NOTE TO USERS
This reproduction is the best copy available.
UMI'
ANTIMICROBIAL RESISTANCE, ANTIMICROBIAL USE AND INFECTION CONTROL IN
COMMUNITY SMALL ANIMAL VETERINARY HOSPITALS IN SOUTHERN ONTARIO
A Thesis
Presented to
The Faculty of Graduate Studies
of
by
COLLEEN P. MURPHY
In partial fulfilment of requirements
for the degree of
Doctor of Philosophy
March, 2010
© Colleen P. Murphy, 2010 Library and Archives Bibliotheque et 1*1 Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition
395 Wellington Street 395, rue Wellington Ottawa ON K1A 0N4 Ottawa ON K1A 0N4 Canada Canada
Your file Votre reference ISBN: 978-0-494-64536-9 Our file Notre reference ISBN: 978-0-494-64536-9
NOTICE: AVIS:
The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library and permettant a la Bibliotheque et Archives Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par I'Internet, preter, telecommunication or on the Internet, distribuer et vendre des theses partout dans le loan, distribute and sell theses monde, a des fins commerciales ou autres, sur worldwide, for commercial or non support microforme, papier, electronique et/ou commercial purposes, in microform, autres formats. paper, electronic and/or any other formats.
The author retains copyright L'auteur conserve la propriete du droit d'auteur ownership and moral rights in this et des droits moraux qui protege cette these. Ni thesis. Neither the thesis nor la these ni des extraits substantiels de celle-ci substantial extracts from it may be ne doivent etre imprimes ou autrement printed or otherwise reproduced reproduits sans son autorisation. without the author's permission.
In compliance with the Canadian Conformement a la loi canadienne sur la Privacy Act some supporting forms protection de la vie privee, quelques may have been removed from this formulaires secondaires ont ete enleves de thesis. cette these.
While these forms may be included Bien que ces formulaires aient inclus dans in the document page count, their la pagination, il n'y aura aucun contenu removal does not represent any loss manquant. of content from the thesis.
1+1 Canada ABSTRACT
ANTIMICROBIAL RESISTANCE, ANTIMICROBIAL USE AND INFECTION
CONTROL IN COMMUNITY SMALL ANIMAL VETERINARY HOSPITALS IN
SOUTHERN ONTARIO
Colleen P. Murphy Advisors: University of Guelph, 2009 Dr. Scott McEwen Dr. Richard Reid-Smith
This thesis is a study of components of the epidemiology of antimicrobial resistance in companion animals. The effect of antimicrobial treatment on the occurrence of antimicrobial resistance in fecal Escherichia coli isolates, antimicrobial use by companion animal veterinarians and the recovery of environmental bacteria fromcompanio n animal veterinary practices were investigated. The effect of antimicrobial treatment on the occurrence of antimicrobial resistance was examined using a cohort design studying in dogs requiring treatment with amoxicillin- clavulanic acid, cephalexin, fluoroquinolones or penicillin. Resistance to amoxicillin-clavulanic acid, cefoxitin, ceftiofur and ceftriaxone in fecal E. coli from dogs was significantly associated with treatment with cephalexin. Also, isolation of fecal Clostridium difficile was significantly associated with treatment with amoxicillin-clavulanic acid. Antimicrobial use by companion animal veterinarians was investigated using journals recording diagnosed new disease events and associated treatments. Antimicrobials were the most frequently prescribed treatment. Non-topical
(oral, parenteral) antimicrobials were the most common type of antimicrobial prescribed, and P- lactams were the most frequentlyprescribe d antimicrobial class. In dogs, 67% of new disease events associated with canine infectious tracheobronchitis were treated with antimicrobials. In cats, 70% and 74% of disease events associated with feline upper respiratory tract disease and feline lower urinary tract disease, respectively, were treated with antimicrobials. The recovery of environmental bacteria from community veterinary hospitals was investigated in a cross-sectional study. The proportion of hospitals with positive environmental samples were: E. coli-92%, C. difficile-58%, methicillin-resistant Staphylococcus aureus -9%, CMY-2 producing E. coli-9%, methicillin-resistant Staphylococcus pseudintermedius-7%, Salmonella-2%. Antimicrobial resistance in E. coli was infrequent, but several important potential pathogens were recovered:
Canadian epidemic strains MRSA-2 and MRSA-5, and C. difficile ribotype 027. An evaluation of infection control practices demonstrated deficiencies that could be improved: development of formal infection control policies, use of isolation for infectious patients, and disinfectant selection for environmental disinfection. The overall study results suggest that use of common antimicrobials in companion animal practice and associated antimicrobial resistance may pose a risk to animal and human health, and that companion animal veterinary hospitals are a reservoir for environmental and antimicrobial resistant pathogens. Acknowledgements
Firstly, I would like to thank my advisors: Drs Richard Reid-Smith and Scott
McEwen. You gently guided me through the various research projects and have provided invaluable assistance in the preparation of this thesis and other manuscripts. Thank you as well to the members of my advisory committee: Scott Weese, Patrick Boerlin and John
Prescott. Your additional support and insight are truly appreciated.
I extend a huge thanks and owe a debt of gratitude to the numerous individuals who worked in the field, the laboratory and the office: Nicol Janecko, Virginia Young,
Joyce Rousseau, Gabriel Jantzi, Meredith Craig, Rebeccah Travis, Gerry Lazarek, Alyssa
Calder, Karlee Thomas, Adriana Sage, Nicole Rolfe, staff of the Canadian Research
Institute for Food Safety, the Laboratory for Foodborne Zoonoses, and Prairie
Diagnostics Services. I need to thank Nicol Janecko especially for managing the projects and working so patiently with me. I am also gratefully for the statistical support that I received from members of the department of Population Medicine: William Sears, Olaf
Berke, David Pearl and Zvonimir Poljak.
Thank you to all the veterinarians, veterinary practices, pet owners and dogs that participated in my research projects. I also need to acknowledge the Ontario Veterinary
College Pet Trust, the Public Health Agency of Canada, Federal Student Work
Employment Program and the Ontario Veterinary College Fellowship for funding the projects and the graduate student stipend. I also thank Denise Coleman of the Laboratory for Foodborne Zoonoses for managing my affairs at PHAC.
Lastly, thanks to David, Hannah, Brian and my other family members for their patience and much needed help that gave me the time I needed to complete this degree.
i Table of Contents Chapter 1 Introduction and Literature Review 1
1.0. Introduction 2 2.0. Antimicrobial resistance in Escherichia coli 2 2.1. Escherichia coli as an opportunistic pathogen in companion animals 3 2.2. Escherichia coli as commensal organisms in companion animals 5 3.0 Salmonella enterica 5 4.0. Beta-lactamase cephamycinase-2 (blacMY-2) 5 5.0. Methicillin-resistant Staphylococcus aureus 6 6.0. Methicillin-resistant Staphylococcus pseudintermedius 8 7.0. Hospital-acquired infections and veterinary hospital-based infection control 9 8.0. Antimicrobial drug use in companion animals 14 P.O. Research objectives 20 Table 47
Chapter 2 Escherichia coli, and selected veterinary and zoonotic pathogens isolated from environmental sites within companion animal veterinary hospitals in southern Ontario: Prevalence and factors associated with recovery 49
Abstract 49 1.0. Introduction 50 2.0. Materials and methods 51 2.1. Sample size calculations 51 2.2. Veterinary hospital selection 51 2.3. Sampling 52 2.4. Microbiology 53 2.5. Antimicrobial susceptibility testing 58 2.6. Generalized linear mixed models of factors potentially associated with environmental recovery of bacteria 58 3.0. Results 60 3.1. Generalized linear mixed models of potential factors associated with environmental recovery of bacteria 62 4.0. Discussion 63 Acknowledgements 70 Figure 80 Tables 81
ii Chapter 3 A prospective cohort study of the effects of antimicrobial treatment on the incidence of antimicrobial resistance in generic fecal Escherichia coli isolates and isolation of Clostridium difficile, Salmonella enterica, blaCMY- 2 positive E. coli, methicillin-resistant Staphylococcus aureus, Staphylococcus pseudintermedius, and vancomycin-resistant Enterococcus spp. from dogs treated in community companion animal practices in southern Ontario 88
Abstract 88 1.0. Introduction 89 2.0. Materials and methods 91 2.1. Veterinary hospital and dog recruitment 91 2.2. Sample size 93 2.3. Laboratory methods 93 2.4. Statistical Analysis 95 3.0. Results 98 3.1. Kaplan Meier survival functions 100 3.2. Cox proportional hazard models 103 4.0. Discussion 104 Acknowledgements Ill References 112 Figure 118 Tables 120
Chapter 4 Out-patient antimicrobial drug use in dogs and cats for new disease events from community companion animal practices in Ontario 124
Abstract 124 1.0. Introduction 125 2.0. Materials and methods 126 2.1. Veterinarian recruitment 126 2.2. Data-entry journal pre-test 127 2.3. Eligibility criteria 127 2.4. Sample size estimation 128 2.5. Data collection 128 2.6. Statistical analysis 129 3.0. Results 131 3.1. Demographics 131 3.2. Journals 132 3.3. Antimicrobial prescription events 133 3.4. Comparison to a formulary referenced dose range (mg/kg) and frequency of administration 136
iii 3.5. Specific disease conditions 137 4.0. Discussion 138 Acknowledgements 142 Reference 143 Tables 146
Chapter 5 Evaluation of specific infection control practices used by companion animal veterinarians in community veterinary practices in southern Ontario 167
Abstract 167 1.0. Introduction 168 2.0. Materials and methods 169 2.1. Statistical methods 171 3.0. Results 172 4.0. Discussion 177 Acknowledgements 185 References 187 Tables 193
Chapter 6 Summary Discussion and Conclusions 198
References 206
Appendices 207
Appendix A: Additional Documents 207 Appendix A.2.1 Recruitment letter mailed to veterinary practices for the studies "Environmental recovery and antimicrobial susceptibility of selected veterinary and zoonotic pathogens, and evaluation of disinfection procedures in private and referral companion-animal veterinary practices in Southern Ontario" and "The effect of antimicrobial therapy on antimicrobial susceptibility and colonization with selected bacteria isolated from dogs presented to private veterinary practices in Southern Ontario 207 Appendix A.2.2 Questionnaire administered to veterinarians for the study "Environmental recovery and antimicrobial susceptibility of selected veterinary and zoonotic pathogens, and evaluation of disinfection procedures in private and referral companion-animal veterinary practices in Southern Ontario" 213
iv Appendix A.2.3. Questionnaire administered to veterinary technicians for the study "Environmental recovery and antimicrobial susceptibility of selected veterinary and zoonotic pathogens, and evaluation of disinfection procedures in private and referral companion-animal veterinary practices in Southern Ontario" 227
Appendix A.3.1 Questionnaire administered to pet owners for the study "The effect of antimicrobial therapy on antimicrobial susceptibility and colonization with selected bacteria isolated from dogs presented to private veterinary practices in Southern Ontario" 233
Appendix A.4.1 Recruitment letter mailed to veterinarians for the "Animal illness and prescription drug use study" 239
Appendix A.4.2 Feedback questionnaire completed by veterinarians who participated in the pretest of the data collection journal for the study "Animal illness and prescription drug use study" 241
Appendix B: Additional Figures 244
Figure A.3.1. Kaplan-Meier estimator of the survival probability of antimicrobial susceptibility of E. coli isolates from dogs treated with antimicrobials and untreated dogs, where none of the estimated survival probabilities were significantly different 244
