) prevalences of Salmonella spp.were recorded in pet snakes, lizards, and tortoises from Poland [5], 64.5% in snakes and lizards from Norwegian zoos [1], and 32.6% in domestic snakes, chameleons, and lizards from central Europe [35], 43.28% of the pet reptiles carried from Western Romania [36], and 50.0% of the lizard from Fernando de Noronha Archipelago (Brazil) [37].Te current study is one of the few studies to isolate Salmonella serovars from wild reptiles.
Te majority of salmonellosis illnesses are associated with a wide range of serotypes of S. enterica subsp.enterica (I) and are primarily transmitted through tainted food and water [30,[38][39][40].In some parts of the world, pet reptiles provide a signifcant source of protein for human populations, and in so doing, a transmission route for Salmonella is established.All reptiles are exploited for human consumption, but turtles are heavily exploited, while crocodiles, snakes, and lizards may be important locally [41,42].Indeed, there have been numerous reports of reptile-associated salmonellosis in humans, especially in children [20,43,44].
Salmonella pathogenicity island 1 is essential for the interaction between Salmonella and host cells.Salmonella invades epithelial cells through SPI-1 (44).Two SPI-1 genes that encode components of the SPI-1 T3SS apparatus, invF and sicA, are directly regulated by the OmpR/ToxR transcriptional regulator HilA [45,46].Moreover, enterocolitis and human intestinal epithelial cell invasion may be infuenced by the regulation of virulence factors including HilA, invA, and SPI-1 efectors such as SipA and SopABD [47,48].
Salmonella's intracellular pathogenicity cycle begins with the invasion of intestinal epithelial cells, controlled by the invA gene [49].Salmonella-specifc gene sequences encode the InvA protein that is essential for gut epithelial invasion [50].Te results showed that all Salmonella isolates tested positive for the invA gene.Tis is in agreement with the fndings of previous studies (12, 13, 21, 22, 37, and 49).It is  4 International Journal of Microbiology not surprising because InvA is used for molecular identifcation of these Salmonella isolates [51].Virulence gene profles showed that all the Salmonella serovars isolated in this study were positive for pagN, hilA, ssrB, prgH, and marT (100%), (96.7%), (96.7%), (86.7%), and (86.7%), respectively.Similar genes were detected in Salmonella species isolated from retail beef samples in selected KwaZulu-Natal municipality areas and in livestock production systems (cattle, sheep, goats, pigs, ducks, and chickens) in the Eastern Cape and KwaZulu-Natal provinces of South Africa [52,53].
Virulence plasmid operons (spvRABCD) are expressed by intracellular environments in host cells and are involved in survival, intracellular growth, and macrophage death [54,55].Te spvR gene was detected in one (3.3%)sample.Tis observation was diferent from the fndings of a study conducted by Derakhshandeh et al. [56] on humans, where they reported that the prevalence of spvB, spvC, and spvR genes was 26 (43.3%), 44 (73.3%), and 28 (46.6%),respectively.Te study on humans and animals reported in 2008 by Amini et al. [57] showed that the spvB and spvC genes were detected in 90% of the isolates.In the current study, the spvB gene was not detected in any of the 30 isolates.In Burkina Faso, Nikiema et al. [58] detected spvR and spvC genes at 36.8% and 48.1%, respectively, from 106 Salmonella isolates (77 human stools and 14 sandwiches).Te spvC gene resides on plasmids and plays an important role in adhesion and systemic infection of host cells [59].Te SipC protein also targets F-actin, which is critical for the internalization and invasion of pathogens [50].In consideration of the low level of detection of the spv gene in wild reptiles, there is a need to expand the surveillance to a broader host range over a larger geographical area.
Of the 17 virulence genes screened in this study, 13 are located on Salmonella pathogenicity islands (SPIs).All Salmonella isolates in this study exhibited high detection rates for virulence genes located on the SPIs, indicating the genes were widely distributed.Te SPI-1 genes sip, hil, and prg encode regulators that produce T3SS efector proteins, assist in Salmonella colonization and invasion of intestinal epithelial cells, and can trigger macrophage necrosis and infammatory responses [16].
Several researchers have recently reported the presence of antibiotic residues in reptiles and antibiotic-resistant bacteria [5][6][7]60].However, drug resistance in reptiles is relatively uncommon in reptile-associated Salmonella [60].Although the prevalence of antimicrobial resistance was not very high in this study, S. Worthington had the widest range of antibiotic resistance (60%).High antibiotic resistance prevalence was observed for nalidixic acid (43.3%) and kanamycin (43.3%).In comparison to Salmonella isolates in water samples in the Philippines, resistance to kanamycin was higher at 75.4% [61].On the other hand, there is a reported high (95.4%)nalidixic acid resistance by Salmonella isolates obtained from broiler and layer chicken farms [62].Tirty-three isolates (33.3%) of Salmonella serovars were resistant to at least one antimicrobial drug.Similar fndings were reported in studies involving Salmonella serovars isolated from reptiles from Taiwan, Trinidad, and Malaysia and their sensitivity to aminoglycosides and quinolones [7,63,64].In the same study by Chen et al. [7], as well as a study from Lithuania, Salmonella serovars isolates from reptiles most frequently displayed resistance to streptomycin and tetracycline [6,7], and in a study from Poland, the highest antibiotic resistance was detected against streptomycin [20].In a study conducted by Dégi et al. [65] in Romania, Salmonella serovars isolated from reptiles were resistant to ceftriaxone, ciprofoxacin, vancomycin, cefoxitin, pristinamycin, ampicillin/sulbactam, and gentamicin.
In contrast to our results, Abrahão et al. [37] have reported 13.3% of isolates from lizard resistant to colistin in Brazil.Given this growing evidence for antibiotic resistance, the importance of reptile-associated Salmonella spp.infections to medical research and public health should not be overlooked.Salmonella enterica subsp.enterica serovar isolates from this study were resistant to aminoglycosides and quinolone classes of antibiotics.Te same antibiotic resistance gene profles were detected in Salmonella serovars isolated from other animals, including commercial chickens, as well as humans in South Africa [66][67][68][69][70]. Similar antibiotic resistance genes (strA, strB, and aadA) were also detected in reptiles in Poland [20].Both strA and strB genes encode aminoglycoside-3″-phosphotransferase (APH(3″)-Ib) and aminoglycoside-6-phosphotransferase (APH(6)-Id) proteins that confer streptomycin resistance, respectively [71].
Strains typically pose a high risk for the spread of resistance genes to other microbiota as well as for the treatment of infections [72].Antimicrobial resistance is rapidly developing and spreading due to interactions between human, animal, and environmental factors [67].Tere was a correlation between the presence of the quinolones (qnrA, qnrS, and parC) genes and the phenotypic susceptibility of the Salmonella serovar strains.Fluoroquinolones are widely used in veterinary practice, but no data involving the incidence of resistance exist [69].Further research is needed to investigate the possible relationships of microorganism transfer between reptiles and other hosts.

