Antimicrobial stewardship in Australia: the role of qualitative research in programme development.

Antimicrobial stewardship (AMS) in Australia is supported by a number of factors, including enabling national policies, sectoral clinical governance frameworks and surveillance programmes, clinician-led educational initiatives and health services research. A One Health research programme undertaken by the National Centre for Antimicrobial Stewardship (NCAS) in Australia has combined antimicrobial prescribing surveillance with qualitative research focused on developing antimicrobial use-related situational analyses and scoping AMS implementation options across healthcare settings, including metropolitan hospitals, regional and rural hospitals, aged care homes, general practice clinics and companion animal and agricultural veterinary practices. Qualitative research involving clinicians across these diverse settings in Australia has contributed to improved understanding of contextual factors that influence antimicrobial prescribing, and barriers and facilitators of AMS implementation. This body of research has been underpinned by a commitment to supplementing 'big data' on antimicrobial prescribing practices, where available, with knowledge of the sociocultural, technical, environmental and other factors that shape prescribing behaviours. NCAS provided a unique opportunity for exchange and cross-pollination across the human and animal health programme domains. It has facilitated synergistic approaches to AMS research and education, and implementation of resources and stewardship activities. The NCAS programme aimed to synergistically combine quantitative and qualitative approaches to AMS research. In this article, we describe the qualitative findings of the first 5 years.

Water Distribution Systems in Pig Farm Buildings: Critical Elements of Design and Management.

Drinking water distribution systems (WDSs) within buildings on pig farms have critical elements of their design and management that impact water provision to pigs, water quality, the efficacy of in-water antimicrobial dosing, and, thus, pig health and performance. We used a mixed-methods approach to survey managers of 25 medium to large single-site and multi-site pig farming enterprises across eastern and southern Australia. We found wide variation in the configuration (looped or branched) and total length of WDSs within buildings across farms and in pipe materials and diameters. Within many conventional buildings and some eco-shelters, WDSs were 'over-sized', comprising large-diameter main pipelines with high holding volumes, resulting in slow velocity water flows through sections of a WDS's main pipeline. In over half of the weaner buildings and one-third of grower/finisher buildings, the number of pigs per drinker exceeded the recommended maximum. Few farms measured flow rates from drinkers quantitatively. WDS sanitization was not practiced on many farms, and few managers were aware of the risks to water quality and pig health. We identified important aspects of water provision to pigs for which valuable recommendations could be added to industry guidelines available to pig farm managers.

Intraspecies Variation in Tetrahymena rostrata.

Two distinct isolates of the facultative parasite, Tetrahymena rostrata were compared, identifying and utilising markers that are useful for studying clonal variation within the species were identified and utilised. The sequences of mitochondrial genomes and several nuclear genes were determined using Illumina short read sequencing. The two T. rostrata isolates had similar morphology. The linear mitogenomes had the gene content and organisation typical of the Tetrahymena genus, comprising 8 tRNA genes, 6 ribosomal RNA genes and 45 protein coding sequences (CDS), twenty-two of which had known function. The two isolates had nucleotide identity within common nuclear markers encoded within the histone H3 and H4 and small subunit ribosomal RNA genes and differed by only 2-4 nucleotides in a region of the characterised actin genes. Variation was observed in several mitochondrial genes and was used to determine intraspecies variation and may reflect the natural history of T. rostrata from different hosts or the geographic origins of the isolates.

Infection of Slugs with Theronts of the Ciliate Protozoan, Tetrahymena rostrata.

Tetrahymena rostrata is a free-living ciliated protozoan and is a facultative parasite of some species of terrestrial mollusks. It is a potential biopesticide of pest slugs, such as the grey field slug, which cause considerable damage to crops. T. rostrata has several developmental forms. Homogeneous preparations of the feeding stage cells (trophonts) and excysted stage cells (theronts) were compared for their ability to infect and kill Deroceras reticulatum slugs. Theronts were more effective and remained viable and infective, even after prolonged starvation.

Effect of Drinking Water Distribution System Design on Antimicrobial Delivery to Pigs.

