1 Introduction

Salmonella is an important ubiquitous human pathogen commonly found in the intestinal tract of captive and wild warm-blooded animals, amphibians, and reptiles [1,2,3]. Reptiles could carry infectious Salmonella symptomatically [4, 5] and asymptomatically as commensal microorganisms of the intestinal tract [5, 6]. Different human-pathogenic Salmonella serovars (Paratyphi B Newport, Typhimurium, Kentucky, Enteritidis, Tennessee, and Poona) have been detected in lizards and snakes (captive and wild) [1, 6,7,8,9]. Recently, the keeping of exotic pet lizards and snakes is a growing global trend. Therefore, a rise in reptile-associated salmonellosis cases has been reported in USA [5, 6, 10,11,12], Japan [13], England [6, 14, 15], Germany, Australia, Netherlands, Canada, and France [6].

During the last decade, exotic pet reptiles (lizards and snakes) have also become quite popular among Saudi citizens. However, pet shop owners and pet keepers remain unaware of pet reptiles-associated Salmonella health hazards. There is also a lack of proper guidelines for the hygienic handling of these pet reptiles. Recently, the Salmonella incidence in pet reptiles (lizards and snakes) in private households in Makkah, Saudi Arabia was reported for the first time [16]. The isolates were phenotypically identified as Salmonella species, but genotypic identification and characterization were lacking. Therefore, molecular characterization of reptile-associated Salmonella isolates was performed during this study.

2 Materials and methods

2.1 Bacterial isolates

Five Salmonella isolates were previously retrieved from pet reptiles in Makkah during January 2022, which included Spalerosophis diadema (local diadem snake), Boa constrictor (imported common boa), and Eumeces schneiderii (imported Schneider’s skink). The isolates were identified as Salmonella spp. using API 20E strips (bioMerieux, Marcy-I’Etoil, France) and Salmonella enterica subsp. enterica by Vitek 2 compact (bioMerieux) [16]. Buffered peptone water (BPW) (Oxoid, UK) was used to culture the isolates for 24 h at 37 °C. Selective enrichment was carried out in Rappaport-Vasilliadis broth (RV) (Molecule-On, New Zealand) for 24 h at 41.5 °C. Positive RV-broth cultures were streaked on Xylose Lysine Deoxycholate (XLD) (Oxoid), Salmonella-Shigella agar (S–S) (Oxoid), and CHROMagar Salmonella Plus (CHROMagar, France) plates and incubated at 37 °C for 24 h [16]. Antimicrobial susceptibility was evaluated during the previous study, which represented the multidrug resistance (MDR) patterns of all the tested isolates [16].

2.2 Bacterial DNA isolation

DNeasy Blood and Tissue kit (QIAGEN, USA) was used to extract Salmonella DNA and its quantity and quality were evaluated spectrophotometrically (Denovix DS-11, Denovix Inc., USA).

2.3 PCR amplification of 16S rRNA gene

16S rRNA gene was amplified using 27F (forward: 5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492 R (reverse: 5′ TACGGYTACCTTGTTACGACTT 3’) primers. The PCR reaction mixture (25 µL) contained 2X DreamTaq Green master mix (12.5 µL, Thermo Fisher Scientific, USA), 10 µM primers (2.0 µL, forward and reverse), bacterial DNA (1.0 µL of 50 ng µL−1), and nuclease-free H2O (9.5 µL). PCR amplification was carried out in Verti™ Thermal Cycler (Applied Biosystems, USA) as follows: 1 cycle of initial denaturation at 95 °C for 3 min, 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 90 s. The final extension was carried out at 72 °C for 7 min (1 cycle) and then reaction tubes were held at 4 °C [17].