Figure A.3.2. Kaplan-Meier estimator of the survival probability of not isolating E. coli isolates with an blacMY-2 phenotype from dogs treated with antimicrobials and untreated dogs 246
Figure A. 3.3. Kaplan-Meier estimator of the survival probability of not isolating Salmonella enterica isolates from dogs treated with antimicrobials and untreated dogs. 247
Appendix C: Additional Tables 248
Table A.3.1. Signalment of dogs in antimicrobial treatment and untreated cohorts 248
Table A.3.2. The dose, frequencyan d duration of treatment for dogs treated with oral amoxicillin-clavulanic acid, oral cephalexin, oral fluoroquinolones and injectable penicillin 249
Table A.3.3. The frequencyo f responses to a questionnaire completed by owners of dogs treated with antimicrobials and untreated dogs 250
v Table A.3.4. Distribution (%) of antimicrobial minimum inhibitory concentrations of fecal Escherichia coli isolates (n=1002) from dogs untreated and treated with antimicrobials from community veterinary practices in southern Ontario 252
Table A.4.1. Categorization of body sites or disease conditions in study dogs that were created by aggregating the incident disease events description reported by veterinarians in the submitted journals 254
Table A.4.2. Categorization of body sites or disease conditions in study cats that were created by aggregating the incident disease events description reported by veterinarians in the submitted journals 256
Table A.4.3. The incident disease event descriptions reported by veterinarians that were aggregated for the evaluation of antimicrobial use in feline lower urinary tract disease, feline upper respiratory tract disease and canine infectious tracheobronchitis 258
Table A.4.4. The age, sex and weight distributions of the dogs and cats with incident disease events treated with non-topical antimicrobials reported in the journals submitted by veterinarians 259
Table A.4.5. Distribution of the description of the incident disease events in dogs and cats treated with non-topical antimicrobials reported by veterinarians in the submitted journals 260
Table A.4.6. Other incident disease events treated with non-topical antimicrobials in dogs 261
Table A.4.7. Other incident disease events treated by non-topical antimicrobials in cats. 263
Appendix D The prevalence of bacterial contamination of surgical cold sterile solutions from community companion animal veterinary practices in southern Ontario 264
Abstract 264 1.0. Introduction 264 2.0. Materials and Methods 266 3.0. Results 267 4.0. Discussion 268 Acknowledgements 270 References 272
vi List of Figures Figure 2.1 Number of sites in the Phase One component of the study where environmental E. coli and C. difficile were recovered from companion animal veterinary hospitals 80
Figure 3.1 Kaplan-Meier estimator of the survival probability of antimicrobial susceptibility of E. coli isolates from dogs treated with antimicrobials and untreated dogs where at least two of the estimated survival probabilities are significantly different 118
Figure 3.2 Kaplan-Meier estimator of the survival probability of not isolating Clostridium difficile fromdog s treated with antimicrobials and untreated dogs 119
vu List of Tables
Table 1.1. Measures of antimicrobial use in companion animal studies 47
Table 2.1 Prevalence of bacterial recovery from environmental sites within companion animals veterinary hospitals 81
Table 2.2 Prevalence of bacterial recovery from equipment within companion animal veterinary hospitals 82
Table 2.3 Ribotypes and toxin profile of environmental Clostridium difficile isolates from companion animal veterinary hospitals 83
Table 2.4 Distribution of antimicrobial minimum inhibitory concentrations of environmental Escherichia coli isolates from veterinary hospitals 85
Table 2.5 Results from a generalized linear mixed model of factors potentially associated with the environmental recovery of Clostridium difficile fromveterinar y hospitals 87
Table 3.1 The frequency of Clostridium difficile ribotypes recovered from dogs treated with antimicrobials and untreated dogs 120
Table 3.2 The incidence rate of resistance in fecal E. coli from dogs treated with antimicrobials and untreated dogs 121
Table 3.3 The incidence rate of isolation of E. coli isolates with a blacm-i phenotype, Clostridium difficile and Salmonella enterica from dogs treated with antimicrobials and untreated dogs 122
Table 3.4 Cox proportional hazard models for antimicrobial resistance in fecal E. coli isolates from dogs to amoxicillin-clavulanic acid, ceftriaxone, ceftiofur and cefoxitin.. 123
Table 4.1 The frequencyo f non-topical antimicrobial prescription events for incident disease events in dogs reported by veterinarians in the submitted journals 146
Table 4.2 The frequency of non-topical antimicrobial prescription events for incident disease events in cats reported by veterinarians in the submitted journals 149
Table 4.3 Percentage of prescription events with non-topical antimicrobials for incident disease events in dogs and cats by recommended use as first, second or third line therapy in veterinary medicine and importance for use to treat human infections 152
Table 4.4 Body-site specific distributions of the duration of antimicrobial therapy for the incident disease events in dogs reported by veterinarians in the submitted journals 153
viii Table 4.5 Body-site-specific distributions of the duration of antimicrobial therapy for the incident disease events in cats reported by veterinarians in the submitted journals 154
Table 4.6 The reported dose and frequency of prescriptions with non-topical antimicrobials for incident disease events in dogs by veterinarians in the submitted journals 155
Table 4.7 The reported dose and frequency of prescriptions with non-topical antimicrobials for incident disease events in cats by veterinarians in the submitted journals 161
Table 4.8 The percent of events of feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis treated with antimicrobials 166
Table 5.1 Clinical signs of gastrointestinal (GI) or respiratory disease, or infectious diseases associated with GI or respiratory tract reported by veterinarians in southern Ontario that would require isolation or other infection control measures 193
Table 5.2 Veterinarian-reported reasons for having appropriate environmental cleaning and disinfection protocols with their veterinary clinic 194
Table 5.3 The reported frequency of specific products used for environmental disinfection by veterinarians and veterinary technicians in veterinary clinics in southern Ontario 195
Table 5.4 The frequency of the methods of preparation of disinfectants for environmental disinfection reported by veterinarians and veterinary technicians from veterinary practices in southern Ontario 196
Table 5.5 Antimicrobials used in clean non-elective surgical procedures reported by veterinarians from veterinary clinics in southern Ontario 197
IX Chapter 1
Introduction and Literature Review
1.0 Introduction
Shortly after the discovery of antimicrobials and their introduction into medical practice, acquired antimicrobial resistance (AMR) was observed to adversely impact clinical outcomes (Forbes, 1949). Currently, in human medicine, the frequency of infection associated with antimicrobial resistant organisms, including those resistant to multiple drugs, is increasing and recognized as a major threat to the treatment of infectious diseases and a major public health concern (Mulvey and Simor, 2009). In companion animal veterinary medicine, antimicrobial resistance is also a clinical and public health concern. Antimicrobial resistant infections in companion animals associated with important multiple-drug resistant organisms, such as methicillin-resistant
Staphylococcus aureus (MRS A), methicillin-resistant Staphylococcus pseudintermedius
(MRSP) and blacMY-2 positive E. coli (Carattoli et al., 2005, Weese and van Duijkeren,
2009) have been described. As humans have close contact with companion animals through social interactions and a shared household environment, it is not unexpected that zoonotic transmission (e.g., MRSA, MRSP, Salmonella enterica) between companion animals and humans have also been documented (Wright et al., 2005, Weese and van
Duijkeren, 2009). However, there has been little systematic study of antimicrobial resistance in companion animals, the factors influencing it and its significance to human health, so there is a gap in knowledge that needs to be addressed as part of the movement to improve antimicrobial stewardship.
1 The following literature review addresses aspects of antimicrobial resistance in companion animals, specifically, antimicrobial resistance in opportunistic pathogens
(e.g., Staphylococcus pseudintermedius, E. coli), commensal bacteria (e.g., fecal E. coli) and zoonotic pathogens (e.g., Salmonella enterica, MRSA), the selective pressure of antimicrobial therapy on the frequency of antimicrobial resistance, and the role of hospital-based infection control programs in the reduction of hospital-acquired infections and associated antimicrobial use to treat these infections.
2.0. Antimicrobial resistance in Escherichia coli.
2.1. Escherichia coli as an opportunistic pathogen in companion animals.
Escherichia coli are common opportunistic pathogens and fecal commensal bacteria of both dogs and cats. Infections in dogs associated with E. coli have been documented in nearly every body site including the urogenital tract, skin, bone, blood, and joints (Gibson et al., 2008). Antimicrobial susceptibility data for E. coli isolated from clinical specimens submitted to diagnostic laboratories are available (Authier et al., 2006,
Pedersen et al., 2007, Ball et al., 2008) and suggest that, in general, the frequency of antimicrobial resistance in this population of E. coli isolates is low. However, there is evidence that in this population the frequency of multi drug resistance may be increasing
(Ball et al., 2008) as well as resistance to ampicillin, cephalothin, chloramphenicol, enrofloxacin and gentamicin (Authier et al., 2006, Ball et al., 2008).
Monitoring antimicrobial susceptibility data derived from diagnostic submissions is useful in identifying possible changes in the frequency of resistance and the emergence of new patterns or multiple-drug resistance, including resistance phenotypes associated with clinically and epidemiologjcally important genotypes (e.g., blacMY-2, blacrx-M)-
2 There are, however, limitations to interpreting and utilizing antimicrobial susceptibility data derived from diagnostic laboratory submissions; for example, companion animal veterinarians infrequently submit diagnostic samples for bacterial culture and susceptibility testing (Reid-Smith et al., 2002). Additionally, much of the reported data is derived from diagnostic laboratories affiliated with veterinary teaching hospitals involved in tertiary care rather than primary care. This biases the data since the source population is either not well defined, not representative of clinical bacterial infections treated in community veterinary practices, or both. Without understanding the sources from which the data are derived it is difficult to interpret them, including changes in resistance patterns and apply the information to the general population of dogs and cats in the community.
Furthermore, data derived from diagnostic laboratory data may represent "worst- case" scenarios, including therapeutic failures or recurrent infections previously treated with antimicrobials. This could over-estimate the true frequency of antimicrobial resistance in isolates associated with clinical infection in general practice. This suggestion is supported by the observation that the number of antimicrobials to which E. coli isolates from canine urine samples were resistant increased significantly over time in recurrent but not in non-recurrent infections (Ball et al., 2008). Depending on the purpose for which the data are being used, such as guidance for antimicrobial drug choice, increasing the frequency of submission of clinical isolates by veterinarians and better understanding of the source population would greatly improve the usefulness of data derived from diagnostic laboratories.
2.2, Escherichia coli as commensal organisms in companion animals.
