Augmented surveillance of antimicrobial resistance with high-throughput robotics detects transnational flow of fluoroquinolone-resistant Escherichia coli strain into poultry.

BACKGROUND: Food animal AMR surveillance programs assess only small numbers of Escherichia coli (from 100 to 600 per animal class) nationally each year, severely limiting the evaluation of public health risk(s). Here we demonstrate an affordable approach for early detection of emerging resistance on a broad scale that can also accurately characterize spatial and temporal changes in resistance. METHODS: Caecal samples (n = 295) obtained from 10 meat poultry were screened using high-throughput robotics. Initial screening via agar dilution (5310 plates) quantified AMR carriage (cfu/g) for each sample. Ciprofloxacin-resistant isolates (n = 91) proceeded to downstream broth microdilution susceptibility testing. A subset of 28 ciprofloxacin-resistant isolates underwent WGS and phylogenetic analysis. RESULTS: Intra- and inter-flock carriage of resistance varied with drug class. Ampicillin and tetracycline resistance was ubiquitous to most birds in all flocks with an average carriage rate of 5.8 log10 cfu/g. Gentamicin and ciprofloxacin-resistant E. coli colonized fewer birds, and had an average carriage rate of 1.2 log10 cfu/g and 1.0 log10 cfu/g of faeces, respectively. Resistance to extended-spectrum cephalosporins was absent. ST354 was the dominant ST among the WGS isolates, but they demonstrated markedly lower resistance gene carriage than their international counterparts. CONCLUSIONS: These data amply demonstrate the ineffectiveness of commonly relied-on approaches to AMR surveillance for achieving early detection of emergence, or for measuring spatial and temporal resistance trends. Genetic analysis suggested there has been transnational flow of a ciprofloxacin-resistant strain into Australian poultry flocks, explaining their detection in a nation that prohibits fluoroquinolone use in poultry.

Genomic profiling of Pasteurella multocida isolated from feedlot cases of bovine respiratory disease.

Pasteurella multocida causes a range of diseases in many host species throughout the world, including bovine respiratory disease (BRD) which is predominantly seen in feedlot cattle. This study assessed genetic diversity among 139 P. multocida isolates obtained from post-mortem lung swabs of BRD-affected feedlot cattle in four Australian states: New South Wales, Queensland, South Australia, and Victoria during 2014-2019. Whole-genome sequencing (WGS) was used to determine capsular serogroup, lipopolysaccharide genotypes, multi-locus sequence types and phylogenetic relationships. Two capsular types (A and D), with most isolates (132/139; 95%) belonging to type A; and three lipopolysaccharide (LPS) genotypes were identified (L1 [6/139; 4.3%], L3 [124/139; 89.2%] and L6 [9/139; 6.4%)]). Multi-locus sequence types (STs) ST9, ST13, ST17, ST20, ST36, ST50, ST58, ST79, ST124, ST125, ST132, ST167, ST185, ST327, ST394, and three novel STs [ST396, ST397, and ST398] were identified, with ST394 (59/139; 42.4%) and ST79 (44/139; 32%) the most prevalent in all four states. Isolates displaying phenotypic resistance to single, dual or multiple antibiotics (macrolide, tetracycline and aminopenicillins) were predominantly ST394 (23/139; 17%). Laterally mobile elements identified in the resistant ST394 isolates included small plasmids, encoding macrolide and/or tetracycline resistance, distributed in all states; and chromosomally located integrative conjugative elements (ICEs) (4 ST394 and 1 ST125) from the same Queensland feedlot. This study highlights the genomic diversity, epidemiological relationships and AMR associations in bovine P. multocida isolates from Australia and provides insight into the unique ST prevalence compared to other major beef-producing countries.

Antimicrobial Resistance of and Genomic Insights into Pasteurella multocida Strains Isolated from Australian Pigs.

