Isolation of Salmonella spp. from black spiny‐tailed iguana (Ctenosaura similis) meat commercialised in markets of León city, Nicaragua

Abstract Background The black spiny‐tailed iguana (Ctenosaura similis) is an endemic animal in Mesoamerica, whose meat is consumed by the local population. Objectives Because the black spiny‐tailed iguana may be potential reservoirs of pathogens, this study aimed to isolate and characterise Salmonella spp. in their meat commercialised in markets of the city of León, Nicaragua. Methods Thirteen specimens were analysed for the isolation of Salmonella spp., as well as their antimicrobial resistance patterns, including the presence of genes encoding extended‐spectrum β‐lactamases. Results Salmonella spp. isolates were found in eight out of 13 samples, with S. enterica serovar Enteritidis being found in six out of eight samples. Moreover, eight Salmonella spp. isolates were resistant to amoxicillin plus clavulanic acid and cephalexin, but sensitive to other tested antibiotics. The bla SHV gene was detected in seven out of eight Salmonella spp. isolates, followed by the bla TEM (two out of eight) and bla CXT‐M (one out of eight) genes. Conclusions These findings represent an important contribution to the implementation of appropriate strategies to prevent foodborne diseases.


Bacteriological analysis
To isolate Salmonella spp., 25.0 g of carcass leg muscle was weighed, by making previously an incision with a sterile scalpel to collect the sample using sterile tweezers. Samples were then placed in a sterile bag and 225 ml of sterile buffered peptone water (OXOID, Waltham, MA, USA) was added, which was further homogenised and incubated at 35 • C for 18 h. A volume of 1 ml of this pre-enrichment phase was transferred to a tube with 10 ml of Rappaport-Vassiliadis broth (OXOID) and incubated at 42 ± 1 • C for 24 h (Donaghy & Madden, 1993 (Carpenter et al., 2018).
For the detection of ESBLs, such as TEM, SHV and CTX-M, the primers described in Table 1  PCR products were visualised by agarose gel electrophoresis (2% w/v) stained with ethidium bromide.

Statistical analysis
The results were analysed as relative frequencies with their respective 95% confidence intervals. Fisher's exact test was applied to determine the significant association between categorical variables.

RESULTS
The frequency of Salmonella spp. isolated using the culture method was eight out of 13 (61.53, CI 95%: 31.57-86.14), regardless of the market.
Salmonella spp. was isolated in the four markets with a higher proportion (three out of four) in the "La Terminal" market, whereas the frequency was two out of three in the Central and "La Estación" markets, respectively. The only sample taken from the "Sutiava" market was positive for Salmonella spp. No significant differences were observed in the isolation of Salmonella between the markets (p ≥ 0.05).
PCR analysis demonstrated that isolates belonging to the genus Salmonella were found in eight out of 13 samples, with S. enterica serovar Enteritidis being found in six out of eight (Table 2). Moreover, the analysis of the resistance patterns showed that eight out of eight Salmonella isolates were resistant to AMC and CL, but sensitive to CIP, TMP/SMX, CN and TET. The bla SHV gene was detected in seven out of eight Salmonella isolates, whereas the bla TEM gene was detected in two out of eight and the bla CXT gene in one out of eight Salmonella isolates.
One isolate from the "La Estación" market was positive for three analysed β-lactamase genes, whereas one isolate from the "Central" market was negative for all analysed β-lactamase genes (Table 2).

DISCUSSION
Salmonella is one of the main bacteria that has been involved in outbreaks due to the consumption of contaminated food. In fact, the Centers for Disease Control and Prevention estimates that causes one million cases of food-related illnesses each year in the USA (Callejón et al., 2015). Moreover, the poor hygiene habits of certain populations may trigger disease episodes (Waldman et al., 2020).
Salmonella spp. was found in 61.53% of the analysed samples, whose data are close to those obtained from previous studies. For instance, it has been reported the presence of Salmonella in 54.1% reptiles (e.g., turtles, lizards and snakes) from Germany and Austria (Geue &