Water consumption and wastage behaviour in pigs: implications for antimicrobial administration and stewardship.

Daily water use and wastage patterns of pigs have major effects on the efficacy of in-water antimicrobial dosing events when conducted for metaphylaxis or to treat clinical disease. However, daily water use and wastage patterns of pigs are not routinely quantified on farms and are not well understood. We conducted a prospective, observational 27-day study of the daily water use and wastage patterns of a pen group of 15 finisher pigs reared in a farm building. We found that the group of pigs wasted a median of 36.5% of the water used per day. We developed models of the patterns of water used and wasted by pigs over each 24-h period using a Bayesian statistical method with the brm() function in the brms package. Both patterns were uni-modal, peaking at 1400-1700, and closely aligned. Wastage was slightly greater during hours of higher water use. We have shown that it is feasible to quantify the water use and wastage patterns of pigs in farm buildings using a system that records and aggregates data, and analyses them using hierarchical generalised additive models. This system could support more efficacious in-water antimicrobial dosing on farms, and better antimicrobial stewardship, by helping to reduce the quantities of antimicrobials used and disseminated into the environment.

Antimicrobial stewardship in companion animal practice: an implementation trial in 135 general practice veterinary clinics.

BACKGROUND: Antimicrobial stewardship programmes (ASPs) have been widely implemented in medical practice to improve antimicrobial prescribing and reduce selection for multidrug-resistant pathogens. OBJECTIVES: To implement different antimicrobial stewardship intervention packages in 135 veterinary practices and assess their impact on antimicrobial prescribing. METHODS: In October 2018, general veterinary clinics were assigned to one of three levels of ASP, education only (CON), intermediate (AMS1) or intensive (AMS2). De-identified prescribing data (1 October 2016 to 31 October 2020), sourced from VetCompass Australia, were analysed and a Poisson regression model fitted to identify the effect of the interventions on the incidence rates of antimicrobial prescribing. RESULTS: The overall incidence rate (IR) of antimicrobial prescribing for dogs and cats prior to the intervention was 3.7/100 consultations, which declined by 36% (2.4/100) in the implementation period, and by 50% (1.9/100) during the post-implementation period. Compared with CON, in AMS2 there was a 4% and 6% reduction in the overall IR of antimicrobial prescribing, and a 24% and 24% reduction in IR of high importance antimicrobial prescribing, attributable to the intervention in the implementation and post-implementation periods, respectively. A greater mean difference in the IR of antimicrobial prescribing was seen in high-prescribing clinics. CONCLUSIONS: These AMS interventions had a positive impact in a large group of general veterinary practices, resulting in a decline in overall antimicrobial use and a shift towards use of antimicrobials rated as low importance, with the greatest impact in high-prescribing clinics.

Review: Water medication of growing pigs: sources of between-animal variability in systemic exposure to antimicrobials.

On many Australian commercial pig farms, groups of growing pigs are mass-medicated through their drinking water with selected antimicrobials for short periods to manage herd health. However, delivery of medication in drinking water cannot be assumed to deliver an equal dose to all animals in a group. There is substantial between-animal variability in systemic exposure to an antimicrobial (i.e. the antimicrobial concentration in plasma), resulting in under-dosing or over-dosing of many pigs. Three sources of this between-animal variability during a water medication dosing event are differences in: (1) concentration of the active constituent of the antimicrobial product in water available to pigs at drinking appliances in each pen over time, (2) medicated water consumption patterns of pigs in each pen over time, and (3) pharmacokinetics (i.e. oral bioavailability, volume of distribution and clearance between pigs and within pigs over time). It is essential that factors operating on each farm that influence the range of systemic exposures of pigs to an antimicrobial are factored into antimicrobial administration regimens to reduce under-dosing and over-dosing.

Appraisal of the Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery in dogs and cats.