3 Antimicrobials are important tools in the successful treatment or prevention of bacterial infections. However, their effect is not limited to the target organisms at the target site; for example, antimicrobials administered for skin infection also exert selective pressure on commensal organisms in other sites, such as the gastrointestinal tract. This is important since the gastrointestinal tract is a reservoir for commensal bacteria and zoonotic and opportunistic pathogens. It is a site where antimicrobial resistant determinants can readily be transmitted between commensal bacteria and pathogens, including transmission between bacterial genera that may be of zoonotic concern (Poppe et al., 2005, Jiang et al., 2006). Previous studies have investigated antimicrobial resistance among generic E. coli isolated from fecal samples or fecal swabs from healthy companion animals (Costa et al., 2008, Murphy et al., 2009). In one study, the prevalence of antimicrobial resistance in E. coli was lower than reported from clinical samples submitted to diagnostic laboratories (Murphy et al., 2009), but in another, the prevalences of resistance to streptomycin and tetracycline were similar to those reported from diagnostic laboratory data (Costa et al., 2008). These variations may be due to differences in source populations or various other factors, including temporal or spatial factors, antimicrobial use patterns, sample types (e.g., feces versus fecal swabs), and isolation methodology. Although the prevalence of antimicrobial resistance in these E. coli populations was generally low, both studies observed some multiple-drug resistance
(up to 8 different antimicrobials) and both demonstrated important genes associated with multiple-drug resistance (e.g., bhcMY-2 and 6/OCTX-M-I), albeit at very low frequencies.
These particular genes are associated with resistance to P-lactam antimicrobials including higher-generation cephalosporins, cephamycins, monobactams and p-lactam inhibitor
4 combinations (Jacoby, 2009), and can be located on plasmids (Jacoby, 2009, Murphy et al., 2009). The mobility of these genetic elements is important in a reservoir site like the gastrointestinal tract because it provides them with the opportunity for transfer to other bacteria, including zoonotic or other pathogens (Poppe et al., 2005, Jiang et al., 2006), and this transfer may be enhanced by antimicrobial selection pressures within the gut environment.
3.0 Salmonella enterica
Salmonella enterica are zoonotic bacteria, and infections in humans have been associated with companion animals (Sato et al., 2000, Cherry et al., 2004, Wright et al.,
2005), dry dog and cat food (CDC 2008a, CDC 2008b), and dog treats (Government of
Canada 2000, CDC 2006). However, the magnitude of risk to human health from exposure to Salmonella from companion animals is unknown. This is worthy of further investigation since the Salmonella serovars recovered from the feces of dogs, in particular S. Heidelberg, are frequently associated with human clinical infections
(Government of Canada 2008).
The estimated prevalence of fecal colonization of healthy dogs and cats by
Salmonella enterica ranges from less than one percent to 10% (Hackett and Lappin,
2003, Kozak et al., 2003, Lefebvre et al., 2006, Tsai et al., 2007, Lefebvre et al., 2008,
Murphy et al., 2009). One study reported a high point prevalence of carriage in healthy dogs (up to 25%) fed a raw food diet (Lefebvre et al. 2008), which has in several studies been associated with fecal shedding of Salmonella enterica in dogs (Morley et al., 2006,
Finley et al., 2007, Lefebvre et al., 2008, Lenz et al., 2009).
4.0Beta-lactamase cephamycinase-2 (W«CMY-2)
5 The p-lactamase CMY-2 is an important determinant of P-lactam resistance in
non-typhoid Salmonella enterica in many countries (Jacoby, 2009). It has primarily been
described in Salmonella enterica from infections in humans (Jacoby, 2009) and animals
(Winokur et al., 2000, Allen and Poppe, 2002, Gray et al., 2004, Diarrassouba et al.,
2007, Daniels et al., 2009, Jacoby, 2009, Rodriguez et al, 2009). blacm-i positive E. coli have been isolated from companion animals, including healthy dogs (Carattoli et al.,
2005, Murphy et al., 2009) and from organs of a dog at necropsy (Carattoli et al., 2005).
Zoonotic transmission of blacm-2 also occurs; WacMY-2 positive Salmonella infection of humans has been associated with cattle (Fey et al., 2000), and pet treats (Pitout et al.,
2003).
5.0 Methicillin resistant Staphylococcus aureus
Recently, the volume of published literature pertaining to MRS A and companion animals has vastly increased. A comprehensive review of this literature is beyond the scope of this review; however, specific epidemiological and clinical features of MRSA will be briefly discussed. Methicillin-resistant Staphylococcus aureus is primarily an opportunistic pathogen of humans, typically categorized into hospital-associated and community-associated infections, and colonization of apparently healthy individuals
(Leonard and Markey 2008). Resistance to p-lactam antimicrobials in MRSA is commonly mediated by the mecA gene encoding for an altered penicillin-binding protein
(PBP2a) with a low affinity for P-lactam antimicrobials (Leonard and Markey 2008, van
Duijkeren et al., 2008). Although primarily associated with humans, MRSA has been reported to colonize and infect many animal species, including companion animals
(Weese and van Duijkeren 2009). Several studies have estimated the prevalence of
6 MRSA community colonization in pets to be approximately one percent, (Baptiste et al.,
2005, Lefebvre et al, 2006, Moodley et al., 2006, Rich and Roberts, 2006, Vengust et al.,
2006, Abbott et al., 2007, Abraham et al., 2007, Boost et al., 2007, Hanselman et al.,
2008, Murphy et al., 2009), which is similar to the estimated prevalence of community colonization in humans without known risk factors (Graham et al., 2006, Kuehnert et al.,
2006, Gorwitz et al., 2008); however, some studies have reported estimates up to 4%
(Abraham et al., 2007, Kottler et al 2008). In addition to the low apparent prevalence of community MRSA colonization in companion animals, the results of one study also suggested that transmission of MRSA between apparently healthy dogs was a rare event
(Loeffler et al., 2009). The reported prevalence of MRSA colonization in companion animal veterinarians was slightly higher (4%) than the general population and the veterinarians were predominately colonized with human strains of MRSA common in the community (Hanselman et al., 2006). Further research is required to characterize the possible occupational risk for MRSA colonization in veterinarians; however, it is possible that close, frequent contact with companion animals, including pets colonized or infected with MRSA, may be the source of colonization for these veterinarians.
Opportunistic MRSA infections affecting most body sites of companion animals have been documented (Tomlin et al., 1999, Baptiste et al., 2005, Leonard et al., 2006,
Morris et al., 2006a, Morris et al., 2006b, Vitale et al., 2006, Weese et al., 2006a, Weese et al., 2007, Griffeth et al., 2008, Schwartz et al., 2009). A case-control study that investigated risk factors associated with MRSA infection (versus methicillin-susceptible
S. aureus (MSSA)) in companion animals demonstrated that fluoroquinolone use was
7 associated with MRS A infection, and the survival of animals with MRS A infections was
not significantly different than those with MSSA infections (Faires, 2008).
Various studies have reported isolation of the same strains of MRS A from
companion animal and human infections in particular geographical regions (Baptiste et
al., 2005, Loeffler et al., 2005,0'Mahony et al., 2005, Leonard et al., 2006, Malik et al.,
2006, Moodley et al., 2006, Weese et al., 2006a, Weese et al., 2007, Grinberg et al.,
2008, Faires et al., 2009). Given this commonality, it is not surprising that probable transmission of MRS A between people and companion animals has been documented
(van Duijkeren et al., 2005, Leonard et al., 2006, Weese et al., 2006a, Nienhoff et al.,
2009), and that within households, people and companion animals may be colonized or infected with indistinguishable strains (Kottler et al 2008, Faires et al., 2009). The frequency of transmission between people and pets is not fully known, however, some data suggest that the frequency is low (Kottler et al 2008, Faires et al., 2009). However, it is also possible that the duration of colonization (pet or human) following transmission is short, resulting in the apparently low transmission frequency observed in studies lacking repeated or longitudinal sampling.
6.0 Methicillin-resistant Staphylococcus pseudintermedius
Methicillin-susceptible Staphylococcus pseudintermedius (formerly S. intermedius) (MSSP) is a common opportunistic pathogen of companion animals, primarily associated with skin and soft tissue infections (Cox et al., 1984). Methicillin- resistance, mediated by the mecA gene, has been documented and reports of multi-drug resistant methicillin-resistant S. pseudintermedius infections are increasing, particularly in Europe (Loeffler et al., 2007, Wettstein et al., 2008, Ruscher et al., 2009). Like MSSP,
8 these MRSP infections are commonly associated with skin and soft tissues (Loeffler et
al., 2007, Ruscher et al., 2009). The estimated prevalence of community colonization is
1.5% to 2% in dogs (Vengust et al., 2006, Griffeth et al., 2008, Hanselman et al., 2008)
and 4% in cats (Abraham et al., 2007).
The reported prevalence of MSSP colonization in humans is from 0.3% to 0.7%
(Talan et al., 1989b, Mahoudeau et al., 1997). Although the origin of some human MSSP
and MRSP infections is unknown (Vandenesch et al., 1995, Gerstadt et al., 1999, Atalay
et al., 2005, Campanile et al., 2007), colonization and infections in humans associated
with (probable) zoonotic transfer of MSSP has occurred (Talan et al., 1989a, Tanner et
al., 2000, Guardabassi et al., 2004, Kikuchi et al., 2004, Kempker et al., 2009).
Colonization associated with multi-drug resistant MRSP has also been reported (Sasaki et
al., 2007, van Duijkeren et al., 2008). Therefore, MRSP is a concern for both animal and human health; however, the role that humans play in the epidemiology of MSSP and
MRSP is poorly understood. In addition, isolates of MSSP and MRSP are sometimes misclassified as MSSA or MRSA, respectively (Talan et al., 1989a, Kempker et al.,
2009).
7.0 Hospital-acquired infections and veterinary hospital-based infection control
In the 1970s the Centres for Disease Control and Prevention (CDC) established the National Nosocomial Infection Surveillance system to integrate human hospital- surveillance activities and to further develop infection control programs. Concurrently, the CDC also initiated the Study on the Efficacy of Nosocomial Infection Control, a national epidemiological study on the impact of hospital infection control and surveillance activities (Hughes, 1987). The Canadian Nosocomial Infection Surveillance
Program was established in 1994 with the objectives to provide national rates and trends on 9 nosocomial infections in Canadian health care facilities, establish "bench mark" data to
enable comparison of rates by Canadian health care facilities, and provide evidence-based
data for the development of national guidelines on clinical issues related to nosocomial
infections. In companion animal medicine, hospital-based infection control has not been the subject of the same degree of investigation (Morley et al., 2005b), research studies on infection control in veterinary medicine are limited in number and scope (Benedict et al.,
2008, Wright et al., 2008), and development and implementation of standards is limited.
Recently, infection control manuals and compendia for veterinary practices have been published (CCAR, 2008, Elchos et al., 2008); however, there are few objective, companion animal-specific data upon which to base guideline development and assessment; many of the recommendations derived in these documents are extrapolated from human data and resources.
Reports of hospital-associated or acquired infections (HAI) in companion animal medicine are primarily limited to outbreaks associated with species-specific or opportunistic pathogens, such as feline calicivirus (Schorr-Evans et al., 2003),
Clostridium difficile (Weese and Armstrong, 2003) and Clostridum perfringens (Kruth et al., 1989), and zoonotic pathogens, such as MRSA (Weese et al., 2006a) and animal and human infections with multi-drug resistant Salmonella (Cherry et al., 2004, Wright et al.,
2005). Outbreak investigations have a role in elucidating the epidemiology of HAI by
^identifying possible pathogens, site or type of infections (e.g., surgical site infections,
(urinary/venous/arterial) catheter associated infections, diarrhea) and factors that may be associated with the outbreak (e.g., groups at risk, risk areas like specific wards). This knowledge may provide insight into potential interventions, as well as areas in need of further investigation, and provide some of the framework required to build infection
10 control policies for HAI in veterinary hospitals. Unfortunately, there are few published
data available on endemic HAI in companion animals; acquiring useful information in
this area is difficult. One challenge is that most community, primary-care veterinary practices are small. Even tertiary-care, referral veterinary practices are relatively small when compared to human hospitals; therefore, they have a very small population at risk.