Limitation of the Study.
Te main drawback of dealing with wild reptiles is how difcult it is to obtain more specimen samples.When it comes to reptile research and surveys, Africa is far less advanced than other continents [73].Areas where reptiles occur in South Africa are usually remote and challenging to work with and sample in, which creates a sampling bias at times, which makes it very difcult for the collection of wild datasets [74].In Barends et al. [74] work in what is irrefutably the most famous park or reserve in South Africa (Kruger National Park) to examine reptile species presence within the 1 km resolution, 92% of KNP would be considered "data defcient" for reptile occurrence.As mentioned in our methods section, Timbavati borders KNP and has the same "big fve" (lion, leopard, rhino, bufalo, and elephant) dangers for feld researchers in terms of sampling [19,74].

Conclusions
According to our knowledge, this is the frst study reporting on the occurrence, antibiotic resistance, and virulence profles of Salmonella serovars from wild reptiles in South Africa.Chameleons had the highest infection rates for Salmonella serovars, followed by lizards, snakes, and turtles.Reptiles can serve as a reservoir for pathogenic bacteria such as Salmonella; hence, precautions should be taken when caring for and transporting them, as well as when keeping them in close contact with other animals.Tere is optimism for efective antibiotic therapy in the case of infection due to the low level of drug resistance of the reptile Salmonella serovars detected in the current study.Te fndings highlight the need for educational eforts aimed at reducing reptilerelated infections.As previous literature cited in this study has mentioned that the prevalence of Salmonella appears higher in captive reptiles elsewhere in the world, we suggest the next logical step would be an investigation of Salmonella prevalence in captive reptiles in the South African pet trade, and with a particular focus on nonnative popular species.International Journal of Microbiology article.OTperformed validation and reviewed and edited the article.All authors have read and agreed to the published version of the manuscript.

Figure 1 :
Figure 1: Distribution of virulence genes in diferent Salmonella serovars recovered from reptiles in South Africa.

Figure 2 :
Figure 2: Heatmap showing the clustering of the antibiotic resistance profles in the Salmonella isolates.Light blue and dark blue indicate the absence and presence of antibiotic and resistance genes, respectively, (https://www.chiPlot.online/#9(accessed on 17 June 2023)).
5 μL nuclease-free water, 12.5 μL PCR, 2X DreamTaq Green Master Mix (Termo-Fisher Scientifc, South Africa), 2 μL template DNA, and 1 μL of each primer.Te following PCR parameters were applied: 94 °C for 5 minutes, 30 cycles of 94 °C for 45 seconds, annealing temperatures (for each gene as shown in Supplementary Table S1) for 45 seconds, and 72 °C for 1 minute; and 72 °C for 10 minutes.2.8.Antimicrobial Susceptibility Testing.Based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI 2023) .2.Detection Rate and Distribution of Virulence Genes in Various Serotypes.A total of 30 Salmonella spp.isolates harbored either one or more diferent virulence genes investigated in this study, with sixteen out of seventeen virulence genes detected in this study (Figure1).Te distribution of virulence genes among each Salmonella isolate is shown on the heatmap (Figure2).