Infection with Pasteurella multocida represents a significant economic threat to Australian pig producers, yet our knowledge of its antimicrobial susceptibilities is lagging, and genomic characterization of P. multocida strains associated with porcine lower respiratory disease is internationally scarce. This study utilized high-throughput robotics to phenotypically and genetically characterize an industry-wide collection of 252 clinical P. multocida isolates that were recovered between 2014 and 2019. Overall, antimicrobial resistance was found to be low, with clinical resistance below 1% for all tested antimicrobials except those from the tetracycline class. Five dominant sequence types, representing 64.8% of all isolates, were identified; they were disseminated across farms and had previously been detected in various animal hosts and countries. P. multocida in Australian farms remain controllable via current antimicrobial therapeutic protocols. The identification of highly dominant, interspecies-infecting strains provides insight into the epidemiology of the opportunistic pathogen, and it highlights a biosecurity threat to the Australian livestock industry. IMPORTANCE Pasteurellosis is rated by the World Animal Health Organisation (OIE) as a high-impact disease in livestock. Although it is well understood in many host-disease contexts, our understanding of the organism in porcine respiratory disease is limited. Given its high frequency of involvement in porcine respiratory disease complex (PRDC), it is important that we are aware of its antimicrobial susceptibilities so that we can respond quickly and appropriately with antimicrobial therapy. Genetic insights about the organism can help us to better understand its epidemiology and inform our biosecurity practices and prophylactic management.

Diversity detected in commensals at host and farm level reveals implications for national antimicrobial resistance surveillance programmes.

BACKGROUND: A key component to control of antimicrobial resistance (AMR) is the surveillance of food animals. Currently, national programmes test only limited isolates per animal species per year, an approach tacitly assuming that heterogeneity of AMR across animal populations is negligible. If the latter assumption is incorrect then the risk to humans from AMR in the food chain is underestimated. OBJECTIVES: To demonstrate the extent of phenotypic and genetic heterogeneity of Escherichia coli in swine to assess the need for improved protocols for AMR surveillance in food animals. METHODS: Eight E. coli isolates were obtained from each of 10 pigs on each of 10 farms. For these 800 isolates, AMR profiles (MIC estimates for six drugs) and PCR-based fingerprinting analysis were performed and used to select a subset (n = 151) for WGS. RESULTS: Heterogeneity in the phenotypic AMR traits of E. coli was observed in 89% of pigs, with 58% of pigs harbouring three or more distinct phenotypes. Similarly, 94% of pigs harboured two or more distinct PCR-fingerprinting profiles. Farm-level heterogeneity was detected, with ciprofloxacin resistance detected in only 60% of pigs from a single farm. Furthermore, 58 STs were identified, with the dominant STs being ST10, ST101, ST542 and ST641. CONCLUSIONS: Phenotypic and genotypic heterogeneity of AMR traits in bacteria from animal populations are real phenomena posing a barrier to correct interpretation of data from AMR surveillance. Evolution towards a more in-depth sampling model is needed to account for heterogeneity and increase the reliability of inferences.

Robotic Antimicrobial Susceptibility Platform (RASP): a next-generation approach to One Health surveillance of antimicrobial resistance.

BACKGROUND: Surveillance of antimicrobial resistance (AMR) is critical to reducing its wide-reaching impact. Its reliance on sample size invites solutions to longstanding constraints regarding scalability. A robotic platform (RASP) was developed for high-throughput AMR surveillance in accordance with internationally recognized standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. METHODS: Experiment A compared RASP's ability to achieve consistent MICs with that of a human technician across eight replicates for four Escherichia coli isolates. Experiment B assessed RASP's agreement with human-performed MICs across 91 E. coli isolates with a diverse range of AMR profiles. Additionally, to demonstrate its real-world applicability, the RASP workflow was then applied to five faecal samples where a minimum of 47 E. coli per animal (239 total) were evaluated using an AMR indexing framework. RESULTS: For each drug-rater-isolate combination in Experiment A, there was a clear consensus of the MIC and deviation from the consensus remained within one doubling dilution (the exception being gentamicin at two dilutions). Experiment B revealed a concordance correlation coefficient of 0.9670 (95% CI: 0.9670-0.9670) between the robot- and human-performed MICs. RASP's application to the five faecal samples highlighted the intra-animal diversity of gut commensal E. coli, identifying between five and nine unique isolate AMR phenotypes per sample. CONCLUSIONS: While adhering to internationally accepted guidelines, RASP was superior in throughput, cost and data resolution when compared with an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One Health AMR endeavours.