The Australian Veterinary Prescribing Guidelines for antimicrobial prophylaxis for surgery on dogs and cats are evidence-based guidelines for veterinary practitioners. Validation of these guidelines is necessary to ensure quality and implementability. Two validated tools, used for medical guideline appraisal, were chosen to assess the guidelines. The terminology from the GuideLine Implementability Appraisal (GLIA) and the Appraisal of Guidelines for Research and Evaluation version 2 (AGREE II) were adapted for use by veterinarians. A two-phase evaluation approach was conducted. In the first phase of the evaluation, the GLIA tool was used by two specialist veterinary surgeons in clinical practice. The results of this phase were then used to modify the guidelines. In the second phase, the AGREE II tool was used by 6 general practitioners and 6 specialists to appraise the guidelines. In phase 1, the specialist surgeons either agreed or strongly agreed that the guidelines were executable, decidable, valid and novel, and that the guidelines would fit within the process of care. The surgeons were neutral on flexibility and measurability. Additional clarity around one common surgical procedure was added to the guidelines, after which the surgeons agreed that the guidelines were sufficiently flexible. In phase 2, 12 veterinarians completed the assessment using the AGREE II tool. In all sections the scaled domain score was greater than 70%. The overall quality of the guidelines was given a global scaled score of 76%. This assessment has demonstrated that the guidelines for antimicrobial prophylaxis for companion animal surgery are valid and appear implementable.

Survey of veterinary prescribing for poultry disease.

BACKGROUND: To enable better antimicrobial stewardship, it is important to know what the patterns of prescribing behaviour are and what diseases or reasons antibiotics are being prescribed. A prescribing guideline (i.e. recommended best practice for writing prescriptions) developed by the Australian poultry veterinarians exists. However, it is not a prescribing guideline detailing treatments for the commonly observed bacterial diseases in commercial poultry. METHODS: An online survey was deployed to all registered veterinarians and members of the Australian Veterinary Poultry Association to identify prescribing behaviours for the most frequently observed bacterial diseases of poultry. RESULTS: A total of 39 survey responses were received. Most surveys were started but not completed; 13 (33%) were completed with 18 (46%) containing some information on prescribing. The most frequent treatment responses were for Escherichia coli in both layers and broilers, chronic respiratory disease (CRD), fowl cholera and spotty liver in layers and necrotic enteritis in broilers. Treatments described were for products registered for poultry use, within the recommended label dose and duration of treatment (Tables 1, 2). Unsurprisingly, tetracyclines and amoxycillin, followed by lincomycin and trimethoprim sulfonamide products were the most frequently reported treatment options. Inappropriate treatments were reported for salmonellosis and one veterinarian recommended the use of enrofloxacin for the treatment of fowl cholera. CONCLUSION: Information provided by respondents will enable the initial development of prescribing guidelines for both commercial and small poultry flocks. Importantly, it identified less than optimal prescribing behaviour for some diseases, a reliance on one class of antibiotic more than others and a failure to utilise all antimicrobial classes potentially available for treatment. Critically, the survey identified a lack of treatment options for bacterial disease in poultry. The most important bacterial diseases of poultry remain the same; effective alternatives for antibiotic treatment are required and old diseases, thought once gone, are reinventing themselves as problems for the future. Surveys of prescribing behaviours are essential for identifying diseases of high priority, changes in treatments and response to treatment and to identify areas for targeted antimicrobial stewardship, and research needs.

Antimicrobial dosing for common equine drugs: a content review and practical advice for veterinarians in Australia.

BACKGROUND: Appropriate dosing with antimicrobial agents is critical for effective treatment and to prevent the development of antimicrobial resistance. METHODS: A review was undertaken of equine journal articles (Equine Veterinary Journal, Equine Veterinary Education, Australian Veterinary Journal, Australian Equine Veterinarian, Journal of Veterinary Internal Medicine and Journal of Equine Veterinary Science) between January 2015 and August 2018. Those with dosing regimens for procaine penicillin G, gentamicin or trimethoprim-sulfonamide in adult horses were examined and evaluated. Pharmacokinetics and -dynamics of these drugs were also reviewed. RESULTS & CONCLUSION: The most frequently reported doses for penicillin, gentamicin and trimethoprim-sulfonamide were 20-25,000 IU/kg, 6.6 mg/kg and 30 mg/kg, respectively. Veterinarians treating equine patients in Australia should be aware of the current recommended doses and inter-dosing intervals to ensure efficacy in therapy and to preserve the usefulness of these antimicrobials for the future.

The mitochondrial genome of Tetrahymena rostrata.

