Further work

While this body of work was able to provide valuable information of the canine commensal bacterial populations in healthy dogs and during and after antimicrobial therapy the small number of dogs recruited in each study potentially reduced the statistical power. In addition, the study populations were either convenience based or clinically led samples, therefore increasing the likelihood of introducing selection bias. Therefore further similar, ideally randomised, studies with more observations are required to validate the findings of this work. In particular, few dogs carried MRSP (10%; 95% CI: 6 – 17%) in the antimicrobial treatment study. While this is a positive finding, due to the number of dogs enrolled, a larger study is needed to corroborate this prevalence (previous studies report carriage in up to 4.5% and 3.5 - 66% of healthy and sick dogs, respectively) and may help to identify potential risk factors. There are few published studies that have examined the detection of MRSP, and in addition, antimicrobial therapy has not been consistently identified as a risk factor.

In addition, a longitudinal study investigating the carriage of mucosal staphylococci, particularly MRSP, in healthy dogs without antimicrobial pressure or veterinary admission would be beneficial. The fitness of MRSP isolates, compared to meticillin susceptible

S. pseudintermedius, in the absence of antimicrobial pressure is currently unknown.

Longitudinal studies are more likely to detect AMR isolates compared to cohort studies due to transient carriage in some individuals. In addition, speciation and/or genotyping would provide information on the carriage of both CoPS and CoNS species in dogs, the later of which has not been previously reported. Finally, assessment of risk factors for AMR in dogs under natural conditions would provide information that could be used to formulate

preventative strategies and used as a baseline for other studies investigating the effects of antimicrobial or veterinary hospital selective pressures.

Further characterisation of the isolates from this work would help to elucidate the findings. For instance, further genotyping, assessment of fitness, phylogenetic grouping and detection of virulence genes with risk factor assessment, particularly in the longitudinal studies, would provide further insight on the carriage of canine gut commensals in healthy dogs versus dogs under antimicrobial therapy. In particular, it would be interesting to investigate the

epidemiology of AMR isolates carried by dogs in multi-dog households and in dogs on raw meat diets.

Furthermore, for all E. coli studies, characterisation of resistance determinants and plasmids amongst phenotypic ESBL- and AmpC-producing E. coli and sequencing of resistance genes and multi-locus sequence typing of (MLST) would provide further information for dogs under different selection pressures e.g. 12% of dogs in the E. coli antimicrobial study carried blaTEM

and/or blaOXA, but these isolates were not sequenced to examine variants and determine the

significance of such genes for phenotypic resistance. In particular MLST may identify certain dominant ESBL clones and allow comparison with other studies. Moreover, blaDHA-1 and blaMOX were detected in dogs in the antimicrobial resistance study; blaDHA-1 is uncommon and

blaMOX has not been previously reported in dogs, so further characterisation would allow comparison with similar isolates detected in humans and other animals. Similarly, for staphylococci, further characterisation of meticillin resistant isolates including determining relative fitness, SCCmec, spa and strain typing would provide epidemiological information for MRSP isolates carried by dogs in the community in the UK.

anaerobic gut bacteria and the duration of any change is of interest. This is being undertaken as an extension of this work and will be reported elsewhere. Metagenomic determination of the mucosal and skin microbiomes has been recently performed in healthy dogs. Similar longitudinal studies in healthy dogs and dogs under antimicrobial pressure, would give further information on the diversity and stability of such populations.

8.3 Conclusions

The prevalence of antimicrobial resistance was high amongst mucosal staphylococci and faecal E. coli in both healthy and sick dogs and the faecal E. coli population structure was diverse and dynamic in healthy dogs. In healthy dogs, these findings are likely to be associated with external influences such as diet, environment and in-contact humans and other animals. In particular, dogs that ate raw meat or animal faeces, lived in multi-dog households or had contact with individuals that had been exposed to health-care environments were at increased risk for AMR commensal bacteria. This highlights the potential of bacterial sharing within households and veterinary premises and may represent a human health risk.

Antimicrobial therapy was also associated with the increased risk of antimicrobial resistance amongst these bacterial populations. In particular, beta-lactam treatment was a risk factor for carriage of MDR and AmpC-producing E. coli, with fluoroquinolone therapy a risk factor for MDR or MRS staphylococci. The percentage of dogs with each resistance outcome generally returned to baseline within three months of finishing treatment and in the multilevel

multivariable models, there was no significant difference for the majority of resistance outcomes and treatment groups between baseline and one month after the end of therapy. Antimicrobial therapy is a risk factor for the detection of antimicrobial resistant commensal bacteria in dogs and recovery to baseline may take between one to three months after the treatment has finished. This highlights the importance of prudent antimicrobial use, which may be aided by antimicrobial prescribing guidelines. However other factors, such as diet, in- contacts, co-selection and bacterial fitness may be involved in the carriage of resistant bacteria and should be considered

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