Summary Poultry meat has been associated frequently and consistently with the transmission of enteric pathogens, including Salmonella and Campylobacter. This association has resulted in the development of HACCP‐based intervention strategies. These strategies (hurdles) begin with elite breeder flocks and filter down the production pyramid. These hurdles include those already established, such as biosecurity, vaccination, competitive exclusion, pre‐ and probiotics, feed and water control, and those more experimental, such as bacteriophage or immunoglobulin therapy. The reduction in enteropathogens entering the processing plant, which employs critical control points, further reduce the exposure of consumers to these organisms. The synergistic application of hurdles will result in an environment that is restrictive and detrimental to enteropathogen colonization and contamination.

In a structured survey of all major chicken-meat producers in Australia, we investigated the antimicrobial resistance (AMR) and genomic characteristics of Campylobacter jejuni (n = 108) and C. coli (n = 96) from cecal samples of chickens at slaughter (n = 200). The majority of the C. jejuni (63%) and C. coli (86.5%) samples were susceptible to all antimicrobials. Fluoroquinolone resistance was detected among both C. jejuni (14.8%) and C. coli (5.2%), although this only included three sequence types (STs) and one ST, respectively. Multidrug resistance among strains of C. jejuni (0.9%) and C. coli (4.1%) was rare, and fluoroquinolone resistance, when present, was never accompanied by resistance to any other agent. Comparative genome analysis demonstrated that Australian isolates were found dispersed on different branches/clusters within the international collection. The major fluoroquinolone-resistant STs of C. jejuni (ST7323, ST2083, and ST2343) and C. coli (ST860) present in Australian chickens were similar to those of international isolates and have been reported previously in humans and animals overseas. The detection of a subpopulation of Campylobacter isolates exclusively resistant to fluoroquinolone was unexpected since most critically important antimicrobials such as fluoroquinolones are excluded from use in Australian livestock. A number of factors, including the low level of resistance to other antimicrobials, the absence of fluoroquinolone use, the adoption of measures for preventing spread of contagion between flocks, and particularly the genomic identities of isolates, all point to humans, pest species, or wild birds as being the most plausible source of organisms. This study also demonstrates the need for vigilance in the form of surveillance for AMR based on robust sampling to manage AMR risks in the food chain. IMPORTANCE Campylobacter is one of the most common causes of gastroenteritis in humans, with infections frequently resulting from exposure to undercooked poultry products. Although human illness is typically self-limiting, a minority of cases do require antimicrobial therapy. Ensuring that Campylobacter originating from meat chickens does not acquire resistance to fluoroquinolones is therefore a valuable outcome for public health. Australia has never legalized the use of fluoroquinolones in commercial chickens and until now fluoroquinolone-resistant Campylobacter has not been detected in the Australian poultry. This structured survey of meat chickens derived from all major Australian producers describes the unexpected emergence of fluoroquinolone resistance in Campylobacter jejuni and C. coli. Genetic characterization suggests that these isolates may have evolved outside the Australian poultry sector and were introduced into poultry by humans, pest species, or wild birds. The findings dramatically underline the critical role of biosecurity in the overall fight against antimicrobial resistance.

Aims:  To validate the effectiveness of a miniaturized most probable number method (mMPN) in enumerating Salmonella from poultry matrices. Methods and Results:  A MPN was developed, based on the ISO 6579:2002 method using modified semi‐solid Rappaport–Vassiliadis media as the sole selective medium. The validation of the mMPN was shown to not differ significantly from, at the 95% confidence level (Student’s t‐test P = 0·357) to, the traditional 9‐tube MPN (tMPN) using pure cultures of Salmonella ser. Typhimurium, Infantis, Montevideo, Muenster and Salmonella subsp II 1,4,12,27:b:[e,n,x] (Sofia). The validation of naturally and artificially contaminated poultry matrices (carcasses, scald tank water, faeces, caeca and feed) showed that detection using the mMPN compared well to the ISO 6572:2002; sensitivity (92%), specificity (97%) and agreement (KAPPA 0·72). The quantitative comparison between the tMPN and mMPN methods showed that 92% of enumerations were less than ±1 log different (Student’s t‐test = 0·13). Financial analysis showed that the mMPN required 64% less media and 56% less labour than the tMPN. Conclusion:  The mMPN is a consistent, easy to automate method for the enumeration of Salmonella from different poultry matrices. Significance and Impact of Study:  The miniaturized MPN reduces the material and labour cost of the method and enables the uniform and accurate measurement of the effectiveness of intervention strategies in the control of Salmonella colonization of poultry.