2.4 Gel electrophoresis

Agarose (Cleaver Scientific, UK) and TBE buffer (1X) were used to prepare the gel (2%) and added with a cyber-safe DNA stain (Invitrogen, USA). PCR products (4.0 µL) were loaded into each well alongside a DNA ladder (100–1000 bp). Gel electrophoresis was carried out at 100 mV for 30 min and a Gel Doc imager system (Thermo Scientific) was used to visualize the DNA fragments.

2.5 Controls

Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028 and Candida Albicans ATCC 14035 were used as positive and negative controls to validate the PCR amplification of the 16S rRNA.

2.6 Sanger sequencing

2.6.1 PCR purification

ExoSAP-IT™ kit (Thermo Fisher Scientific) was used to purify the amplicons. ExoSAP-IT™ (2 µL) was added to PCR products (5 µL) and the mixture was incubated for 15 min at 37 °C to remove the remaining nucleotides and primers. ExoSAP-IT™ reagent was inactivated by incubating the mixture for 15 min at 80 °C.

2.6.2 Cycle sequencing PCR

BigDye Terminator v3.1 cycle sequencing kit (LifeTechnologies, USA) was used for cycle sequencing PCR reaction whereas PCR product concentration was estimated using nanodrop. Cycle sequencing mixture (10 µL) consisted of 5 × BigDye buffer (1 µL), BigDye (2 µL), 3.2 µM primer reverse (1 µL), purified PCR product (quantity varied with the sample concentration), and nuclease-free H2O (10 µL). The cycle sequence PCR program consisted of 25 denaturation cycles (96 °C for 10 s) followed by annealing (58 °C for 10 s), and extension (60 °C for 4 min).

2.6.3 Purification of cycle sequencing PCR

BigDye Xterminator purification kit (Applied Biosystems) was used to purify cycle sequencing PCR. SAM solution (45 µL) was added to the cycle sequencing reaction followed by the addition of XTerminator beads (10 µL). The mixture was vortexed at 3500 rpm for 45 min followed by centrifugation for 2 min at 14,000 rpm. A 30 µL aliquot of the upper layer was carefully transferred into optical 96-well plate wells. The plate was sealed and spun.

2.6.4 Sequencing platform

SeqStudio (Life Technologies) was used for sequencing at a medium module.

2.7 Sequence alignment

SNapGene (version 6.0.2, GSL Biotech LLC, USA) was employed for sequence analysis and retrieving the FASTA format. Sequences were aligned through the NCBI blast tool whereas the phylogenetic tree was constructed in Molecular Evolutionary Genetics Analysis software (MEGA 11.0).

2.8 Phylogenetic tree analysis

16S rRNA sequences of Salmonella enterica subsp. enterica serovar Typhimurium isolates from reptiles (A1, B1, B2, B3, and G2) were added to FASTA files and compared using Geneious Prime ® 2023.1.1. Escherichia coli (accession number: J01859.1) was obtained through a DNA blast at NCBI, which served as the outgroup. The sequences were grouped after manually removing the low-quality sequences and subjected to MUSCLE alignment (MUSCLE 5.1). The alignment of six sequences was used to construct a phylogenetic tree using PAUP* plugin and following the maximum parsimony algorithm, FastStep search, heuristic tree search, and 1000 bootstrap (fixed seeds 100) replications.

3 Results

During the previous study, five Salmonella isolates from exotic pet reptiles were identified by API (Salmonella spp.) and Vitek 2 compact system (Salmonella enterica subsp. enterica). 16S rRNA gene sequences confirmed these isolates as Salmonella enterica subsp. enterica serovar Typhimurium (Table 1).

Table 1 16S rRNA sequences of reptilian Salmonella isolates
Full size table

The phylogenetic tree revealed a close relationship and common ancestry of Salmonella enterica subsp. enterica serovar Typhimurium isolates (Fig. 1). Isolates from G2 (Schneider’s skink), B1 (diadem snake), A1 (common boa), B2 (diadem snake), and B3 (diadem snakes) formed a clade to represent a subgroup of A1, B2, and B3 with a 58% bootstrap value. Despite an older common ancestor, a close relationship between B1, A1, B2, and B3 isolates was noted with a 78% bootstrap value.