On many pig farms, growing pigs are mass-medicated for short periods with antimicrobial drugs through their drinking water for metaphylaxis and to treat clinical disease. We conducted a series of four prospective observational cohort studies of routine metaphylactic in-water antibiotic dosing events on a commercial pig farm, to assess the concentration of antimicrobial available to pigs throughout a building over time. Each dosing event was conducted by the farm manager with a differently designed looped water distribution system (WDS). We found that the antimicrobial concentration in water delivered to pigs at drinkers in each pen by a building's WDS over time was profoundly influenced by the design of the WDS and the pigs' water usage and drinking pattern, and that differences in the antimicrobial concentration in water over time at drinkers throughout a building could be eliminated through use of a circulator pump in a looped WDS. We also used a hydraulic WDS modelling tool to predict the antimicrobial concentration at drinkers over time during and after a dosing event. Our approach could be used to evaluate alternative in-water dosing regimens for pigs in a specific building in terms of their clinical efficacy and ability to suppress the emergence of antimicrobial resistance, and to determine the optimal regimen. The approach is applicable to all additives administered through drinking water for which the degree of efficacy is dependent on the dose administered.

In-Water Antibiotic Dosing Practices on Pig Farms.

Pigs reared on many farms are mass-medicated for short periods with antibiotics through their drinking water to control bacterial pathogen loads and, if a disease outbreak occurs, to treat pigs until clinical signs are eliminated. Farm managers are responsible for conducting in-water antibiotic dosing events, but little is known about their dosing practices. We surveyed managers of 25 medium to large single-site and multi-site pig farming enterprises across eastern and southern Australia, using a mixed methods approach (online questionnaire followed by a one-on-one semi-structured interview). We found wide variation in the antibiotics administered, the choice and use of dosing equipment, the methods for performing dosing calculations and preparing antibiotic stock solutions, the commencement time and duration of each daily dosing event, and the frequency of administration of metaphylaxis. Farm managers lacked data on pigs' daily water usage patterns and wastage and the understanding of pharmacology and population pharmacometrics necessary to optimize in-water dosing calculations and regimens and control major sources of between-animal variability in systemic exposure of pigs to antibiotics. There is considerable scope to increase the effectiveness of in-water dosing and reduce antibiotic use (and cost) on pig farms by providing farm managers with measurement systems, technical guidelines, and training programs.

Faecal microbiota and antimicrobial resistance gene profiles of healthy foals.

BACKGROUND: The human and domestic animal faecal microbiota can carry various antimicrobial resistance genes (ARGs), especially if they have been exposed to antimicrobials. However, little is known about the ARG profile of the faecal microbiota of healthy foals. A high-throughput qPCR array was used to detect ARGs in the faecal microbiota of healthy foals. OBJECTIVES: To characterise the faecal microbiota and ARG profiles in healthy Australian foals aged less than 1 month. STUDY DESIGN: Observational study. METHODS: The faecal microbiota and ARG profiles of 37 Thoroughbred foals with no known gastrointestinal disease or antimicrobial treatment were determined using 16S rRNA gene sequencing and a high-throughput ARG qPCR array. Each foal was sampled on one occasion. RESULTS: Firmicutes and Bacteroidetes were dominant in the faecal microbiota. Foals aged 1-2 weeks had significantly lower microbiota richness than older foals. Tetracycline resistance genes were the most common ARGs in the majority of foals, regardless of age. ARGs of high clinical concern were rarely detected in the faeces. The presence of ARGs was associated with the presence of class I integron genes. MAIN LIMITATIONS: Samples were collected for a case-control study so foals were not sampled longitudinally, and thus the development of the microbiota as individual foals aged could not be proven. The history of antimicrobial treatment of the dams was not collected and may have affected the microbiota of the foals. CONCLUSION: The ARGs in foal faeces varied concomitantly with age-related microbiota shifts. The high abundance of tetracycline resistance genes was likely due to the dominance of Bacteroides spp.

Colonization of a hand washing sink in a veterinary hospital by an Enterobacter hormaechei strain carrying multiple resistances to high importance antimicrobials.

