Integron-associated antimicrobial resistance in Australian beef cattle

A consequence of antimicrobial use in food production systems is the potential for antimicrobial resistant (AMR) bacteria to develop and transfer to the human population via the food chain. Integrons have been identified as critical factors in the development of AMR. Despite Australia being amongst the world’s largest exporters of beef, there is a general lack of data on the prevalence of AMR in bacteria from Australian beef cattle. Consequently, the aim of this study has been to contribute to research strategies and knowledge of AMR by investigating integron-associated antimicrobial resistance in Australian beef cattle production systems. This study developed a protocol that targets resistance integrons. The protocol was trialled on 50 bovine faecal samples with a total of 39 integron-containing isolates recovered. Characterisation of the integrons was performed and it was determined that the majority of integrons harboured genes encoding resistance to trimethoprim (dfr) and streptomycin / spectinomycin (aad). The protocol provides an opportunity to rapidly interrogate populations of bacteria within a defined sample for resistance integrons. The protocol was used to investigate integron-associated AMR in Australian beef cattle production systems. Each production system was investigated for resistance integrons to determine if production practices were impacting on the prevalence and types of AMR present. The investigation found that the prevalence of integron-containing bacteria was higher in lot-fed cattle than grass-fed cattle which in turn were higher than organically produced cattle. However, the types of AMR differed very little across production systems and suggested that the higher prevalence of integrons in lot-fed samples may be a function of the intensive nature of this type of production system rather than a result of selective pressure caused by antibiotic use. Although there appeared to be no obvious trends in the types of gene cassettes carried by integrons from differing production systems, if lot-fed cattle continually arrive at slaughter with a higher prevalence of integron-containing bacteria then these cattle may be more likely to contribute to contamination of the final product. Samples from lot-fed, grass-fed and organically produced cattle at slaughter were collected. Despite the apparent unrelatedness of the cattle herds, there was little difference in the PCR prevalence of class 1 and class 2 integrase, the gene cassettes harboured by the integrons, and the host organism for the integron. Genes encoding resistance to streptomycin and chloramphenicol (catB8) dominated the majority of arrays regardless of production system, although two novel arrays were identified. One of the arrays, cmlA5-blaoxa-10-aadA1, was found in A. veronii biovar Sobria isolates from organic cattle thereby confirming the ability of multi-resistant integrons to persist in environments that have no obvious antimicrobial selection. The abattoir study revealed an unusually high prevalence of Aeromonas isolates carrying integrons. It appeared to implicate the abattoir environment as a direct contributor to the presence of integron-containing bacteria in each herd. Characterisation of each Aeromonas isolate determined that the isolates were not clonal in nature and not a result of persistent contamination at the abattoir. It seemed more probable that the Aeromonas isolates were present in the cattle prior to arrival and may have been acquired from the environment. To explore this further, soil samples from cattle associated and non-cattle associated areas were tested for the presence of resistance integrons. The prevalence of class 1 integrons was higher in cattle-associated soil samples than in non-cattle-associated soil samples, however the diversity of gene cassettes harboured by the integrons was greater in non-cattle-associated samples than cattle-associated samples. An array harbouring blaoxa-30 was isolated from a non-cattle-associated soil sample. Its presence continues to highlight the potential for multi-resistant integrons to exist in environments with no obvious antimicrobial selection pressure.The detection of seldom reported class 1 integron arrays in this study indicates the potential of the developed protocol to interrogate populations of bacteria for resistance integrons. This is highlighted further by the isolation of a novel class 2 integron. This novel class 2 integron from Providencia stuartii possesses a class 2 integrase that is predicted to be fully functional and has a variable region comprising nine ORFs that do not encode AMR genes. Overall, this study demonstrated that integrons are present in all cattle production systems employed in Australia and although the prevalence of integrons appeared to align with the anticipated use of antimicrobials in each system, differences in the integrons from each production system were not evident. As the similarities observed between integrons extend to isolates from organically produced cattle and from non-cattle associated soil samples it is suggested that the majority of integrons identified in this study are not present as a direct result of antimicrobial use in cattle production. Nevertheless, the potential of integrons to capture AMR genes remains and their presence in beef cattle highlights the need for the continued prudent use of antimicrobials