Detailed molecular analysis of antibiotic resistance regions within a collection of multi-drug resistant Salmonella spp. from Australian sources

Salmonella spp., in particular Salmonella Typhimurium is an important zoonotic pathogen both here in Australia and internationally. Over the past few decades the use of antimicrobials in human, agricultural and aquacultural settings has created significant selection pressures, giving rise to multiply antibiotic resistant bacteria, including Salmonella. The acquisition and dissemination of the genes responsible for antimicrobial resistance has been largely attributed to mobile genetic elements, including Class 1 integrons and the gene cassettes they contain. The initial aim of this study was to examine a collection of 136 multiply resistant Salmonella of different serovars from varying Australian sources (predominately animal and to a lesser extent human) for the presence of Class 1 integrons and to identify the gene cassettes present. Using PCR to amplify up specific regions within the Class 1 integron structure, 51.4% of the isolates examined were found to contain the Class 1 integron associated intI1 integrase. All of these, apart from 11 isolates, contained cassette arrays which were characterised using restriction enzyme analysis and DNA sequencing. The gene cassettes identified among the collection were almost solely responsible for resistance to trimethoprim and aminoglycosides. The dfrA5 gene cassette (responsible for resistance to trimethoprim) was the most prevalent cassette, particularly among the bovine isolates. Three new gene cassettes responsible for resistance to aminoglycosides, trimethoprim and lincosamides (aacCA5, dfrB6 and linG, respectively) were identified. SGI1 (Salmonella Genomic Island 1), a 43 kb chromosomal island known to contain a large multi-drug resistance integron, In104 was found to be present in 10 experimental isolates and associated with 4 new serovars, namely Kiambu, Dusseldorf, Cerro and Emek. The integron in the Salmonella Emek strain was found to have inserted via transposition at a unique site within the island backbone and this structure has been named SGI2. This unique insertion site suggests that SGI2 has evolved independently of SGI1. A second collection of multi-drug resistant almonella Paratyphi BdT+ isolates sourced from human infections in Melbourne and the corresponding home aquaria of infected patients were examined for clonality and the presence of SGI1. All S. Paratyphi BdT+ from infected individuals were indistinguishable from the isolates from their respective fish tanks, using IS200 profiling techniques and pulse field gel electrophoresis of XbaI digested chromosomal DNA. SGI1 (containing the aadA2 and blaP1 gene cassettes) was found to be present in all the S. Paratyphi BdT+ isolates examined. This is the first definitive molecular study showing that ornamental fish tanks are a reservoir for multiply resistant Salmonella aratyphi BdT+. Studies examining the molecular mechanisms involved in antimicrobial resistance, and the way in which mobile elements are incorporated and clustered into large multi-drug resistance regions such as SGI1, provide useful information needed for the ongoing surveillance of multiply resistant Salmonella and other bacterial pathogens involved in outbreaks domestically and internationally.