BACKGROUND: Hospital intensive care units (ICUs) are known reservoirs of multidrug resistant nosocomial bacteria. Targeted environmental monitoring of these organisms in health care facilities can strengthen infection control procedures. A routine surveillance of extended spectrum beta-lactamase (ESBL) producers in a large Australian veterinary teaching hospital detected the opportunistic pathogen Enterobacter hormaechei in a hand washing sink of the ICU. The organism persisted for several weeks, despite two disinfection attempts. Four isolates were characterized in this study. METHODS: Brilliance-ESBL selective plates were inoculated from environmental swabs collected throughout the hospital. Presumptive identification was done by conventional biochemistry. Genomes of multidrug resistant Enterobacter were entirely sequenced with Illumina and Nanopore platforms. Phylogenetic markers, mobile genetic elements and antimicrobial resistance genes were identified in silico. Antibiograms of isolates and transconjugants were established with Sensititre microdilution plates. RESULTS: The isolates possessed a chromosomal Tn7-associated silver/copper resistance locus and a large IncH12 conjugative plasmid encoding resistance against tellurium, arsenic, mercury and nine classes of antimicrobials. Clusters of antimicrobial resistance genes were associated with class 1 integrons and IS26, IS903 and ISCR transposable elements. The blaSHV-12, qnrB2 and mcr-9.1 genes, respectively conferring resistance to cephalosporins, quinolones and colistin, were present in a locus flanked by two IS903 copies. ESBL production and enrofloxacin resistance were confirmed phenotypically. The isolates appeared susceptible to colistin, possibly reflecting the inducible nature of mcr-9.1. CONCLUSIONS: The persistence of this strain in the veterinary hospital represented a risk of further accumulation and dissemination of antimicrobial resistance, prompting a thorough disinfection of the ICU. The organism was not recovered from subsequent environmental swabs, and nosocomial Enterobacter infections were not observed in the hospital during that period. This study shows that targeted routine environmental surveillance programs to track organisms with major resistance phenotypes, coupled with disinfection procedures and follow-up microbiological cultures are useful to control these risks in sensitive areas of large veterinary hospitals.

Use of cefovecin in dogs and cats attending first-opinion veterinary practices in Australia.

BACKGROUND: Cefovecin is a long-acting third-generation cephalosporin commonly used in veterinary medicine. Third-generation cephalosporins are critically important antimicrobials that should only be used after culture and susceptibility testing. The authors describe the common indications for cefovecin use in dogs and cats, and the frequency of culture and susceptibility testing. MATERIALS AND METHODS: A cross-sectional study was performed using clinical records extracted from VetCompass Australia. A previously described method was used to identify records containing cefovecin. The reason for cefovecin use was annotated in situ in each consultation text. RESULTS: Over a six-month period (February and September 2018), 5180 (0.4 per cent) consultations involved cefovecin administration, of which 151 were excluded. Cats were administered cefovecin more frequently than dogs (1.9 per cent of cat consultations and 0.1 per cent of dog consultations). The most common reasons for cefovecin administration to cats were cat fight injuries and abscesses (28 per cent) and dermatitis (13 per cent). For dogs, the most common reasons for cefovecin administration were surgical prophylaxis (24 per cent) and dermatitis (19 per cent). Culture and susceptibility testing were reported in 16 cases (0.3 per cent). CONCLUSION: Cefovecin is used in many scenarios in dogs and cats where antimicrobials may be either not indicated or where an antimicrobial of lower importance to human health is recommended.

Impact of insertion sequences on convergent evolution of Shigella species.

Shigella species are specialised lineages of Escherichia coli that have converged to become human-adapted and cause dysentery by invading human gut epithelial cells. Most studies of Shigella evolution have been restricted to comparisons of single representatives of each species; and population genomic studies of individual Shigella species have focused on genomic variation caused by single nucleotide variants and ignored the contribution of insertion sequences (IS) which are highly prevalent in Shigella genomes. Here, we investigate the distribution and evolutionary dynamics of IS within populations of Shigella dysenteriae Sd1, Shigella sonnei and Shigella flexneri. We find that five IS (IS1, IS2, IS4, IS600 and IS911) have undergone expansion in all Shigella species, creating substantial strain-to-strain variation within each population and contributing to convergent patterns of functional gene loss within and between species. We find that IS expansion and genome degradation are most advanced in S. dysenteriae and least advanced in S. sonnei; and using genome-scale models of metabolism we show that Shigella species display convergent loss of core E. coli metabolic capabilities, with S. sonnei and S. flexneri following a similar trajectory of metabolic streamlining to that of S. dysenteriae. This study highlights the importance of IS to the evolution of Shigella and provides a framework for the investigation of IS dynamics and metabolic reduction in other bacterial species.

Antibiotic Resistance Genes in Antibiotic-Free Chicken Farms.

Rising concern about the use of antibiotics in food production has resulted in many studies on the occurrence of antibiotic resistance genes (ARGs) in animal-associated bacterial communities. There are few baseline data on the abundance of ARGs on farms where chickens are intensively raised with little or no use of antibiotics. This study used a high-throughput quantitative PCR array to survey two antibiotic-free chicken farms for the occurrence of ARGs and mobile genetic elements known to enhance the spread of ARGs. No antibiotics had been used on the study farms for five years prior to this study. The results provide a baseline for the occurrence of resistance genes in the chicken production system without direct selective pressure.

Z/I1 Hybrid Virulence Plasmids Carrying Antimicrobial Resistance genes in S. Typhimurium from Australian Food Animal Production.