Since infection control is an emerging discipline in companion animal medicine, most general practice veterinarians do not have the training, skills and resources to investigate
HAI outbreaks, to develop or implement surveillance systems that may be required to gather endemic HAI data, or to interpret and fully use any acquired data. Furthermore, there is no established network in Ontario for companion animal infection control to support further development in the area.
Investigations into endemic HAI in companion animals have mostly involved urinary-catheter-associated urinary tract infections (UTI) (Smarick et al., 2004, Ogeer-
Gyles et al., 2006b, Bubenik et al., 2007) or post-operative surgical site infections (SSI)
(Olmstead et al., 1983, Vasseur et al., 1985, Vasseur et al., 1988, Whittem et al., 1999,
Bergh et al., 2006, Weese and Hailing, 2006b, Lafaver et al., 2007). Estimates of the incidence of UTI in hospitalized dogs with urinary catheters, predominately associated with E. coli, range from 10% to 86% of dogs (Smarick et al., 2004, Ogeer-Gyles et al.,
2006b). Smarick et al. (2004) reported an incidence of 5 UTI per 100 dog days and observed that antimicrobial administration was protective for its development. Bubnik et al. (2007) investigated risk factors for UTI in hospitalized dogs with urinary catheters and found that age, duration of catheterization and antimicrobial administration were positively associated with UTI. The study was cross-sectional in design, therefore,
11 causality cannot be inferred, especially when interpreting the association between antimicrobial administration and UTI, and the authors did not distinguish between hospital-acquired and hospital-expressed UTI. Another limitation to these studies was the absence of UTI case definition, including HAI-UTI.
The reported incidence of SSI in companion animals ranges from <1-18%
(Olmstead et al., 1983, Parker et al 1984, Vasseur et al., 1985, Paul et al 1987, Vasseur et al., 1988, Brown et al., 1997, Whittem et al., 1999, Bergh et al., 2006, Weese and
Hailing, 2006, Lafaver et al., 2007). This variation is partially due to differences in case definitions for SSI and study populations (e.g., all types of surgical procedures, cranial cruciate surgery, only dirty surgeries). Surgical site infections in companion animals have been positively associated with duration of surgery or anaesthesia (Brown et al., 1997,
Heldmann et al., 1999, Nicholson et al., 2002, Eugster et al., 2004), sex (higher in males),
(Nicholson et al., 2002, Bergh et al., 2006), and negatively associated with antimicrobial prophylaxis (Vasseur et al., 1988, Eugster et al., 2004). Other factors have also been identified in cross-sectional studies: increased body weight, higher ASA (American
Society of Anesthesiologists) score, the number of people present during surgery, dirty wounds, length of post-operative stay in ICU, placement of a drain (Eugster et al., 2004), concurrent endocrinopathy (Nicholson et al., 2002), propofol use, and time from clipping to surgery (Brown et al., 1997, Heldmann et al., 1999). The data in these studies were all obtained from referral, tertiary care veterinary hospitals, sometimes involving surgical procedures infrequently or never performed in community, companion animal practice
(e.g., total hip replacements or tibial tuberosity advancement). Currently, no published data are available describing the frequency of SSI in community animal practice or
12 associated risk factors, which is important since the patient population in this type of
practice is likely to be different from that in referral institutions; and differences are
likely in other possibly important factors, such as the types of surgical procedures,
anaesthesia protocols, duration of hospitalization, and experience of surgeons and other
veterinary personnel that may affect SSI. Surgical site infections are a possible adverse
event following surgery, but understanding the factors that are positively or negatively
associated with SSI is important to reducing the frequency of this complication.
Infection control in veterinary hospitals may also be important for the protection
of animal and human health. Recently, results of surveys addressing zoonotic diseases
among veterinarians, veterinary practices and characteristics of infection control programs have been published (Benedict et al., 2008, Lipton et al., 2008, Wright et al.,
2008). In general, these surveys identified possible deficiencies in infection control within veterinary hospitals, particularly with zoonotic disease prevention, and highlight possible inconsistencies between risk perceptions by veterinarians and their preventive and risk reduction actions. For example, only 23% of companion animals veterinarians self-reported a current rabies titre status (Wright et al., 2008), 92% reported that they recap needles prior to disposal (Wright et al., 2008), and 50% of the respondents described eating and drinking in animal handling areas (Lipton et al., 2008, Wright et ah,
2008). Furthermore, Wright et al. (2008) reported that approximately 71% of respondents that were concerned about rabies, gastrointestinal zoonoses or dermatophytosis did not use appropriate protective personal equipment when examining patients with clinical signs compatible with these diseases (e.g., neurologic signs, diarrhea, or skin lesions).
13 Lipton et al. (2008) reported that 28% of veterinarian respondents had at some
time in the past experienced a zoonotic disease, the most frequently reported of which were dermatophytosis, cat scratch fever and animal bites and that the length of time in
clinical practice was positively associated with having a zoonotic disease. Benedict et al.
(2008) reported that 50% of responding veterinary teaching hospitals reported significant health problems associated with zoonotic infections in hospital personnel in the previous
2 years, most frequently Cryptosporidium parvum, MRSA, S. enterica and unindentified cutaneous dermatophytes. The differences in the findings of these two studies are possibly related to different study populations (community veterinary practices versus veterinary teaching hospitals) and focus on infectious agents versus disease (e.g., cat scratch fever).
8.0 Antimicrobial drug use in companion animals
Antimicrobial drug use in companion animals has been described in several studies or surveillance programs (Table 1.1) (Watson and Maddison, 2001, Odensvik et al., 2001, Prescott et al., 2002, Rantala et al., 2004a, Holso et al., 2005, Weese 2006,
DANMAP 2007). Although these studies were geographically and temporally distinct, and varied vastly in terms of design, study populations and measure of outcome, most reported (3-lactams as the predominant class of antimicrobials used in companion animals. First generation cephalosporins, amoxicillin and amoxicillin-clavulanic acid were consistently the P-lactams most frequently used in companion animals. However, in
Norway, where first generation cephalosporins were not approved for use in companion animals, trimethoprim/sulphonamide combinations were the predominant antimicrobials used (Odensvik et al., 2001).
14 In Denmark, antimicrobials are available only under prescription and are
dispensed mainly through pharmacies. Antimicrobial use data are systematically
collected through either VetStat (veterinary use) or the Danish Medicines Agency
(human use) (DANMAP 2007) and compiled in DANMAP annual reports. Relative to
other species, antimicrobial use in companion animals (mainly dogs and cats) in
Denmark in 2007 was among the lowest in terms of the quantity (tonnes active compound) of antimicrobials used: pigs;91, humans;50, cattle;15, aquaculture;4, fur- bearing species;2, companion animals;2, poultry;0.6 and horses;0.2. Notwithstanding the relatively low overall tonnes of active compound, companion animal use constituted 53% and 30% of the total veterinary consumption of cephalosporins and fluoroquinolones, respectively. DANMAP further compared antimicrobial use among food-producing species using standardized units: "Animal Defined Daily Doses" (ADDxx), average daily maintenance dose for a "standard animal" (defined by the assumed average bodyweight of the relevant age group within the particular animal species) or "Animal Dose Course"
(ADC, per kilogram live weight or age group) to adjust for the duration of treatment
(DANMAP 2007). These use measures were not calculated for companion animals, possibly because of difficulty in defining an average bodyweight for companion animals, although other authors have described antimicrobial use in dogs in terms of ADD
(Prescott et al., 2002). Advantages of this type of metric include its inherent adjustment for concentrations of antimicrobials, dosing and the ability to compare antimicrobial use across animal species, among different studies and over time.
Three studies described changes over time in antimicrobial use in companion animals (Odensvik et al., 2001, Prescott et al., 2002, Weese 2006). Only one (from a
15 tertiary care teaching veterinary hospital) described statistically significant changes in antimicrobial use during the study period (1995-2004), including a significant decrease in the number of prescriptions per 1000 admissions with significant reductions over time in prescriptions for first generation cephalosporin, fluoroquinolone, penicillin and trimethoprim-sulfonamide and an increase for metronidazole (Weese 2006). However, the study did not identify any factor(s) associated with the observed changes, or whether the changes were attributable to differing source populations from which the data were derived.
No published studies have as yet described the frequency of inappropriate or imprudent use of antimicrobials by companion animals. Guidelines for the prudent use of antimicrobials have been published by many professional veterinary organisations
(CVMA 2000, AAFP 2001, Morley et al., 2005, AAFP/AAHA 2006, AVMA 2006).
These guidelines are typically very general in nature (e.g., recommend use of antimicrobials only when an infection is present or likely present, promote use of culture and susceptibility testing as a guide to therapy) and do not provide specific recommendations on use of particular antimicrobials. Numerous other resources are available to veterinarians (e.g., formularies, textbooks, studies or reviews in peer- reviewed journals, on-line written materials, manufacturer resources) to assist in empiric antimicrobial selection and use, including recommendations for antimicrobial use for specific diseases, bacterial organism or body sites. These resources do not, however, typically include consideration of antimicrobial stewardship or prudent use; their recommendations are primarily derived from clinical susceptibility patterns (which as
16 noted earlier can be misleading) and pharmacokinetic/pharmacodynamic properties and presumably were not developed with prudent use principles in mind.
Use of susceptibility patterns from diagnostic laboratory data to derive recommendations on antimicrobial selection is, in part, based on the assumption that these data are representative of the population to which the recommendations will be applied. The validity of this assumption is, however, usually unknown. In a survey of
Ontario companion animal veterinarians conducted in 2002, respondents reported infrequent submission of samples for bacterial culture and antimicrobial susceptibility testing (Reid-Smith et al., 2002). Additionally, antimicrobial susceptibility data in published studies are often derived from diagnostic laboratories affiliated with tertiary- care teaching hospitals (Authier et al., 2006, Pedersen et al., 2007, Ball et al., 2008). The extent to which isolates from these settings are representative of the infectious agents and associated susceptibility patterns encountered in general practice is unknown, but some lack of unrepresentativeness can be assumed.
The prudent use guidelines published by the American College of Veterinary
Internal Medicine (ACVIM) (Morley et al., 2005) recommended development of a hierarchy of antimicrobial selection. One author has proposed such a hierarchy for companion animal practice based on the above guidelines, comprising first, second, and third-line antimicrobials (Weese 2006). The proposed hierarchy deems that first-line antimicrobials (e.g., penicillins including potentiated penicillin, first and second generation cephalosporins) are appropriate for empirical therapy, second-line antimicrobials (e.g., fluoroquinolones, third generation cephalosporins) are appropriate for use when bacterial culture and antimicrobial susceptibility data indicate resistance to
17 first-line antimicrobials, and third-line antimicrobials (e.g., carbapenems and
vancomycin) are appropriate to use only when bacterial culture and antimicrobial
susceptibility data indicate resistance to both first and second-line antimicrobials.
