Recessive mutations in the Mlo gene confer broad spectrum resistance in barley (Hordeum vulgare) to powdery mildew (Blumeria graminis f. sp. hordei), a widespread and damaging disease. However, all alleles discovered to date also display deleterious pleiotropic effects, including the naturally occurring mlo-11 mutant which is widely deployed in Europe. Recessive resistance was discovered in Eth295, an Ethiopian landrace, which was developmentally controlled and quantitative without spontaneous cell wall appositions or extensive necrosis and loss of photosynthetic tissue. This resistance is determined by two copies of the mlo-11 repeat units, that occur upstream to the wild-type Mlo gene, compared to 11–12 in commonly grown cultivars and was designated mlo-11 (cnv2). mlo-11 repeat unit copy number-dependent DNA methylation corresponded with cytological and macroscopic phenotypic differences between copy number variants. Sequence data indicated mlo-11 (cnv2) formed via recombination between progenitor mlo-11 repeat units and the 3′ end of an adjacent stowaway MITE containing region. mlo-11 (cnv2) is the only example of a moderated mlo variant discovered to date and may have arisen by natural selection against the deleterious effects of the progenitor mlo-11 repeat unit configuration.

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.

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.

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 recognised standards (CLSI and ISO 20776-1:2019) and validated through a series of experiments. Methods: Experiment A compared RASPs ability to achieve consistent MICs to that of a human technician across eight replicates for four E. coli isolates. Experiment B assessed RASPs 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. RASPs 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 to an experienced human technician. Integration of robotics platforms in the microbiology laboratory is a necessary advancement for future One-Health AMR endeavours.

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.