Fig. 1.
figure 1

16S rRNA sequences based phylogenetic comparison of Salmonella enterica subsp. enterica serovar Typhimurium isolates of reptile origin (A1: imported common boa, B1, B2, B3: local diadem snake, G2: Schnieders’ skink). The sequences of all the isolates were compared using Geneious Prime 2023. MUSCLE sequence alignment was performed and the phylogenetic tree was constructed using PAUP* plugin following the maximum parsimony algorithm with 1000 replicates at each corresponding node. E. coli (J01859.1) from the NCBI database served as the outgroup

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4 Discussion

The captive reptiles (zoo and private household) could carry various Salmonella enterica serovars. Reptilian species often have exotic Salmonella enterica subsp. salame (II), arizonae (IIIa), diarizonae (IIIb). Similarly, human pathogenic Salmonella enterica serovars are also commonly detected in reptiles [1]. During this study, Salmonella enterica subsp. enterica serovar Typhimurium was detected in pet snakes (common boa and diadem snake) and a lizard (Schneider’s skink) in private households. Salmonella enterica serovars’ abundance in reptiles (lizards and snakes) has been reported [1]. During the current study, Salmonella typhimurium was found in imported common boa (Boa constrictor), local diadem snake (Spalerosophis diadema), and imported Schneider’s skink (Eumeces schneiderii). Contrarily, Salmonella typhimurium presence in common boa (Boa constrictor) has never been reported. Boa constrictor asymptomatically carries Salmonella enterica subsp. arizonae IIIa [18], untypable Salmonella enterica subsp. diarizonae IIIb [4], Salmonella enterica subsp. enterica serovar Muenchen [19], Salmonella enterica subsp. salame (II) and Salmonella enterica subsp. diarizonae (IIIb) [20], Salmonella enterica subsp. enterica serovar Oranienburg and Muenchen, Salmonella enterica subsp. IIIa and subsp. IIIb [21], and Salmonella enterica subsp. enterica serovar Memphis [22]. Schneider’s skink (Eumeces schneiderii) has never been extensively studied for the detection of Salmonella serovars. However, Ebani et al. [22] have isolated Salmonella enterica subsp. enterica serovar Ebrie from Eumeces schneiderii. Furthermore, Salmonella spp. presence in diadem snake has never been explored until recently when Degi et al. [23] detected Salmonella spp. from diadem snake in Romania. Given that the identification of Salmonella spp. in Saudi Arabian local and exotic diadem snakes was not previously known [16]. Therefore, we believe that this is the first report of Salmonella enterica subsp. enterica serovar Typhimurium asymptomatic presence in Saudi Arabian pet reptiles [imported common boa (Boa constrictor), local diadem snake (Spalerosophis diadema), and imported Schneider’s skink (Eumeces schneiderii)].

Phylogenetic analysis revealed the presence of four related strains. The strains were assigned to genetic clades based on their 16S rRNA sequences. S. enterica serovar Typhimurium was commonly detected in tested local and imported reptiles [snakes (common boa and diadem snake) and lizards (Schneider’s skink)]. All the Salmonella enterica subsp. enterica serovar Typhimurium isolates were closely related and shared a common ancestor. The isolates were mainly grouped based on their host reservoir. For instance, strains B1, A1, B2, and B3 isolated from geographically distant snakes were more related to each other than lizard-originated strain G2. These findings suggest that particular Salmonella strains/serovars are associated with a specific reptilian host species regardless of their origin. The close relatedness of snakes-originated Salmonella typhimurium highlights the role of diet as a Salmonella spp. source. The snakes were fed on small rodents whereas the skink was fed on small insects.

