Rebecca Abraham

Objectives
Enterococci are opportunistic, sometimes life-threatening pathogens with increasing antimicrobial resistance, particularly among clinical human isolates in Australia. While recent studies have ruled out pigs and chickens as major reservoirs of resistant enterococci, the role of cattle remains unclear. This study examines the antimicrobial resistance (AMR) profiles of enterococci from Australian cattle and explores the phylogenetic relationship of bovine E. faecium with isolates from other livestock and human sepsis cases.

Methods
A total of 1001 bovine faecal samples were tested, yielding E. faecium (n=343), E. faecalis (n=92), and E. hirae (n=284). Minimum inhibitory concentration assays were conducted against 15 antimicrobials. A subset of 67 isolates underwent whole genome sequencing (WGS).

Results
Most isolates were wild-type to all tested antimicrobials. Resistance was most common to erythromycin in E. faecium (18.7%), daptomycin in E. faecalis (12.1%), and tetracycline in E. hirae (13.3%). A single E. faecalis isolate was non-wild-type to vancomycin, and nine isolates (E. faecium n=4, E. faecalis n=2, E. hirae n=3) showed linezolid resistance. However, WGS did not detect known resistance genes or mutations for vancomycin or linezolid. Phylogenetic analysis revealed that bovine E. faecium clustered with other livestock isolates and vancomycin-negative human isolates.

Conclusion
Antimicrobial resistance among enterococci from Australian cattle is low. These strains are genetically distinct from vancomycin-resistant E. faecium circulating in hospitals, suggesting that cattle are not a significant source of clinically relevant AMR enterococci in Australia.

The increasing resistance of bacteria to antimicrobials is a major threat to public health. This study investigates the prevalence of antimicrobial resistance, both phenotypic and genotypic, among Campylobacter isolates from Australian meat chickens in 2022, as a follow up to investigate trends since the last national surveillance undertaken in 2016. Isolates (n = 186) were obtained at slaughter from 200 pooled cecal samples taken from 1,000 meat chickens. The majority of C. jejuni (68.7%) and C. coli (88.9%) isolates were susceptible to all the antibiotics that were tested, and no multi-drug resistance was found. Resistance to ciprofloxacin (fluoroquinolone) was detected in 24.4% of the C. jejuni and 3.2% of the C. coli isolates. Whole genome sequencing revealed a diverse range of sequence types (STs). These included 32 previously reported STs for C. jejuni and 13 for C. coli, as well as four and seven previously undescribed STs for each species, respectively. The STs containing fluoroquinolone-resistant isolates were ST2083, ST10130, ST2895, ST7323, ST2398, and ST1078 for C. jejuni, and ST860 and ST894 for C. coli. Although fluoroquinolones are not used in animal production in Australia, resistance amongst C. jejuni isolates was high (24.4%). This finding emphasizes the need for enhanced surveillance and regular sampling along the food chain to understand the source of the isolates and to mitigate risks of antimicrobial resistance to protect public health.

There is growing recognition of the significant role food production animals play in the dissemination of antimicrobial-resistant bacteria. This issue is particularly relevant in the realm of food production, where the administration of antibiotics of importance to human health to animals can foster the emergence of resistant bacterial strains that may be transmitted to humans through the food supply chain. Further, resistance can develop to antibiotics of importance for animal health which can create health and productivity issues. Cross-sectional surveys of Australian food animal production systems have revealed that very low levels of antimicrobial resistance (AMR) are present in key bacterial species. Expansion of AMR surveillance in food production systems is, however, needed to improve the detection sensitivity of emerging resistances of importance to human and animal health. Scalable high throughput technologies, such as the robotic antimicrobial susceptibility platform (RASP), represents a step-fold change in the capacity to accurately detect AMR. Enhanced surveillance along with improved biosecurity, industry engagement and antimicrobial stewardship will contribute to maintaining low levels of AMR in Australia’s food production systems.

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.

Human population and activities play an important role in dissemination of antimicrobial resistant bacteria. This study investigated the relationship between carriage rates of critically important antimicrobial-resistant (CIA-R) Escherichia coli and Klebsiella pneumoniae by Silver Gulls and their proximity to human populations. Faecal swabs (n = 229) were collected from Silver Gulls across 10 southern coastline locations in Western Australia (WA) traversing 650 kms. The sampling locations included main town centres and remote areas. Fluoroquinolone and extended-spectrum cephalosporin-resistant E. coli and K. pneumoniae were isolated and tested for antimicrobial sensitivity. Genome sequencing was performed on n = 40 subset out of 98 E. coli and n = 14 subset out of 27 K. pneumoniae isolates to validate phenotypic resistance profiles and determine the molecular characteristics of strains. CIA-R E. coli and K. pneumoniae were detected in 69 (30.1 %) and 20 (8.73 %) of the faecal swabs respectively. Two large urban locations tested positive for CIA-R E. coli (frequency ranging from 34.3 % to 84.3 %), and/or for CIA-R K. pneumoniae (frequency ranging from 12.5 % to 50.0 %). A small number of CIA-R E. coli (3/31, 9.7 %) were identified at a small tourist town, but no CIA-R bacteria were recovered from gulls at remote sites. Commonly detected E. coli sequence types (STs) included ST131 (12.5 %) and ST1193 (10.0 %). Five K. pneumoniae STs were detected which included ST4568, ST6, ST485, ST967 and ST307. Resistance genes including blaCTX-M-3, blaCTX-M-15 and blaCTX-M-27 were identified in both bacterial species. High-level colonisation of CIA-R E. coli and K. pneumoniae in Silver Gulls in and around urban areas compared to remote locations substantiates that anthropogenic activities are strongly associated with acquisition of resistant bacteria by gulls.