Tetrahymena rostrata is a ciliated protist which can parasitize the gray field slug, Deroceras reticulatum. Here, we report the sequence of the mitochondrial genome (mt genome) of a strain of T. rostrata that was isolated from the egg of D. reticulatum. Whole cell genomic DNA was sequenced using Illumina® MiSeq and the mitochondrial DNA sequence reads were extracted and assembled. The resulting 47,235 bp assembly contained rRNAs, tRNAs, and 45 protein coding DNA sequences of which 21 encoded proteins of unknown function. Phylogenetic analysis showed T. rostrata clustered with Tetrahymena thermophila, Tetrahymena pigmentosa, Tetrahymena pyriformis, Tetrahymena paravorax, and Tetrahymena malaccensis.

Antimicrobial labelling in Australia: a threat to antimicrobial stewardship?

Antimicrobial resistance is a public health emergency, placing veterinary antimicrobial use under growing scrutiny. Antimicrobial stewardship, through appropriate use of antimicrobials, is a response to this threat. The need for antimicrobial stewardship in Australian veterinary practices has had limited investigation. A 2016 survey undertaken to investigate antimicrobial usage patterns by Australian veterinarians found that antimicrobial dose rates were varied and often inappropriate. Doses of procaine penicillin in horses and cattle were often low, with 68% and 90% of respondents, respectively, reporting doses that were unlikely to result in plasma concentrations above minimum inhibitory concentrations for common equine and bovine pathogens. Frequency of penicillin administration was also often inappropriate. Gentamicin doses in horses were largely appropriate (89% of dose rates appropriate), but 9% of respondents reported twice daily dosing. Amoxycillin and amoxycillin-clavulanate were administered at the appropriate doses, or above, to dogs and cats by 54% and 70% of respondents, respectively. Here, we explore the potential reasons for inappropriate antimicrobial dose regimens and report that antimicrobial labels often recommend incorrect dose rates and thus may be contributing to poor prescribing practices. Changes to legislation are needed to ensure that antimicrobial drug labels are regularly updated to reflect the dose needed to effectively and safely treat common veterinary pathogens. This will be especially true if changes in legislation restrict antimicrobial use by veterinarians to the uses and doses specified on the label, thus hampering the current momentum towards improved antimicrobial stewardship.

Antimicrobial susceptibility testing by Australian veterinary diagnostic laboratories.

BACKGROUND: The national strategy for tackling antimicrobial resistance highlights the need for antimicrobial stewardship in veterinary practice and for surveillance of antimicrobial susceptibility in veterinary pathogens. Diagnostic laboratories have an important role in facilitating both of these processes, but it is unclear whether data from veterinary diagnostic laboratories are similar enough to allow for compilation and if there is consistent promotion of appropriate antimicrobial use embedded in the approaches of different laboratories to susceptibility testing. METHODS: A cross-sectional study of antimicrobial susceptibility testing and reporting procedures by Australian veterinary diagnostic laboratories was conducted in 2017 using an online questionnaire. All 18 veterinary diagnostic laboratories in Australia completed the questionnaire. RESULTS: Kirby-Bauer disc diffusion was the method predominantly used for antimicrobial susceptibility testing and was used to evaluate 86% of all isolates, although two different protocols were used across the 18 laboratories (CLSI 15/18, CDS 3/18). Minimum inhibitory concentrations were never reported by 61% of laboratories. Common isolates were consistently reported on across all species, except for gram-negative isolates in pigs, for which there was some variation in the approach to reporting. There was considerable diversity in the panels of antimicrobials used for susceptibility testing on common isolates and no consistency was apparent between laboratories for any bacterial species. CONCLUSION: We recommend that nationally agreed and consistent antimicrobial panels for routine susceptibility testing should be developed and a uniform set of guidelines should be adopted by veterinary diagnostic laboratories in Australia.

Barriers to and enablers of implementing antimicrobial stewardship programs in veterinary practices.

BACKGROUND: Antimicrobial stewardship (AMS) programs are yet to be widely implemented in veterinary practice and medical programs are unlikely to be directly applicable to veterinary settings. OBJECTIVE: To gain an in-depth understanding of the factors that influence effective AMS in veterinary practices in Australia. METHODS: A concurrent explanatory mixed methods design was used. The quantitative phase of the study consisted of an online questionnaire to assess veterinarians' attitudes to antimicrobial resistance (AMR) and antimicrobial use in animals, and the extent to which AMS currently is implemented (knowingly or unknowingly). The qualitative phase used semi-structured interviews to gain an understanding of the barriers to and enablers of AMS in veterinary practices. Data were collected and entered into NVivo v.11, openly coded and analyzed according to mixed methods data analysis principles. RESULTS: Companion animal, equine, and bovine veterinarians participated in the study. Veterinary practices rarely had antimicrobial prescribing policies. The key barriers were a lack of AMS governance structures, client expectations and competition between practices, cost of microbiological testing, and lack of access to education, training and AMS resources. The enablers were concern for the role of veterinary antimicrobial use in development of AMR in humans, a sense of pride in the service provided, and preparedness to change prescribing practices. CONCLUSION AND CLINICAL IMPORTANCE: Our study can guide development and establishment of AMS programs in veterinary practices by defining the major issues that influence the prescribing behavior of veterinarians.