Unfortunately, this is the only specific published classification that is currently available to aid companion animal veterinarians with antimicrobial selection in terms that are explicitly intended to enhance prudent use. Critical assessment of this classification system is not possible since the methods and criteria used to derive the classification system were not defined. The author referred to the ACVIM recommendations (Morley et al., 2005) to classify antimicrobials into primary, secondary or tertiary categories.
Primary antimicrobials were defined as older antimicrobials and those with a narrow spectrum (e.g., simple penicillins, tetracyclines, sulfonamides). Secondary antimicrobials were newer, with an extended spectrum (compared to primary antimicrobials) and greater importance to the treatment of serious or frequently antimicrobial-resistant infections in humans. Tertiary antimicrobials were defined as antimicrobials that are very important for human and animal health care, with extended spectra of coverage and useful against most resistant bacteria. The AC VIM also recommended considering objective data regarding the efficacy, toxicity and predisposition for use, effect of antimicrobial use on the prevalence of antimicrobial resistance in target and bystander bacterial populations, and importance to human health as a guide to antimicrobial selection. However, some of the recommended objective data are not publicly available (e.g., effect of antimicrobial use on the prevalence of antimicrobial resistance in target and bystander bacterial populations), while availability of other data, such as efficacy, are limited due to the lack of research in this area and in available diagnostic laboratory data.
18 Antimicrobial selection is a component of prudent use of antimicrobials; however, other factors such as dose and duration of therapy also contribute. Numerous clinical and experimental studies have investigated antimicrobial use in cats an dogs, including comparisons between antimicrobials for specific infections (Bywater et al., 1985, Frank and Kunkle 1993, Cotard et al., 1995, Dossin et al., 1998, Sturgess et al., 2001, Gerhardt et al., 2006, Stegemann et al., 2007a, Stegemann et al., 2007b, Ruch-Gallie et al., 2008,
Six et al., 2009) and different dosing regimes for individual antimicrobials (Senior et al.,
1985, Lloyd et al., 1997, Toma et al., 2008). The results of experimental trials suggested that short-duration treatment (1-3 days) was insufficient to clear urinary tract infection in dogs (Turnwald et al., 1986, Rogers et al., 1988); however, no published clinical trials have been conducted to determine optimal therapeutic regimens, including antimicrobial selection, dose, duration for elimination of any bacterial infection of dogs, nor how such optimal therapy affects antimicrobial resistance in pathogens or commensals from target or non-target sites (e.g., gastrointestinal tract). Some epidemiological studies have demonstrated that previous antimicrobial exposure (not necessarily optimal therapy) was associated with the prevalence of resistance to certain antimicrobials in commensal E. coli (Ogeer-Gyles et al. 2006a, Murphy et al., 2009), opportunistic pathogens (Medleau et al., 1986, Rantala et al., 2004b), including MRSA (Faires, 2008), and with colonization with C. difficile (Clooten et al., 2008, Lefebvre et al., 2009). An experimental study demonstrated that dogs treated with enrofloxacin were more effectively colonized with multidrug- resistant E. coli (Trott et al., 2004). However, caution is needed in drawing inferences from some of these studies. Cross-sectional and case-control studies can be subject to reverse-causation for factors that are time-variant, such as antimicrobial
19 exposure, and study subjects and conditions in experimental trials may not be representative of community populations.
In conclusion, this literature review shows the need for studies of common infections in companion animal practice, including associated bacterial agents and their antimicrobial susceptibility patterns and optimal therapies including dose, frequency of administration and duration of therapy. Such studies could provide evidence to support antimicrobial selection for empiric therapy and increase understanding of the effects of treatment-related factors on the occurrence of antimicrobial resistance.
9.0 Research objectives
To better understand the epidemiology of antimicrobial resistance in selected bacteria from companion animals presented to community veterinary hospitals and the role of infection control in community veterinary hospitals on the epidemiology of antimicrobial resistance, the following research objectives were established: 1) to compare the incidence of antimicrobial resistance in generic fecal E. coli isolates and the isolation of C. difficile, Salmonella enterica, blacMY-2 positive E. coli, MRS A, MRSP and vancomycin-resistant Enterococcus spp. (VRE) from dogs treated with antimicrobials to untreated, healthy dogs; 2) to determine the associations between antimicrobial treatment and the risk of antimicrobial resistance in fecal E. coli isolates and the isolation of fecal
C. difficile, Salmonella enterica, blacMY-2 positive E. coli, MRSA, MRSP and VRE; 3) to describe non-topical antimicrobial (oral and parenteral) use in dogs and cats; 4) to compare antimicrobial use in dogs and cats in general veterinary practice against published formulary doses (mg/kg) and frequencies of administration; 5) to evaluate antimicrobial use in feline upper respiratory tract disease, feline lower urinary tract
20 disease and canine infectious tracheobronchitis (diseases where antimicrobial use
generally is not indicated); 6) to determine the environmental recovery of E. coli, C. difficile, Salmonella enterica, MRSA, MRSP, feline calicivirus and canine parvovirus from general practice community veterinary hospitals; and 7 ) to evaluate specific infection control practices in veterinary practices including environmental disinfection, management of infectious patients and antimicrobial use in clean surgical procedures.
Objectives 1 and 2 were investigated through a cohort study of dogs that were treated with antimicrobials and healthy dogs with no recent antimicrobial treatment. The research hypotheses were that treatment with antimicrobials would increase the frequency of antimicrobial resistance in fecal E. coli isolates and the recovery of C. difficile,
Salmonella enterica, blacwt-2 positive E. coli, MRSA, MRSP and VRE (H0 tested: that antimicrobial therapy was not associated with the above). Objectives 3-5 were investigated using a cross-sectional study in which companion animal veterinarians completed journals describing incident disease events and associated prescription treatments including antimicrobials. The hypotheses were that antimicrobials (in particular pMactams) would be used commonly for the treatment of incident disease events, and that antimicrobials may be used for disease conditions where antimicrobial treatment was not generally indicated. Objectives 6 and 7 were investigated using a cross-sectional study of companion animal veterinary hospitals where environmental sites were swabbed for various microorganisms, and veterinarians and veterinary technicians completed a survey on specific infection control practices. The hypotheses were that bacteria can be recovered from environmental sites within veterinary practices and deficiencies in specific infection control practices are associated with the recovery of
21 environmental of bacteria (H0 tested: specific infection control practices were not associated with recovery of environmental bacteria).
22 References
Abbott, Y, N Leonard, B Markey, A Rossney, J Dunne. 2007. Persistence of MRS A
infection. Vet Rec, v. 160, p. 851-2.
Abraham, JL, D O Morris, G C Griffeth, F S Shofer, S C Rankin. 2007. Surveillance of
healthy cats and cats with inflammatory skin disease for colonization of the skin by
methicillin-resistant coagulase-positive staphylococci and Staphylococcus schleiferi ssp.
schleiferi. Vet Dermatol, v. 18, p. 252-9.
Allen, KJ, C Poppe. 2002. Occurrence and characterization of resistance to extended-
spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from
food-producing animals in Canada. Can J Vet Res, v. 66, p. 137-44.
American Association of Feline Practitioners (AAFP). 2001. Basic guidelines of judicious therapeutic use of antimicrobials in cats.
http://www.catvets.com/uploads/PDF/antimicrobials.pdf. Accessed September 2009.
American Association of Feline Practitioners/American Animal Hospital Association
(AAFP/AAHA). 2006. Basic guidelines of judicious therapeutic use of antimicrobials. http://www.aahanet.org/PublicDocuments/AAFP_AAHA_AntimicrobialGuidelines.pdf.
Accessed September 2009.
American Veterinary Medical Association (AVMA). 2006. American association of feline practitioners/American animal hospital association basic guidelines of judicious
23 therapeutic use of antimicrobials. Revised 2009. http://www.avma.org.subzero.lib.uoguelph.ca/issues/policy/jtua_aaQ)_aaha.asp. Accessed
September 2009.
Atalay, B, F Ergin, M Cekinmez, H Caner, N Altinors. 2005. Brain abscess caused by
Staphylococcus intermedins. Acta Neurochir v. 147, p. 347-8.
Authier, S, D Paquette, O Labrecque, S Messier. 2006. Comparison of susceptibility to
antimicrobials of bacterial isolates from companion animals in a veterinary diagnostic
laboratory in Canada between 2 time points 10 years apart. Can Vet J, v. 47, p. 774-8.
Ball, KR, J E Rubin, M Chirino-Trejo, P M Dowling. 2008. Antimicrobial resistance and
prevalence of canine uropathogens at the Western College of Veterinary Medicine
Veterinary Teaching Hospital, 2002-2007. Can Vet J, v. 49, p. 985-90.
Baptiste, KE, K Williams, N J Willams, A Wattret, P D Clegg, S Dawson, J E Corkill, T
O'Neill, C A Hart. 2005. Methicillin-resistant staphylococci in companion animals.
Emerg Infect Dis, v. 11, p. 1942-4.
Benedict, KM, P S Morley, D C Van Metre. 2008. Characteristics of biosecurity and
infection control programs at veterinary teaching hospitals. J Am Vet Med Assoc, v. 233,
p. 767-73.
Bergh, MS, R S Gilley, F S Shofer, A S Kapatkin. 2006. Complications and radiographic
24 findings following cemented total hip replacement: a retrospective evaluation of 97 dogs.
Vet Comp Orthop Traumatol. v. 19, p. 172-9.
Boost, MV, M M O'Donoghue, K H Siu. 2007. Characterisation of methicillin-resistant
Staphylococcus aureus isolates from dogs and their owners. Clin Microbiol Infect, v. 13,
p. 731-3.
Brown, DC, M G Conzemius, F Shofer, H Swann. 1997. Epidemiologic evaluation of postoperative wound infections in dogs and cats. J Am Vet Med Assoc, v. 210, p. 1302-6.
Bubenik, LJ, G L Hosgood, D R Waldron, L A Snow. 2007. Frequency of urinary tract infection in catheterized dogs and comparison of bacterial culture and susceptibility testing results for catheterized and noncatheterized dogs with urinary tract infections. J
Am Vet Med Assoc, v. 231, p. 893-9.
Bywater, RJ, G R Hewett, A B Marshall, B West. 1985. Efficacy of clavulanate- potentiated amoxycillin in experimental and clinical skin infections. Vet Rec, v. 116, p.
177-9.
Campanile, F, D Bongiorno, S Borbone, M Venditti, M Giannella, C Franchi, S Stefani.
2007. Characterization of a variant of the SCCmec element in a bloodstream isolate of
Staphylococcus intermedius. Microb Drug Resist, v. 13, p. 7-10.
25 Canadian Committee on Antibiotic Resistance (CCAR). 2008. Infection Prevention and
Control Best Practices for Small Animal Veterinary Clinics.
Canadian Veterinary Medical Association (CVMA), 2000, Guidelines on the prudent use of antimicrobial drugs in animals. http://canadianveterinarians.net/Documents/Resources/Files/85_Resources_Prudent-Use- of-Antimicrobial-Drugs-in-Animals.pdf. Accessed September 2009.
Carattoli, A, S Lovari, A Franco, G Cordaro, P Di Matteo, A Battisti. 2005. Extended- spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy, from 2001 to 2003. Antimicrob Agents Chemother, v. 49, p. 833-5.