Emergence of Fluoroquinolone-Resistant Campylobacter jejuni and Campylobacter coli among Australian Chickens in the Absence of Fluoroquinolone Use.

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.IMPORTANCECampylobacter 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.

Resistance to critically important antimicrobials in Australian silver gulls (Chroicocephalus novaehollandiae) and evidence of anthropogenic origins.

OBJECTIVES: Antimicrobial resistance (AMR) to critically important antimicrobials (CIAs) amongst Gram-negative bacteria can feasibly be transferred amongst wildlife, humans and domestic animals. This study investigated the ecology, epidemiology and origins of CIA-resistant Escherichia coli carried by Australian silver gulls (Chroicocephalus novaehollandiae), a gregarious avian wildlife species that is a common inhabitant of coastal areas with high levels of human contact. METHODS: Sampling locations were widely dispersed around the perimeter of the Australian continent, with sites separated by up to 3500 km. WGS was used to study the diversity and molecular characteristics of resistant isolates to ascertain their epidemiological origin. RESULTS: Investigation of 562 faecal samples revealed widespread occurrence of extended-spectrum cephalosporin-resistant (21.7%) and fluoroquinolone-resistant (23.8%) E. coli. Genome sequencing revealed that CIA-resistant E. coli isolates (n = 284) from gulls predominantly belonged to human-associated extra-intestinal pathogenic E. coli (ExPEC) clones, including ST131 (17%), ST10 (8%), ST1193 (6%), ST69 (5%) and ST38 (4%). Genomic analysis revealed that gulls carry pandemic ExPEC-ST131 clades (O25:H4 H30-R and H30-Rx) and globally emerging fluoroquinolone-resistant ST1193 identified among humans worldwide. Comparative analysis revealed that ST131 and ST1193 isolates from gulls overlapped extensively with human clinical isolates from Australia and overseas. The present study also detected single isolates of carbapenem-resistant E. coli (ST410-blaOXA-48) and colistin-resistant E. coli (ST345-mcr-1). CONCLUSIONS: The carriage of diverse CIA-resistant E. coli clones that strongly resemble pathogenic clones from humans suggests that gulls can act as ecological sponges indiscriminately accumulating and disseminating CIA-resistant bacteria over vast distances.

Examination of Australian Streptococcus suis isolates from clinically affected pigs in a global context and the genomic characterisation of ST1 as a predictor of virulence.

Streptococcus suis is a major zoonotic pathogen that causes severe disease in both humans and pigs. Australia's pig herd has been quarantined for over 30 years, however S. suis remains a significant cause of disease. In this study, we investigated S. suis from 148 cases of clinical disease in pigs from 46 pig herds over a period of seven years, to determine the level of genetic difference from international isolates that may have arisen over the 30 years of separation. Isolates underwent whole genome sequencing, genome analysis and antimicrobial susceptibility testing. Data was compared at the core genome level to clinical isolates from overseas. Results demonstrated five predominant multi-locus sequence types and two major cps gene types (cps2 and 3). At the core genome level Australian isolates clustered predominantly within one large clade consisting of isolates from the UK, Canada and North America. A small proportion of Australian swine isolates (5%) were phylogenetically associated with south-east Asian and UK isolates, many of which were classified as causing systemic disease, and derived from cases of human and swine disease. Based on this dataset we provide a comprehensive outline of the current S. suis clones associated with disease in Australian pigs and their global context, with the main finding being that, despite three decades of separation, Australian S. suis are genomically similar to overseas strains. In addition, we show that ST1 clones carry a constellation of putative virulence genes not present in other Australian STs.