Antimicrobials used for surgical prophylaxis by equine veterinary practitioners in Australia.

BACKGROUND: Antimicrobials are widely used in Australian veterinary practices, but no investigation into the classes of antimicrobials used, or the appropriateness of use in horses, has been conducted. OBJECTIVES: The aim of the study was to describe antimicrobial use for surgical prophylaxis in equine practice in Australia. STUDY DESIGN: Cross-sectional questionnaire survey. METHODS: An online questionnaire was used to document antimicrobial usage patterns. Information solicited in the questionnaire included demographic details of the respondents, the frequency with which antimicrobials were used for specific surgical conditions (including the dose, timing and duration of therapy) and practice antimicrobial use policies and sources of information about antimicrobials and their uses. RESULTS: A total of 337 members of the Australian veterinary profession completed the survey. Generally, the choice of antimicrobial was appropriate for the specified equine surgical condition, but the dose and duration of therapy varied greatly. While there was poor optimal compliance with British Equine Veterinary Association guidelines in all scenarios (range 1-15%), except removal of a nonulcerated dermal mass (42%), suboptimal compliance (compliant antimicrobial drug selection but inappropriate timing, dose or duration of therapy) was moderate for all scenarios (range 48-68%), except for an uninfected contaminated wound over the thorax, where both optimal and suboptimal compliance was very poor (1%). Veterinarians practicing at a university hospital had higher odds of compliance than general practice veterinarians (Odds ratio 3.2, 95% CI, 1.1-8.9, P = 0.03). MAIN LIMITATIONS: Many survey responses were collected at conferences which may introduce selection bias, as veterinarians attending conferences may be more likely to have been exposed to contemporary antimicrobial prescribing recommendations. CONCLUSIONS: Antimicrobial use guidelines need to be developed and promoted to improve the responsible use of antimicrobials in equine practice in Australia. An emphasis should be placed on antimicrobial therapy for wounds and appropriate dosing for procaine penicillin.

Longitudinal study of Salmonella 1,4,[5],12:i:- shedding in five Australian pig herds.

The shedding patterns of Salmonella spp. and MLVA profiles of Salmonella enterica subspecies enterica (I) serotype 1,4,[5],12:i:- were monitored in a 12-month longitudinal observational study of five pig herds to inform management; provide indications of potential hazard load at slaughter; and assist evaluation of MLVA for use by animal and public health practitioners. Twenty pooled faecal samples, stratified by age group, were collected quarterly. When Salmonella was cultured, multiple colonies were characterized by serotyping and where S. Typhimurium-like serovars were confirmed, isolates were further characterized by phage typing and multiple locus variable number tandem repeat analysis (MLVA). Salmonella was detected in 43% of samples. Salmonella 1,4,[5],12:i- was one of several serovars that persisted within the herds and was found among colonies from each production stage. Virtually all Salmonella 1,4,[5],12:i:- isolates were phage type 193, but exhibited 12 different, closely-related MLVA profiles. Salmonella 1,4,[5],12:i:- diversity within herds was low and MLVA profiles were stable indicating colonization throughout the herds and suggesting each farm had an endemic strain. High prevalence of S. 1,4,[5],12:i:- specific shedding among terminal animals indicated high hazard load at slaughter, suggesting that primary production may be an important pathway of S. 1,4,[5],12:i:- into the human food chain, this has implications for on-farm management and the application and targeting control measures and further evidence of the need for effective process control procedures to be in place during slaughter and in pork boning rooms. These findings have implications for animal health and food safety risk mitigation and risk management.

Antibody responses to infections with strains of Plasmodium falciparum expressing diverse forms of merozoite surface protein 2.