Centers for Disease Control and Prevention (CDC). 2008. Multistate outbreak of human
Salmonella infections caused by contaminated dry dog food—United States, 2006-2007.
Morb Mortal Wkly Rep, v. 57, p. 521-4.
Centers for Disease Control and Prevention (CDC). 2008b. Update: recall of dry dog and cat food products associated with human Salmonella Schwarzengrund infections—United
States, 2008. Morb Mortal Wkly Rep, v. 57, p. 1200-2.
Centers for Disease Control and Prevention (CDC). 2006. Human salmonellosis associated with animal-derived pet treats—United States and Canada, 2005. Morb Mortal
Wkly Rep, v. 55, p. 702-5.
26 Cherry, B, A Burns, G S Johnson, H Pfeiffer, N Dumas, D Barrett, P L McDonough, M
Eidson. 2004. Salmonella Typhimurium outbreak associated with veterinary clinic.
Emerg Infect Dis, v. 10, p. 2249-51.
Clooten, J, S Kruth, L Arroyo, J S Weese. 2008. Prevalence and risk factors for
Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit.
Vet Microbiol, v. 129, p. 209-14.
Costa, D, P Poeta, Y Saenz, A C Coelho, M Matos, L Vinue, J Rodrigues, C Torres.
2008. Prevalence of antimicrobial resistance and resistance genes in faecal Escherichia
coli isolates recovered from healthy pets. Vet Microbiol, v. 127, p. 97-105.
Cotard, JP, P Gruet, D Pechereau, P Moreau, J P Pages, E Thomas, J Deleforge. 1995.
Comparative study of marbofloxacin and amoxicillin-clavulanic acid in the treatment of
urinary tract infections in dogs. J Small Mm Pract, v. 36, p. 349-53.
Cox, HU, S S Newman, A F Roy, J D Hoskins.1984. Species of Staphylococcus isolated from animal infections. Cornell Vet. v. 74, p. 124-35.
Daniels, JB, D R Call, D Hancock, W M Sischo, K Baker, T E Besser. 2009. Role of ceftiofur in selection and dissemination of WacMY-2-mediated cephalosporin resistance in
Salmonella enterica and commensal Escherichia coli isolates from cattle. Appl Environ
Microbiol, v. 75, p. 3648-55.
27 DANMAP. 2007. Use of antimicrobial agents and occurrence of antimicrobial resistance
in bacteria from food animals, foods and humans in Denmark, http://www.danmap.org.
Accessed September 2009.
Diarrassouba, F, M S Diarra, S Bach, P Delaquis, J Pritchard, E Topp, B J Skura. 2007.
Antibiotic resistance and virulence genes in commensal Escherichia coli and Salmonella
isolates from commercial broiler chicken farms. J Food Prot, v. 70, p. 1316-27.
Dossin, O, P Gruet, E Thomas. 1998. Comparative field evaluation of marbofloxacin
tablets in the treatment of feline upper respiratory infections. J Small Anim Pract, v. 39, p. 286-9.
Elchos, BL, J M Scheftel, B Cherry, E E DeBess, S G Hopkins, J F Levine, C J Williams.
2008. Compendium of veterinary standard precautions for zoonotic disease prevention in veterinary personnel. J Am Vet Med Assoc, v. 233, p. 415-32,415-32.
Eugster, S, P Schawalder, F Gaschen, P Boerlin. 2004. A prospective study of postoperative surgical site infections in dogs and cats. Vet Surg. v. 33, p. 542-50.
Faires, MC. 2008. Evaluation of methicillin-resistant Staphylococcus aureus and characterization of S. pseudintermedius in dogs and cats. (A thesis presented to the
Faculty of Graduate Studies of the University of Guelph in partial fulfillment of requirements for the degree of Master of Science) University of Guelph, Guelph.
28 Fairest MC, K C Tater, J S Weese. 2009. An investigation of methicillin-resistant
Staphylococcus aureus colonization in people and pets in the same household with an
infected person or infected pet. J Am Vet Med Assoc, v. 235, p. 540-3.
Fey, PD, T J Safranek, M E Rupp, E F Dunne, E Ribot, P C Iwen, P A Bradford, F J
Angulo, S H Hinrichs. 2000. Ceftriaxone-resistant salmonella infection acquired by a
child from cattle. N Engl J Med, v. 342, p. 1242-9.
Finley, R, C Ribble, J Aramini, M Vandermeer, M Popa, M Litman, R Reid-Smith. 2007.
The risk of salmonellae shedding by dogs fed iSW/wowe//a-contaminated commercial raw food diets. Can Vet J, v. 48, p. 69-75.
Forbes, GB. 1949. Infection with penicillin-resistant staphylococci in hospital and general practice. Br Med J, v. 2, p. 569-71.
Frank, LA, G A Kunkle. 1993. Comparison of the efficacy of cefadroxil and generic and proprietary cephalexin in the treatment of pyoderma in dogs. J Am Vet Med Assoc, v.
203, p. 530-3.
Gerhardt, N, B S Schulz, C Werckenthin, K Hartmann. 2006. Pharmacokinetics of enrofloxacin and its efficacy in comparison with doxycycline in the treatment of
Chlamydophilafelis infection in cats with conjunctivitis. Vet Rec, v. 159, p. 591-4.
Gerstadt, K, J S Daly, M Mitchell, M Wessolossky, S H Cheeseman. 1999. Methicillin-
29 resistant Staphylococcus intermedins pneumonia following coronary artery bypass
grafting. Clin Infect Dis. v. 29, p. 218-9.
Gibson, JS, J M Morton, R N Cobbold, H E Sidjabat, L J Filippich, D J Trott. 2008.,
Multidrug-resistant E. coli and enterobacter extraintestinal infection in 37 dogs. J Vet
Intern Med, v. 22, p. 844-50.
Gorwitz, RJ, D Kruszon-Moran, S K McAllister, G McQuillan, L K McDougal, G E
Fosheim, B J Jensen, G Killgore, F C Tenover, M J Kuehnert. 2008. Changes in the
prevalence of nasal colonization with Staphylococcus aureus in the United States, 2001-
2004. J Infect Dis. v. 197, p. 1226-34.
Government of Canada. 2000. Human health risk from exposure to natural dog treats.
Canada Communicable Disease Report, v. 26.
Government of Canada. 2008. Canadian Integrated Program for Antimicrobial Resistance
Surveillance (CIPARS) 2007- Preliminary Results. Guelph (Ontario): Public Health
Agency of Canada.
Graham, PL 3rd, S X Lin, E L Larson. 2006. A U.S. population-based survey of
Staphylococcus aureus colonization. Ann Intern Med. v. 144, p. 318-25.
Gray, JT, L L Hungerford, P J Fedorka-Cray, M L Headrick. 2004. Extended-spectrum- cephalosporin resistance in Salmonella enterica isolates of animal origin. Antimicrob
30 Agents Chemother, v. 48, p. 3179-81.
Griffeth, GC, D O Morris, J L Abraham, F S Shofer, S C Rankin. 2008. Screening for
skin carriage of methicillin-resistant coagulase-positive staphylococci and
Staphylococcus schleiferi in dogs with healthy and inflamed skin: Vet Dermatol, v. 19, p.
142-9.
Grinberg, A, D D Kingsbury, IR Gibson, B M Kirby, H J Mack, D Morrison. 2008.
Clinically overt infections with methicillin-resistant Staphylococcus aureus in animals in
New Zealand: a pilot study. N Z Vet J, v. 56, p. 237-42.
Guardabassi, L, M E Loeber, A Jacobson. 2004. Transmission of multiple antimicrobial- resistant Staphylococcus intermedius between dogs affected by deep pyoderma and their owners. Vet Microbiol, v. 98, p. 23-7.
Hackett, T, M R Lappin. 2003. Prevalence of enteric pathogens in dogs of north-central
Colorado. J Am Anim Hosp Assoc, v. 39, p. 52-6.
Hanselman, BA, S Kruth, J S Weese. 2008. Methicillin-resistant staphylococcal colonization in dogs entering a veterinary teaching hospital. Vet Microbiol, v. 126, p.
277-81.
Hanselman, BA, S A Kruth, J Rousseau, D E Low, B M Willey, A McGeer, J S Weese.
2006. Methicillin-resistant Staphylococcus aureus colonization in veterinary personnel.
31 Emerg Infect Dis. v. 12, p. 1933-8.
Heldmann, E, D C Brown, F Shofer. 1999. The association of propofol usage with
postoperative wound infection rate in clean wounds: a retrospective study. Vet Surg. v.
28, p. 256-9.
Holso, K, M Rantala, A Lillas, S Eerikainen, P Huovinen, L Kaartinen. 2005. Prescribing antimicrobial agents for dogs and cats via university pharmacies in Finland—patterns and quality of information. Acta Vet Scand, v. 46, p. 87-93.
Hughes, JM. 1987. Nosocomial infection surveillance in the United States: historical perspective. Infect Control, v. 8, p. 450-3.
Jacoby, GA. 2009. AmpC beta-lactamases. Clin Microbiol Rev, v. 22, p. 161-82.
Jiang, X, H Yang, B Dettman, M P Doyle. 2006. Analysis of fecal microbial flora for antibiotic resistance in ceftiofur-treated calves. Foodborne Pathog Dis, v. 3, p. 355-65.
Kempker, R, D Mangalat, T Kongphet-Tran, M Eaton. 2009. Beware of the Pet Dog: A
Case of Staphylococcus intermedius Infection. Am J Med Sci. Epub ahead of print.
Kikuchi, K, T Karasawa, C Piao, I Itoda, H Hidai, H Yamaura, K Totsuka, T Morikawa,
M Takayama. 2004. Molecular confirmation of transmission route of Staphylococcus intermedius in mastoid cavity infection from dog saliva. J Infect Chemother, v. 10, p. 46-
32 8.
Kottler, S, J R Middleton, J Perry, J S Weese, L A Cohon. In press. Prevalence of
Staphylococcus aureus and MRSA carriage in three populations. JVIM.
Kozak, M, K Horosova, V Lasanda, J Bilek, J Kyselova. 2003. Do dogs and cats present a risk of transmission of salmonellosis to humans? Bratisl Lek Listy, v. 104, p. 323-8.
Kruth, SA, J F Prescott, M K Welch, M H Brodsky. 1989. Nosocomial diarrhea associated with enterotoxigenic Clostridium perfringens infection in dogs. J Am Vet Med
Assoc, v. 195, p. 331-4.
Kuehnert, MJ, D Kruszon-Moran, H A Hill, G McQuillan, S K McAllister, G Fosheim, L
K McDougal, J Chaitram, B Jensen, S K Fridkin, G Killgore, F C Tenover. 2006.
Prevalence of Staphylococcus aureus nasal colonization in the United States, 2001-2002.
J Infect Dis.v. 193,172-9.
Lafaver, S, N A Miller, W P Stubbs, R A Taylor, R J Boudrieau. 2006. Tibial tuberosity advancement for stabilization of the canine cranial cruciate ligament-deficient stifle joint: surgical technique, early results, and complications in 101 dogs. Vet Surg. v. 36, p. 573-
86.
