Year

2009

Degree Name

Doctor of Philosophy

Department

School of Biological Sciences - Faculty of Science

Abstract

One hundred and thirty-six Salmonella enterica strains, isolated from humans, animals, environmental and plant sources in Australia from 23 serovars, were examined for the streptomycin resistance gene strA and strB, the sulfonamide resistance gene sul2, and the tetracycline resistance gene tetA(A) and tetA(B). Thirteen strains were identified as containing the strA-strB genes located on the transposon Tn5393. S. enterica serovar Hadar accounted for 11 of these strains, 6 of which were isolated from humans and 5 were from ducks. This investigation is therefore the first report of the Tn5393 transposon being detected in bacterial strains from a human source in Australia.

RSF1010 plasmids were identified and extracted from 4 S. enterica strains, and were further confirmed by restriction enzyme profiling using PstI, SspI and EcoRV. Small non-conjugative plasmid p9123 was extracted and characterised from 3 of the S. enterica strains and also confirmed by restriction enzyme digestion. An RSF1010-like plasmid was also identified in 3 of the strains. This plasmid was found to be approximately 2.6 kb larger than RSF1010, and possibly derived from the RSF1010 plasmid via insertion of the tetracycline resistance gene tetA(A) between strB and mobC genes.

An IS26-strB-strA-sul2-repC-repA-IS26 antibiotic resistance region was identified in 33 S. enterica strains, among these were 23 serovar Typhimurium isolates, 8 serovar Bovismorbificans, 1 serovar Senftenberg and 1 isolate where the serovar could not be conclusively identified. The 23 Typhimurium strains were further characterised by PCR and Southern hybridisation analysis using a blaTEM gene probe. The analysis identified two classes of antibiotic resistance gene clusters. Eleven S. enterica serovar Typhimurium strains harboured an IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 antibiotic resistance gene cluster and another 10 S. enterica serovar Typhimurium strains contained an IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1 gene cluster, without the IS26 element downstream of the blaTEM-1 gene. Two strains contain elements of these gene clusters but further investigation is needed to fully identify these.

Further linkage PCR amplifications revealed that the IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 antibiotic resistance gene cluster was possibly inserted into the 3P-CS of a class 1 integron (In4 type) and truncated the 3P-CS region. Three derivatives were identified, of which the dfrA5-intI1 type was most commonly found downstream of the blaTEM-1-IS26 region. Southern hybridisation analysis using an IS200 gene probe revealed that strains which contain different antibiotic resistance gene clusters also display different but related IS200 profiles.

The antibiotic resistance gene clusters of 19 S. enterica serovar Typhimurium strains were transferred to an E. coli 294 Rifr recipient either by direct mating or triparental mating methods. These experiments confirmed that the antibiotic resistance gene clusters were located on conjugative or mobilisable plasmids. The antibiotic resistance gene clusters of 4 S. enterica serovar Typhimurium strains could not be transferred to the E. coli 294 Rifr recipient. These experimental results suggest that the antibiotic resistance gene cluster of IS26-strB-strA-sul2-repC-repA-IS26-blaTEM-1-IS26 might move as one genetic element between distinct plasmid backbones.

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