Knowledge of mobile genetic elements that capture and disseminate antimicrobial resistance genes between diverse environments, particularly across human-animal boundaries, is key to understanding the role anthropogenic activities have in the evolution of antimicrobial resistance. Plasmids that circulate within the Enterobacteriaceae and the Proteobacteria more broadly are well placed to acquire resistance genes sourced from separate niche environments and provide a platform for smaller mobile elements such as IS26 to assemble these genes into large, complex genomic structures. Here, we characterised two atypical Z/I1 hybrid plasmids, pSTM32-108 and pSTM37-118, hosting antimicrobial resistance and virulence associated genes within endemic pathogen Salmonella enterica serovar Typhimurium 1,4,[5],12:i:-, sourced from Australian swine production facilities during 2013. We showed that the plasmids found in S. Typhimurium 1,4,[5],12:i:- are close relatives of two plasmids identified from Escherichia coli of human and bovine origin in Australia circa 1998. The older plasmids, pO26-CRL125 and pO111-CRL115, encoded a putative serine protease autotransporter and were host to a complex resistance region composed of a hybrid Tn21-Tn1721 mercury resistance transposon and composite IS26 transposon Tn6026. This gave a broad antimicrobial resistance profile keyed towards first generation antimicrobials used in Australian agriculture but also included a class 1 integron hosting the trimethoprim resistance gene dfrA5. Genes encoding resistance to ampicillin, trimethoprim, sulphonamides, streptomycin, aminoglycosides, tetracyclines and mercury were a feature of these plasmids. Phylogenetic analyses showed very little genetic drift in the sequences of these plasmids over the past 15 years; however, some alterations within the complex resistance regions present on each plasmid have led to the loss of various resistance genes, presumably as a result of the activity of IS26. These alterations may reflect the specific selective pressures placed on the host strains over time. Our studies suggest that these plasmids and variants of them are endemic in Australian food production systems.

Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198.

Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S.enterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S.enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. enterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. enterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.

Exploration of antibiotic resistance risks in a veterinary teaching hospital with Oxford Nanopore long read sequencing.

The Oxford Nanopore MinION DNA sequencing device can produce large amounts of long sequences, typically several kilobases, within a few hours. This long read capacity was exploited to detect antimicrobial resistance genes (ARGs) in a large veterinary teaching hospital environment, and to assess their taxonomic origin, genetic organisation and association with mobilisation markers concurrently. Samples were collected on eight occasions between November 2016 and May 2017 (inclusive) in a longitudinal study. Nanopore sequencing was performed on total DNA extracted from the samples after a minimal enrichment step in broth. Many ARGs present in the veterinary hospital environment could potentially confer resistance to antimicrobials widely used in treating infections of companion animals, including aminoglycosides, extended-spectrum beta-lactams, sulphonamides, macrolides, and tetracyclines. High-risk ARGs, defined here as single or multiple ARGs associated with pathogenic bacterial species or with mobile genetic elements, were shared between the intensive care unit (ICU) patient cages, a dedicated laundry trolley and a floor cleaning mop-bucket. By contrast, a floor surface from an office corridor without animal contact and located outside the veterinary hospital did not contain such high-risk ARGs. Relative abundances of high-risk ARGs and co-localisation of these genes on the same sequence read were higher in the laundry trolley and mop bucket samples, compared to the ICU cages, suggesting that amplification of ARGs is likely to occur in the collection points for hospital waste. These findings have prompted the implementation of targeted intervention measures in the veterinary hospital to mitigate the risks of transferring clinically important ARGs between sites and to improve biosecurity practices in the facility.

Population wide assessment of antimicrobial use in dogs and cats using a novel data source - A cohort study using pet insurance data.

Antimicrobial use in veterinary practice is under increasing scrutiny as a contributor to the rising risk of multidrug resistant bacterial pathogens. Surveillance of antimicrobial use in food animals is extensive globally, but population level data is lacking for companion animals. Lack of census data means cohorts are usually restricted to those attending veterinary practices, which precludes aggregating data from large cohorts of animals, independent of their need for veterinary intervention. The objective of this study was to investigate the exposure of dogs and cats to antimicrobials at a population level. A retrospective cohort study was performed using a novel data source; a pet insurance database. The rate of antimicrobial prescribing, and the rate of prescribing of critically important antimicrobials, was measured in a large population of dogs (813,172 dog-years) and cats (129,232 cat-years) from 2013 - 2017. The incidence rate of antimicrobial prescribing was 5.8 prescriptions per 10 dog years (95% CI 5.8-5.9 per 10 dog years) and 3.1 prescriptions per 10 cat years (95% CI 3.1-3.2 per 10 cat years). Critically important antimicrobials accounted for 8% of all the antimicrobials prescribed over the 4-year study. Cats were 4.8-fold more likely than dogs to be prescribed 3rd-generation cephalosporins. The level of antimicrobial exposure in dogs and cats was less than half that for the coincident human community. Data such as this provides a unique opportunity to monitor antimicrobial prescribing in veterinary medicine, which is a critical component of optimal antimicrobial stewardship.