Reptiles (captive and wild) could acquire Salmonella through various transmission routes. Salmonella typhimurium transmission to reptilian species during this study could be attributed to animal feed. The snakes were fed on small rodents (mice and rats) obtained from pet shops, which are known carriers of Salmonella serovars including Salmonella typhimurium [24]. It explains the specific serovar presence in snakes. Schneider’s skink was fed on small insects (crickets and cockroaches). However, the same Salmonella serovar was also found in the lizard during this study. Cockroaches are known carriers of human-pathogenic Salmonella serovars [25] and might have served as a source in this study. The owner usually collected small insects from the immediate vicinity to feed the skink, which might have Salmonella contamination. Moreover, the reptile owner used only one cage to keep all the feed animals (small rodents and insects) before feeding to pets. The feed collection cage could be previously contaminated with Salmonella typhimurium to serve as a bacterial source. Drozdz et al. [26] have already discussed that the immediate environment of pet reptiles or feed storage places can also contribute to Salmonella spp. transmission. Salmonella could also be transmitted if the pet reptiles are kept in a shared cage. However, it can be ruled out in this study as the owner confirmed that pet reptiles were never kept together.

5 Zoonotic potential of identified Salmonella typhimurium

Salmonella are important zoonotic microorganisms with global prevalence [26]. The zoonotic potential and pathogenicity of pet reptile-associated Salmonella vary between different strains, serovars, and subspecies [2]. The pathogenicity of Salmonella typhimurium isolates was not assessed in this study. Therefore, their zoonotic potential should be carefully examined. Highly zoonotic S. enterica subsp. enterica serovar Typhimurium was isolated during this study. Salmonella enterica subsp. enterica serovars (Typhimurium and Enteritidis) are responsible for 99% of global human salmonellosis [20]. Moreover, Salmonella typhimurium serovar is commonly associated with human salmonellosis in Saudi Arabia [27. 28]. These strains have also been reported to exhibit MDR patterns [16]. Reptile-associated Salmonella infection data from Saudi Arabia are not available. Therefore, the scale of the problem remains unknown, and unreported reptile-associated salmonellosis cannot be ruled out due to the emerging popularity of pet reptiles in private households in Saudi Arabia.

6 Recommendations for pet reptile owners and handlers

The results of current and previous study [16] have revealed valuable data regarding Salmonella spp. prevalence in pet reptiles and their drug susceptibility in Makkah, Saudi Arabia. This is the first report of human-pathogenic Salmonella serovar Typhimurium occurrence in pet reptiles of Saudi Arabia. Salmonella reservoir identification is crucial to assess the spreading risk of this zoonotic agent and related infection. Pet reptiles are a potent source of Salmonella transmission. Therefore, cage-cleaning and animal handling should be performed hygienically. The washing of hands is highly recommended after each contact with reptiles or their surrounding objects. The free roaming of pet reptiles in the house particularly in kitchens should be restricted to avoid potential Salmonella contaminations. The use of disposable gloves and disinfectants is recommended while cleaning their cages. Immunocompromised and elderly individuals and children under the age of five should avoid touching reptiles or their environment. Public and reptile owners’ education is necessary to prevent salmonellosis.

7 Conclusion

Reptiles are a potential source of zoonotic Salmonella spp. and their MDR strains could further worsen the situation. The increasing trend of keeping exotic pet reptiles in households has led to a rise in Salmonella spp. transmission. Salmonella typhimurium isolation and molecular characterization from pet reptiles indicate the zoonotic potential of pet reptiles-to-human transmission. Therefore, reptile-pet owners should be well-aware of Salmonella spp. potential transmission routes and associated human health repercussions. One limitation of the study is that samples were taken from one household, various attempts were made to collect samples from other households and also from pet shops, however, all attempts were unsuccessful. Further important epidemiological data regarding the diversity and prevalence of Salmonella serovars in pet reptiles kept in private households within the city, as well as sampling reptile collection in pet shops could have been revealed if more samples were analyzed.