Lefebvre, SL, R Reid-Smith, P Boerlin, J S Weese. 2008. Evaluation of the risks of shedding Salmonellae and other potential pathogens by therapy dogs fed raw diets in
33 Ontario and Alberta. Zoonoses Public Health, v. 55, p. 470-80.
Lefebvre, SL, R J Reid-Smith, D Waltner-Toews, J S Weese. 2009. Incidence of
acquisition of methicillin-resistant Staphylococcus aureus, Clostridium difficile, and other
health-care-associated pathogens by dogs that participate in animal-assisted interventions.
J Am Vet Med Assoc, v. 234, p. 1404-17.
Lefebvre, SL, D Waltner-Toews, A S Peregrine, R Reid-Smith, L Hodge, L G Arroyo, J
S Weese. 2006. Prevalence of zoonotic agents in dogs visiting hospitalized people in
Ontario: implications for infection control. J Hosp Infect, v. 62, p. 458-66.
Lenz, J, D Joffe, M Kauffman, Y Zhang, J LeJeune. 2009. Perceptions, practices, and consequences associated with foodborne pathogens and the feeding of raw meat to dogs.
Can Vet J, v. 50, p. 637-43.
Leonard, FC, Y Abbott, A Rossney, P J Quinn, R O'Mahony, B K Markey. 2006.
Methicillin-resistant Staphylococcus aureus isolated from a veterinary surgeon and five dogs in one practice: Vet Rec, v. 158, p. 155-9.
Leonard, FC, B K Markey. 2008. Methicillin-resistant Staphylococcus aureus in animals: a review. Vet J, v. 175, p. 27-36.
Lipton, BA, S G Hopkins, J E Koehler, R F DiGiacomo. 2008. A survey of veterinarian involvement in zoonotic disease prevention practices. J Am Vet Med Assoc, v.233, p.
34 1242-9.
Lloyd, DH, D N Carlotti, H J Koch, A H Van den Broek. 1997. Treatment of canine
pyoderma with co-amoxyclav: a comparison of two dose rates. Vet Rec, v. 141, p. 439-
41.
Loeffler, A, A K Boag, J Sung, J A Lindsay, L Guardabassi, A Dalsgaard, H Smith, K B
Stevens, D H Lloyd. 2005. Prevalence of methicillin-resistant Staphylococcus aureus
among staff and pets in a small animal referral hospital in the UK. J Antimicrob
Chemother, v. 56, p. 692-7.
Loeffler, A, M Linek, A Moodley, L Guardabassi, J M Sung, M Winkler, R Weiss, D H
Lloyd. 2007. First report of multiresistant, mecA-positive Staphylococcus intermedius in
Europe: 12 cases from a veterinary dermatology referral clinic in Germany: Vet
Dermatol, v. 18, p. 412-21.
Loeffler, A, D U Pfeiffer, J A Lindsay, R Soares-Magalhaes, D H Lloyd. 2009. Lack of transmission of methicillin-resistant Staphylococcus aureus (MRS A) between apparently healthy dogs in a rescue kennel. Vet Microbiol. Epub ahead of print.
Mahoudeau, I, X Delabranche, G Prevost, H Monteil, Y Piemont. 1997. Frequency of isolation of Staphylococcus intermedius from humans. J Clin Microbiol, v. 35, p. 2153-4.
Malik, S, G W Coombs, F G O'Brien, H Peng, M D Barton. 2006. Molecular typing of
35 methicillin-resistant staphylococci isolated from cats and dogs. J Antimicrob Chemother,
v. 58, p. 428-31.
Medleau, L, R E Long, J Brown, W H Miller. 1986. Frequency and antimicrobial
susceptibility of Staphylococcus species isolated from canine pyodermas: Am J Vet Res,
v. 47, p. 229-31.
Moodley, A, M Stegger, A F Bagcigil, K E Baptiste, A Loeffler, D H Lloyd, N J
Williams, N Leonard, Y Abbott, R Skov, L Guardabassi. 2006. spa typing of methicillin-
resistant Staphylococcus aureus isolated from domestic animals and veterinary staff in
the UK and Ireland. J Antimicrob Chemother, v. 58, p. 1118-23.
Morley, PS, M D Apley, T E Besser, D P Burney, P J Fedorka-Cray, M G Papich, J L
Traub-Dargatz, J S Weese. 2005. Antimicrobial drug use in veterinary medicine. J Vet
Intern Med, v. 19, p. 617-29.
Morley, PS, R A Strohmeyer, J D Tankson, D R Hyatt, D A Dargatz, P J Fedorka-Cray.
2006. Evaluation of the association between feeding raw meat and Salmonella enterica infections at a Greyhound breeding facility. J Am Vet Med Assoc, v. 228, p. 1524-32.
Morris, DO, E A Mauldin, K O'Shea, F S Shofer, S C Rankin. 2006a. Clinical, microbiological, and molecular characterization of methicillin-resistant Staphylococcus aureus infections of cats. Am J Vet Res, v. 67, p. 1421-5.
36 Morris, DO, K A Rook, F S Shofer, S C Rankin. 2006b. Screening of Staphylococcus
aureus, Staphylococcus intermedius, and Staphylococcus schleiferi isolates obtained from
small companion animals for antimicrobial resistance: a retrospective review of 749
isolates (2003-04). Vet Dermatol, v. 17, p. 332-7.
Mulvey, MR, A E Simor. 2009. Antimicrobial resistance in hospitals: how concerned
should we be? CMAJ. v. 180, p. 408-15.
Murphy, CP, R J Reid-Smith, J F Prescott, B N Bonnett, C Poppe, P Boerlin, J S Weese,
N Janecko, S A McEwen. 2009. Occurence of antimicrobial resistance in selected bacteria in healthy dogs and cats presented to private veterinary clinics in Southern
Ontario. Can Vet J. v.50, p. 1047-53.
Nicholson, M, M Beal, F Shofer, D C Brown. 2002. Epidemiologic evaluation of postoperative wound infection in clean-contaminated wounds: A retrospective study of
239 dogs and cats. Vet Surg. v. 31, p. 577-81.
Nienhoff, U, K Kadlec, IF Chaberny, J Verspohl, G F Gerlach, S Schwarz, D Simon, I
Nolte. 2009. Transmission of methicillin-resistant Staphylococcus aureus strains between humans and dogs: two case reports. J Antimicrob Chemother, v. 64, p. 660-2.
O'Mahony, R, Y Abbott, F C Leonard, B K Markey, P J Quinn, P J Pollock, S Fanning, A
S Rossney. 2005. Methicillin-resistant Staphylococcus aureus (MRSA) isolated from animals and veterinary personnel in Ireland. Vet Microbiol, v. 109, p. 285-96.
37 Odensvik, K, K Grave, C Greko. 2001. Antibacterial drugs prescribed for dogs and cats
in Sweden and Norway 1990-1998. Acta Vet Scand, v. 42, p. 189-98.
Ogeer-Gyles, J, K A Mathews, W Sears, J F Prescott, J S Weese, P Boerlin. 2006a.
Development of antimicrobial drug resistance in rectal Escherichia coli isolates
from dogs hospitalized in an intensive care unit. JAVMA, v. 229, p.694-9.
Ogeer-Gyles, J, K Mathews, J S Weese, J F Prescott, P Boerlin. 2006b. Evaluation of
catheter-associated urinary tract infections and multi-drug-resistant Escherichia coli
isolates from the urine of dogs with indwelling urinary catheters. J Am Vet Med Assoc,
v. 229, p. 1584-90.
Olmstead, ML, R B Hohn, T M Turner. 1983. A five-year study of 221 total hip replacements in the dog. J Am Vet Med Assoc, v. 183, p. 191-4.
Paul HA, Bargar WL.1987. A modified technique for canine total hip replacement. J Am
Animal Hosp Assoc, v. 23, p. 16-17.
Parker RB, Bloomberg MS, Bitetto W. 1984. Canine total hip arthroplasty: a clinical review of 20 cases. J Am Animal Hosp Assoc, v. 20 p. 97-103.
Pedersen, K, K Pedersen, H Jensen, K Finster, V F Jensen, O E Heuer. 2007. Occurrence of antimicrobial resistance in bacteria from diagnostic samples from dogs. J Antimicrob
Chemother, v. 60, p. 775-81.
38 Pitout, JD, M D Reisbig, M Mulvey, L Chui, M Louie, L Crowe, D L Church, S Elsayed,
D Gregson, R Ahmed, P Tilley, N D Hanson. 2003. Association between handling of pet
treats and infection with Salmonella enterica serotype Newport expressing the AmpC
beta-lactamase, CMY-2. J Clin Microbiol, v. 41, p. 4578-82.
Poppe, C, L C Martin, C L Gyles, R Reid-Smith, P Boerlin, S A McEwen, J F Prescott, K
R Forward. 2005. Acquisition of resistance to extended-spectrum cephalosporins by
Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey
poult intestinal tract. Appl Environ Microbiol, v. 71, p. 1184-92.
Prescott, JF, W J Hanna, R Reid-Smith, K Drost. 2002. Antimicrobial drug use and resistance in dogs. Can Vet J, v. 43, p. 107-16.
Rantala, M, K Holso, A Lillas, P Huovinen, L Kaartinen. 2004a. Survey of condition- based prescribing of antimicrobial drugs for dogs at a veterinary teaching hospital. Vet
Rec.v. 155, p. 259-62.
Rantala, M, E Lahti, J Kuhalampil, S Pesonen, A K Jarvinen, Saijonmaa-Koulumies, T
Honkanen-Buzalski.2004b.Antimicrobial resistance in Staphylococcus enterica,
Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs.Acta Vet Scand, v. 45, p. 37-45.
Reid-Smith, R, E Sifton, N Rinkardt, D Ford, R Zoutmann, B Bonnett. 2002. ntimicrobial
39 drug use by Canadian small animal veterinarians. National Antimicrobial Resistance
Monitoring System Meeting.
Rich, M, L Roberts. 2006. MRSA in companion animals. Vet Rec. v. 159, p. 535-6.
Rodriguez, I, W Barownick, R Helmuth, M C Mendoza, M R Rodicio, A Schroeter, B
Guerra. 2009. Extended-spectrum {beta}-lactamases and AmpC {beta}-lactamases in
ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in
Germany during 2003-07. J Antimicrob Chemother, v. 64, p. 301-9.
Rogers, KS, G E Lees, R B Simpson. 1988. Effects of single-dose and three-day
trimethoprim-sulfadiazine and amikacin treatment of induced Escherichia coli urinary
tract infections in dogs. Am J Vet Res, v. 49, p. 345-9.
Ruch-Gallie, RA, J K Veir, M E Spindel, M R Lappin. 2008. Efficacy of amoxycillin and
azithromycin for the empirical treatment of shelter cats with suspected bacterial upper respiratory infections. J Feline Med Surg, v. 10, p. 542-50.
Ruscher, C, A Lubke-Becker, C G Wleklinski, A Soba, L H Wieler, B Walther. 2009.
Prevalence of Methicillin-resistant Staphylococcus pseudintermedius isolated from clinical samples of companion animals and equidaes. Vet Microbiol, v. 136, p. 197-201.
Sasaki, T, K Kikuchi, Y Tanaka, N Takahashi, S Kamata, K Hiramatsu. 2007.