The World Health Organisation has defined “highest priority critically important antimicrobials” (CIAs) as those requiring the greatest control during food production. Evidence demonstrating that restricted antimicrobial usage prevents the emergence of resistance to CIA’s amongst pathogenic and commensal organisms on a production system-wide scale would strengthen international efforts to control antimicrobial resistance (AMR). Therefore, in a designed survey of all major chicken-meat producers in Australia, we investigated the phenotypic AMR of E. coli (n = 206) and Salmonella (n = 53) from caecal samples of chickens at slaughter (n = 200). A large proportion of E. coli isolates (63.1%) were susceptible to all tested antimicrobials. With regards to CIA resistance, only two E.coli isolates demonstrated resistance to fluoroquinolones, attributed to mutations in the quinolone resistance-determining regions of gyrA. Antimicrobial resistance was observed for trimethoprim/sulfamethoxazole (8.7%), streptomycin (9.7%), ampicillin (14.1%), tetracycline (19.4%) and cefoxitin (0.5%). All Salmonella isolates were susceptible to ceftiofur, chloramphenicol, ciprofloxacin, colistin, florfenicol, gentamicin and tetracycline. A low frequency of Salmonella isolates exhibited resistance to streptomycin (1.9%), ampicillin (3.8%), and cefoxitin (11.3%). AMR was only observed among Salmonella Sofia serovars. None of the Salmonella isolates exhibited a multi-class-resistant phenotype. Whole genome sequencing did not identify any known resistance mechanisms for the Salmonella isolates demonstrating resistance to cefoxitin. The results provide strong evidence that resistance to highest priority CIA’s is absent in commensal E. coli and Salmonella isolated from Australian meat chickens, and demonstrates low levels of resistance to compounds with less critical ratings such as cefoxitin, trimethoprim/sulfamethoxazole, and tetracycline. Apart from regulated exclusion of CIAs from most aspects of livestock production, vaccination against key bacterial pathogens and stringent biosecurity are likely to have contributed to the favorable AMR status of the Australian chicken meat industry. Nevertheless, industry and government need to proactively monitor AMR and antimicrobial stewardship practices to ensure the long-term protection of both animal and human health.

ObjectiveIn Australia, Salmonella serovar Typhimurium (S. Typhimurium) is the predominant zoonotic serovar in humans and is frequently isolated from layer hens. Vaccination against this serovar has been previously shown to be effective in broilers and the aim of this current study was to assess and determine the best vaccination strategy (live or inactivated) to minimise caecal colonisation by S. Typhimurium.MethodsA long‐term experiment (56 weeks) was conducted on ISABROWN pullets using a commercial live aroA deleted mutant S. Typhimurium vaccine and an autogenous inactivated multivalent Salmonella vaccine (containing serovars Typhimurium, Infantis, Montevideo and Zanzibar). These vaccines were administered PO or by SC or IM injection, either alone or in combination. Pullets were vaccinated throughout rearing (to 18 weeks of age) and sequentially bled for antibody titre levels. The birds, vaccinated and controls, were challenged orally with a field isolate of S. Typhimurium at different ages, held for 21 days post‐challenge, then euthanased and their caeca cultured for the presence of Salmonella. ResultsNone of the oral live‐vaccinated groups exhibited lasting protection. When administered twice, the inactivated vaccine gave significant protection at 17 weeks of age and the live vaccine given by SC injection given twice produced significant protection at 17, 25 and 34 weeks.ConclusionsVaccination regimens that included parenteral administration of live or inactivated vaccines and thus achieved positive serum antibody levels were able to provide protection against challenge. Hence, vaccination may play a useful role in a management strategy for Salmonella carriage in layer flocks.

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Due to Australia’s management of antimicrobial use in poultry, particularly the discontinued use of avoparcin for nearly 20 years, it is hypothesized that vancomycin-resistant enterococci associated with human disease are not derived from poultry isolates. This study evaluated antimicrobial resistance (AMR) of five enterococcal species isolated from Australian meat chickens, genomic features of Enterococcus faecium and Enterococcus faecalis, and the phylogenetic relationship of the poultry-derived E. faecium with isolates from human sepsis cases. All enterococcal isolates from chicken ceca were subjected to antimicrobial susceptibility testing. E. faecium and E. faecalis underwent whole-genome sequencing. E. faecium was compared at the core genome level to a collection of human isolates (n = 677) obtained from cases of sepsis over a 2-year period spanning 2015 to 2016. Overall, 205 enterococci were isolated consisting of five different species. E. faecium was the most frequently isolated species (37.6%), followed by E. durans (29.7%), E. faecalis (20%), E. hirae (12.2%), and E. gallinarum (0.5%). All isolates were susceptible to vancomycin and gentamicin, while one isolate was linezolid resistant (MIC 16 mg/liter). Core genome analysis of the E. faecium demonstrated two clades consisting predominantly of human or chicken isolates in each clade, with minimal overlap. Principal component analysis for total gene content revealed three clusters comprised of vanA-positive, vanB-positive, and both vanA- and vanB-negative E. faecium populations. The results of this study provide strong evidence that Australian chicken E. faecium isolates are unlikely to be precursor strains to the currently circulating vancomycin-resistant strains being isolated in Australian hospitals.

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