Metodologías, fuentes, cajas de herramientas y trajes del investigador

The propensity of reptiles and amphibians to carry pathogenic bacteria, particularly

Salmonella, is well recognized (Aguirre et al., 2006). Outbreaks associated with pet turtles have

led to regulatory changes affecting the ownership of these animals in the United States (Cohen, 1980; Bosch et al., 2015; FDA, 2017b). While zoonotic transmission from exotic pets has been examined, the role of reptile meat in the food supply has not been studied. Interestingly, relatively little attention has been paid to the role of reptile/amphibian derived products in the food supply to the epidemiology of foodborne illness or the dissemination of resistance. In the United States, there are reports of pathogenic Salmonella Pomona isolated from alligator meat and a case of recurrent Salmonella Arizona infection in a patient who consumed rattlesnake (Cortes et al., 1992; Sakaguchi et al., 2017). Imported reptile and amphibian products have largely escaped study despite the large scale of production; the soft shell turtle production reached 344 800 tonnes in 2014 (FAO, 2014). Interestingly, the role of the international pet trade in the dissemination of resistance was recently highlighted. A mcr-1 producing E. coli was isolated from Asian grass lizards imported into Germany from Vietnam leading the authors to suggest that the international pet trade may be a vehicle for the dissemination of resistant organisms (Unger et al., 2017).

The emergence of resistance to last line of defense drugs mediated by the carbapenemases and mcr family of genes has ignited interest in identifying organisms possessing these genes on food products imported from regions of the world where these resistance mechanisms may be endemic. Since the initial detection of the VIM-2 carbapenemase gene from a Korean squid imported into Canada in 2014, several more studies have detected carbapenemase genes from a variety of Enterobacteriaceae and non-Enterobacteriaceae isolated from imported seafood and vegetables from Southeast Asian countries (Rubin et al., 2014; Morrison and Rubin, 2015; Mangat

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et al., 2016; Janecko et al., 2016; Ceccarelli et al., 2017). Also, a few studies have detected the

mcr-1 gene isolated from E. coli in imported vegetables and chicken from Thailand, Vietnam and

South America (Hasman et al., 2015; Zurfuh et al., 2016). Although the overall prevalence of these resistance genes in imported food remains low, it is worrisome as the consumption of raw or improperly cooked food provide the ideal condition for transmission and spread of resistant bacteria or their respective mobile genetic elements to resident intestinal flora. These studies emphasize the need for further monitoring of antimicrobial resistance and inclusion of imported products into national surveillance programs.

In the current investigation some food safety hazards unique to the products tested compared to foods derived from major agricultural species were identified. We were very surprised to find that one of the soft shell turtles that were included in this investigation was sold un-gutted. The opportunities for cross contamination are substantially greater when whole animals, including intestines with visible fecal material, are dressed and prepared in the kitchen than when other meats are used. It was also observed that the dried products, including the geckos which is where all

Salmonella isolates were recovered from, were not handled in a manner which was consistent with

a high level of meat hygiene. These products were uniformly displayed without packaging in the open air at the retail location and were handled with bare hands.

Reptile and amphibian meat products are not just a source of protein but are also used for traditional medicinal reasons and religious purposes. The meat, fat, skin, blood and bones are used as raw materials in the preparation of powdered medicines, oils, and salves for the treatment and prevention of numerous illnesses, such as sore throat, skin issues, aches, inflammation, burns, asthma, bleeding disorders, rheumatism, cancer and many more (Alves et al., 2008; Vats and Thomas, 2015). Many parts of these animals are used in rituals, magic spells or used as amulets

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for protection (Alves et al., 2009). Public health professionals investigating outbreaks should be cognisant of this when designing questionnaires to capture the different types of products that people may have consumed as they are not necessarily considered meat.

In this investigation we have demonstrated that reptile and amphibian derived products are an underappreciated source of resistance. Future studies should seek to identify at what stage of the production cycle (on farm, at slaughter, during processing/packaging or at the retail level) resistant organisms enter the food products. Although the sample size was small, the Salmonella serotypes and CTX-M variants identified are not inconsistent with the organisms originating in Asia. Of the Salmonella isolated in this study, Urbana and Poona are reptile associated serotypes that have been linked to outbreaks in fresh produce and pet reptiles (Jackson et al., 2013; Walters et al., 2016). Salmonella Weltevreden is the most common serotype associated with imported seafood and aquatic production systems and is a frequent and increasing cause of human infection predominating Southeast Asia (Ponce et al., 2008; Makendi et al., 2016). Of the CTX-M enzymes identified, CTX-M-55, -83 and -27 are found most commonly in Asia especially China, but also in North Africa; while CTX-M-15 is the most prominent variant found worldwide (Zhao and Hu, 2013; Tong et al., 2015).

In this study the use of a taxa independent resistance selective culture media allowed for the identification of clinically relevant resistance in bacterial species which are not targeted by resistance surveillance programs. The breadth of non-Escherichia or Salmonella genera of Enterobacteriaceae possessing ESBLs, and the NDM-1 producing Acinetobacter sp. highlight the gaps of traditional surveillance approached. The presumptive location of these broad spectrum β- lactamases on transmissible plasmids in the context of these findings indicates that consideration

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of the resistome of the whole bacterial community is required for a holistic understanding of the epidemiology of antimicrobial resistance.