pSTM6-275, a Conjugative IncHI2 Plasmid of Salmonella enterica That Confers Antibiotic and Heavy-Metal Resistance under Changing Physiological Conditions.

Detailed annotation of an IncHI2 plasmid, pSTM6-275, from Salmonella enterica serotype 1,4,5,12:i:- strain TW-Stm6 revealed a composite structure, including antimicrobial resistance genes on mobile genetic elements. The plasmid was thermosensitive for transfer to Escherichia coli and conferred reduced susceptibility to antibiotics, copper sulfate, and silver nitrate. Metal ion susceptibility was dependent on physiological conditions, giving an insight into the environments where this trait might confer a fitness advantage.

Cross-sectional study of antimicrobials used for surgical prophylaxis by bovine veterinary practitioners in Australia.

Antimicrobials are widely used in veterinary practices, but there has been no investigation of antimicrobial classes used or the appropriateness of their use in bovine practice. This study investigated antimicrobial use for surgical prophylaxis in bovine practice in Australia. A cross-sectional study of veterinarian antimicrobial usage patterns was conducted using an online questionnaire. Information solicited included respondent's details, the frequency with which antimicrobials were used for specific surgical conditions (including the dose, timing and duration of therapy) and details of practice antimicrobial use policies and sources of information about antimicrobials. In total, 212 members of the Australian veterinary profession working in bovine practice completed the survey. Antimicrobials were always or frequently used by more than 75 per cent of respondents in all scenarios. Generally, antimicrobial drug choice was appropriate for the reported surgical conditions. Procaine penicillin and oxytetracycline accounted for 93 per cent of use. However, there was a wide range of doses used, with underdosing and inappropriate timing of administration being common reasons for inappropriate prophylactic treatment. There was very low use of critically important antimicrobials (3.3 per cent of antimicrobials reported). Antimicrobial use guidelines need to be developed and promoted to improve the responsible use of antimicrobials in bovine practice.

Genome Sequence of an Australian Monophasic Salmonella enterica subsp. enterica Typhimurium Isolate (TW-Stm6) Carrying a Large Plasmid with Multiple Antimicrobial Resistance Genes.

We report the genome sequence of a monophasic Salmonella enterica subsp. enterica Typhimurium strain (TW-Stm6) isolated in Australia that is similar to epidemic multidrug-resistant strains from Europe and elsewhere. This strain carries additional antibiotic and heavy-metal resistance genes on a large (275-kb) IncHI2 plasmid.

Antimicrobials used for surgical prophylaxis by companion animal veterinarians in Australia.

Antimicrobials are widely used in veterinary practices, but there has been no investigation into the classes of antimicrobials used or the appropriateness of their use in surgical prophylaxis. Antimicrobial usage guidelines were published by the Australian Infectious Disease Advisory Panel (AIDAP) in 2013, but there has been no investigation of compliance with them. This study aimed to investigate antimicrobial use for surgical prophylaxis in companion animal practice and assess compliance with AIDAP guidelines for selected conditions by conducting a cross-sectional study of antimicrobial usage patterns of Australian veterinarians using an online questionnaire. Information solicited included: details of the respondent, the frequency with which antimicrobials were used for specific surgical conditions (including dose and duration) and practice antimicrobial use policies and sources of information about antimicrobial drugs and their uses. A total of 886 members of the Australian veterinary profession completed the survey. Few (22%) reported that their practice that had an antimicrobial use policy. Generally, the choice of antimicrobial drug was appropriate for the given surgical conditions. There was poor compliance with AIDAP guidelines for non-use of antimicrobials for routine neutering. Veterinarians caring solely for companion animals had higher odds of optimal compliance with guidelines than veterinarians in mixed species practices (OR 1.4, 95%CI 1.1-1.9). Recent graduates (>2011) had lower odds of compliance than older graduates (OR 0.8, 95%CI 0.6-0.9). The findings suggest that antimicrobial use guidelines need to be expanded and promoted to improve the responsible use of antimicrobials in small animal practice in Australia.