Individuals living in areas where Plasmodium falciparum is endemic experience numerous episodes of infection. These episodes may or may not be symptomatic, with the outcome depending on a combination of parasite and host factors, several of which are poorly understood. One factor is believed to be the particular alleles of several parasite proteins to which the host is capable of mounting protective immune responses. We report a study examining antibody responses to MSP2 in 15 semi-immune teenagers and adults living in the Khanh-Hoa area of southern-central Vietnam, where P. falciparum is highly endemic; subjects were serially infected with multiple strains of P. falciparum. The MSP2 alleles infecting these subjects were determined by nucleotide sequencing. A total of 62 MSP2 genes belonging to both dimorphic families were identified, of which 33 contained distinct alleles, with 61% of the alleles being detected once. Clear changes in the repertoire occurred between infections. Most infections contained a mixture of parasites expressing MSP2 alleles from both dimorphic families. Two examples of reinfection with a strain expressing a previously encountered allele were detected. Significant changes in antibody levels to various regions of MSP2 were detected over the course of the experiment. There was no clear relation between the infecting form of MSP2 and the ensuing antibody response. This study highlights the complexity of host-parasite relationship for this important human pathogen.

Quantitative determination of the biodegradable polymer Poly(beta-hydroxybutyrate) in a recombinant Escherichia coli strain by use of mid-infrared spectroscopy and multivariative statistics.

Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(beta-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.

Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis.

Glycopeptidolipids (GPLs) are major components of the cell walls of several species of mycobacteria. We have isolated a transposon mutant of Mycobacterium smegmatis that is unable to synthesize mature GPLs and that displays a rough colony morphology. The disrupted gene, mtf1, shares a high degree of homology with several S-adenosylmethionine-dependent methyltransferases. The enzyme encoded by mtf1 is required for the methylation of a single rhamnose residue that forms part of the conserved GPL core structure. This conclusion is supported by the finding that (a) the mutant synthesized only GPLs with undermethylated (either mono- or nonmethylated instead of di- or trimethylated) rhamnose residues; (b) complementation of the mutant with a wild-type copy of mtf1 restored high levels of synthesis of GPLs containing di- and trimethylated rhamnose; and (c) S-adenosylmethionine-dependent methylation of rhamnosylated GPLs could be detected in cell lysates of wild-type cells and mtf1-complemented mutant cells, but not in mutant cells lacking intact mtf1. Structural analysis of wild-type and mutant GPLs suggests that disruption of mtf1 specifically inhibits addition of O-methyl groups to the 3 (or 2)-position of the rhamnose. In the absence of 3-O-methylation, further methylation of GPL rhamnose is apparently inhibited, and overall GPL synthesis is down-regulated by 90%.

Characterization of a Mycobacterium smegmatis mutant lacking penicillin binding protein 1.

The ponA gene of Mycobacterium smegmatis encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to beta-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.

Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis.

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.

Temporal variation of the merozoite surface protein-2 gene of Plasmodium falciparum.

Extensive polymorphism of key parasite antigens is likely to hamper the effectiveness of subunit vaccines against Plasmodium falciparum infection. However, little is known about the extent of the antigenic repertoire of naturally circulating strains in different areas where malaria is endemic. To address this question, we conducted a study in which blood samples were collected from parasitemic individuals living within a small hamlet in Western Irian Jaya and subjected to PCR amplification using primers that would allow amplification of the gene encoding merozoite surface protein-2 (MSP2). We determined the nucleotide sequence of the amplified product and compared the deduced amino acid sequences to sequences obtained from samples collected in the same hamlet 29 months previously. MSP2 genes belonging to both major allelic families were observed at both time points. In the case of the FC27 MSP2 family, we observed that the majority of individuals were infected by parasites expressing the same form of MSP2. Infections with parasites expressing 3D7 MSP2 family alleles were more heterogeneous. No MSP2 alleles observed at the earlier time point were detectable at the later time point, either for the population as a whole or for individuals who were assayed at both time points. We examined a subset of the infected patients by using blood samples taken between the two major surveys. In no patients could we detect reinfection by a parasite expressing a previously encountered form of MSP2. Our results are consistent with the possibility that infection induces a form of strain-specific immune response against the MSP2 antigen that biases against reinfection by parasites bearing identical forms of MSP2.

Expression in bacteria other than Escherichia coli.

Although many bacteria are used for the expression of foreign genes, there is still a need to develop better expression systems. Advances have been made in the stabilization of gene maintenance and in the control of expression, therefore increasing the potential usefulness of some of these bacteria.