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.

Giardia duodenalis is the causative agent of the neglected diarrhoeal disease giardiasis. While often self-limiting, giardiasis is ubiquitous and impacts hundreds of millions of people annually. It is also a common gastro-intestinal disease of domestic pets, wildlife, and livestock animals. However, despite this impact, there is no vaccine for Giardia currently available. In addition, treatment relies on chemotherapies that are associated with increasing failure rates. To identify new treatment options for giardiasis we recently screened the Compounds Australia Scaffold Library for new chemotypes with selective anti-Giardia activity, identifying three compounds with sub-μM activity and promising selectivity. Here we extended these studies by examining the anti-Giardia activity of series CL9569 compounds. This compound series was of interest given the promising activity (IC50 1.2 μM) and selectivity demonstrated by representative compound, SN00798525 (1). Data from this work has identified an additional three thieno [3,2-b]pyrrole 5-carboxamides with anti-Giardia activity, including 2 which displayed potent cytocidal (IC50 ≤ 10 nM) and selective activity against multiple Giardia strains, including representatives from both human-infecting assemblages and metronidazole resistant parasites. Preclinical studies in mice also demonstrated that 2 is well-tolerated, does not impact the normal gut microbiota and can reduce Giardia parasite burden in these animals.

Controlling the use of the most critically important antimicrobials (CIAs) in food animals has been identified as one of the key measures required to curb the transmission of antimicrobial resistant bacteria from animals to humans. Expanding the evidence demonstrating the effectiveness of restricting CIA usage for preventing the emergence of resistance to key drugs amongst commensal organisms in animal production would do much to strengthen international efforts to control antimicrobial resistance (AMR). As Australia has strict controls on antimicrobial use in layer hens, and internationally comparatively low levels of poultry disease due to strict national biosecurity measures, we investigated whether these circumstances have resulted in curtailing development of critical forms of AMR. The work comprised a cross-sectional national survey of 62 commercial layer farms with each assessed for AMR in Escherichia coli isolates recovered from faeces. Minimum inhibitory concentration analysis using a panel of 13 antimicrobials was performed on 296 isolates, with those exhibiting phenotypic resistance to fluoroquinolones (a CIA) or multi-class drug resistance (MCR) subjected to whole genome sequencing. Overall, 53.0% of isolates were susceptible to all antimicrobials tested, and all isolates were susceptible to cefoxitin, ceftiofur, ceftriaxone, chloramphenicol and colistin. Resistance was observed for amoxicillin-clavulanate (9.1%), ampicillin (16.2%), ciprofloxacin (2.7%), florfenicol (2.4%), gentamicin (1.0%), streptomycin (4.7%), tetracycline (37.8%) and trimethoprim/sulfamethoxazole (9.5%). MCR was observed in 21 isolates (7.0%), with two isolates exhibiting resistance to four antimicrobial classes. Whole genome sequencing revealed that ciprofloxacin-resistant (fluoroquinolone) isolates were devoid of both known chromosomal mutations in the quinolone resistance determinant regions and plasmid-mediated quinolone resistance genes (qnr)—other than in one isolate (ST155) which carried the qnrS gene. Two MCR E. coli isolates with ciprofloxacin-resistance were found to be carrying known resistance genes including aadA1, dfrA1, strA, strB, sul1, sul2, tet(A), blaTEM-1B, qnrS1 and tet(A). Overall, this study found that E. coli from layer hens in Australia have low rates of AMR, likely due to strict control on antimicrobial usage achieved by the sum of regulation and voluntary measures.

Extended-spectrum cephalosporin-resistant (ESC-R) Escherichia coli have disseminated in food-producing animals globally, attributed to horizontal transmission of blaCTX-M variants, as seen in the InCI1-blaCTX-M-1 plasmid. This ease of transmission, coupled with its demonstrated long-term persistence, presents a significant One Health antimicrobial resistance (AMR) risk. Bacteriophage (phage) therapy is a potential strategy in eliminating ESC-R E. coli in food-producing animals; however, it is hindered by the development of phage-resistant bacteria and phage biosafety concerns. Another alternative to antimicrobials is probiotics, with this study demonstrating that AMR-free commensal E. coli, termed competitive exclusion clones (CECs), can be used to competitively exclude ESC-R E. coli. This study isolated and characterized phages that lysed E. coli clones harboring the InCI1-blaCTX-M-1 plasmid, before investigation of the effect and synergy of phage therapy and competitive exclusion as a novel strategy for decolonizing ESC-resistant E. coli. In vitro testing demonstrated superiority in the combined therapy, reducing and possibly eliminating ESC-R E. coli through phage-mediated lysis coupled with simultaneous prevention of regrowth of phage-resistant mutants due to competitive exclusion with the CEC. Further investigation into this combined therapy in vivo is warranted, with on-farm application possibly reducing ESC-R prevalence, while constricting newly emergent ESC-R E. coli outbreaks prior to their dissemination throughout food-producing animals or humans.

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.