Methicillin-resistant Staphylococcus pseudintermedius in a veterinary teaching hospital. J
40 Clin Microbiol, v. 45, p. 1118-25.
Sato, Y, T Mori, T Koyama, H Nagase. 2000. Salmonella virchow infection in an infant transmitted by household dogs. J Vet Med Sci, v. 62, p. 767-9.
Schorr-Evans, EM, A Poland, W E Johnson, N C Pedersen. 2003. An epizootic of highly virulent feline calicivirus disease in a hospital setting in New England. J Feline Med
Surg,v. 5, p. 217-26.
Schwartz, M, I C Boettcher, S Kramer, A Tipold. 2009. Two dogs with iatrogenic discospondylitis caused by meticillin-resistant Staphylococcus aureus. J Small Anim
Pract,v. 50, p. 201-5.
Senior, DF, J M Gaskin, C D Buergelt, P P Franks, T J Keefe. 1985. Amoxycillin and clavulanic acid combination in the treatment of experimentally induced bacterial cystitis in cats. Res Vet Sci, v. 39, p. 42-6.
Six, R, D M Cleaver, C J Lindeman, J Cherni, R Chesebrough, G Papp, T L Skogerboe,
D J Weigel, J F Boucher, M R Stegemann. 2009. Effectiveness and safety of cefovecin sodium, an extended-spectrum injectable cephalosporin, in the treatment of cats with abscesses and infected wounds. J Am Vet Med Assoc, v. 234, p. 81-7.
Smarick, SD, S C Haskins, J Aldrich, J E Foley, P H Kass, M Fudge, G V Ling. 2004.
Incidence of catheter-associated urinary tract infection among dogs in a small animal
41 intensive care unit. J Am Vet Med Assoc, v. 224, p. 1936-40.
Stegemann, MR, N Coati, C A Passmore, J Sherington. 2007a. Clinical efficacy and safety of cefovecin in the treatment of canine pyoderma and wound infections. J Small
Anim Pract, v. 48, p. 378-86.
Stegemann, MR, J Sherington, C Passmore. 2007b. The efficacy and safety of cefovecin in the treatment of feline abscesses and infected wounds. J Small Anim Pract, v. 48, p.
683-9.
Sturgess, CP, T J Gruffydd-Jones, D A Harbour, R L Jones. 2001.Controlled study of the efficacy of clavulanic acid-potentiated amoxycillin in the treatment of Chlamydia psittaci in cats. Vet Rec, v. 149, p. 73-6.
Talan, DA, E J Goldstein, D Staatz, G D Overturf. 1989a. Staphylococcus intermedins: clinical presentation of a new human dog bite pathogen. Ann Emerg Med. v. 18, p. 410-3,
410-3.
Talan, DA, D Staatz, A Staatz, G D Overturf. 1989b. Frequency of Staphylococcus intermedins as human nasopharyngeal flora. J Clin Microbiol, v. 27, p. 2393.
Tanner, MA, C L Everett, D C Youvan. 2000. Molecular phylogenetic evidence for noninvasive zoonotic transmission of Staphylococcus intermedins from a canine pet to a human. J Clin Microbiol, v. 3 8, p. 1628-31.
42 Toma, S, S Colombo, L Cornegliani, P Persico, M Galzerano, M M Gianino, C Noli.
2008. Efficacy and tolerability of once-daily cephalexin in canine superficial pyoderma: an open controlled study. J Small Anim Pract, v. 49, p. 384-91.
Tomlin, J, M J Pead, D H Lloyd, S Howell, F Hartmann, H A Jackson, P Muir. 1999.
Methicillin-resistant Staphylococcus aureus infections in 11 dogs. Vet Rec, v. 144, p. 60-
4.
Trott, DJ, L J Filippich, J C Bensink, M T Downs, S E McKenzie, K M Townsend, S M
Moss, J J Chin. 2004. Canine model for investigating the impact of oral enrofloxacin on commensal coliforms and colonization with multidrug-resistant Escherichia coli. J Med
Microbiol, v. 53, p. 439-43.
Tsai, HJ, H C Huang, C M Lin, Y Y Lien, C H Chou. 2007. Salmonellae and
Campylobacters in household and stray dogs in northern Taiwan. Vet Res Commun, v.
31, p. 931-9.
Turnwald, GH, K A Gossett, H U Cox, M T Kearney, A F Roy, D E Thomas, G C Troy.
1986. Comparison of single-dose and conventional trimethoprim-sulfadiazine therapy in experimental Staphylococcus intermedins cystitis in the female dog. Am J Vet Res, v. 47, p. 2621-3.
van Duijkeren, E, D J Houwers, A Schoormans, M J Broekhuizen-Stins, R Dcawaty, A C
Fluit, J A Wagenaar. 2008. Transmission of methicillin-resistant Staphylococcus
43 intermedins between humans and animals. Vet Microbiol, v. 128, p. 213-5.
van Duijkeren, E, M J Wolfhagen, M E Heck, W J Wannet. 2005. Transmission of a
Panton-Valentine leucocidin-positive, methicillin-resistant Staphylococcus aureus strain
between humans and a dog. J Clin Microbiol, v. 43, p. 6209-11.
Vandenesch, F, M Celard, D Arpin, M Bes, T Greenland, J Etienne. 1995. Catheter- related bacteremia associated with coagulase-positive Staphylococcus intermedius. J Clin
Microbiol, v. 33, p. 2508-10.
Vasseur, PB, J Levy, E Dowd, J Eliot. 1988. Surgical wound infection rates in dogs and cats. Data from a teaching hospital. Vet Surg. v. 17, p. 60-4.
Vasseur, PB, H A Paul, L R Enos, D C Hirsh. 1985. Infection rates in clean surgical procedures: a comparison of ampicillin prophylaxis vs a placebo. J Am Vet Med Assoc, v. 187, p. 825-7.
Vengust, M, M E Anderson, J Rousseau, J S Weese. 2006. Methicillin-resistant staphylococcal colonization in clinically normal dogs and horses in the community. Lett
Appl Microbiol, v. 43, p. 602-6.
Vitale, CB, T L Gross, J S Weese. 2006. Methicillin-resistant Staphylococcus aureus in cat and owner. Emerg Infect Dis, v. 12, p. 1998-2000.
44 Watson, AD, J E Maddison. 2001. Systemic antibacterial drug use in dogs in Australia.
Aust Vet J, v. 79, p. 740-6.
Weese, JS. 2006. Investigation of antimicrobial use and the impact of antimicrobial use
guidelines in a small animal veterinary teaching hospital. 1995-2004: J Am Vet Med
Assoc, v. 228, p. 553-8.
Weese, JS, J Armstrong. 2003. Outbreak of Clostridium difficile-associated disease in a
small animal veterinary teaching hospital. J Vet Intern Med. v. 17, p. 813-6.
Weese, JS, H Dick, B M Willey, A McGeer, B N Kreiswirth, B Innis, D E Low. 2006c.
Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol, v. 115, p. 148-
55.
Weese, JS, M Faires, J Rousseau, A M Bersenas, K A Mathews. 2007. Cluster of methicillin-resistant Staphylococcus aureus colonization in a small animal intensive care unit. J Am Vet Med Assoc, v. 231, p. 1361-4.
Weese, JS, K B Hailing. 2006b. Perioperative administration of antimicrobials associated with elective surgery for cranial cruciate ligament rupture in dogs: 83 cases (2003-2005).
J Am Vet Med Assoc, v. 229, p. 92-5.
Weese, JS, E van Duijkeren. 2009. Methicillin-resistant Staphylococcus aureus and
45 Staphylococcus pseudintermedius in veterinary medicine. Vet Microbiol. Epub ahead of
print.
Wettstein, K, S Descloux, A Rossano, V Perreten. 2008. Emergence of methicillin-
resistant Staphylococcus pseudintermedius in Switzerland: three cases of urinary tract
infections in cats. Schweiz Arch Tierheilkd, v. 150, p. 339-43.
Whittem, TL, A L Johnson, C W Smith, D J Schaeffer, B R Coolman, S M Averill, T K
Cooper, G R Merkin. 1999. Effect of perioperative prophylactic antimicrobial treatment
in dogs undergoing elective orthopedic surgery. J Am Vet Med Assoc, v. 215, p. 212-6.
Winokur, PL, A Brueggemann, D L DeSalvo, L Hoffmann, M D Apley, E K Uhlenhopp,
M A Pfaller, G V Doern. 2000. Animal and human multidrug-resistant, cephalosporin- resistant salmonella isolates expressing a plasmid-mediated CMY-2 AmpC beta-
lactamase. Antimicrob Agents Chemother, v. 44, p. 2777-83.
Wright, JG, S Jung, R C Holman, N N Marano, J H McQuiston. 2008. Infection control practices and zoonotic disease risks among veterinarians in the United States: J Am Vet
Med Assoc, v. 232, p. 1863-72.
Wright, JG, L A Tengelsen, K E Smith, J B Bender, R K Frank, J H Grendon, D H Rice,
A M Thiessen, C J Gilbertson, S Sivapalasingam, T J Barrett, T E Besser, D D Hancock,
F J Angulo. 2005. Multidrug-resistant Salmonella Typhimurium in four animal facilities:
Emerg Infect Dis, v. 11, p. 1235-41.
46 Table 1.1 Measures of antimicrobial use in companion animal studies
Reference Country Data Source Study period Species Measure of Outcomes
DANMAP Denmark National monitoring program 2007 Pets, primarily Kilogram active compound
2007 dogs and cats
Holso et al Finland Drug dispensing data from April 2001 Dog and cat Percentage of total prescriptions
2005 university pharmacy records Divided by species
Odensviket Sweden and Wholesalers 1990-1998 Dog and cat Kilogram active substance, al 2001 Norway Number of packages sold
Aggregated dog and cat data
Prescott et al Canada Drug dispensing data from 1990-1999 Dog Dog daily dose defined as:
2002 pharmacy records from a Total number of grams dispensed by the
tertiary care, veterinary pharmacy (dog only)/number of grams
teaching hospital3 given to a 20kg dog for 1 day of treatment
at recommended dosages.
47 Reference Country Data Source Study period Species Measure of Outcomes
Rantala et al Finland Drug dispensing data from Nov. 2000- Apr. Dog Percentage of total prescriptions
2004 pharmacy records from a 2001
tertiary care, veterinary
teaching hospital
Watson and Australia Questionnaire distributed to 1997 Dog A 4-rater ranking of antimicrobial use (e.g.,
Maddison veterinarians used frequently to never used).
2001
Weese 2006 Canada Drug dispensing data from 1995-2005 Dog and cat Prescriptions/1000 admission per year,
pharmacy records from a Percentage of total prescriptions,
tertiary care, veterinary milligrams antimicrobial dispensed to
teaching hospital8 specific hospital area (e.g. surgery)/1000
admissions
Aggregated dog and cat data
"Studies by Prescott and Weese are from the same institution
48 Chapter 2
Escherichia coli, and selected veterinary and zoonotic pathogens
isolated from environmental sites within companion animal veterinary
hospitals in southern Ontario: Prevalence and factors associated with
recovery.
As accepted for publication by the Canadian Veterinary Journal
Abstract
Hospital-based infection control in veterinary medicine is emerging and the role of the environment in hospital-acquired infections (HAT) in veterinary hospitals is largely unknown. This study determined the environmental recovery of E. coli and selected veterinary and zoonotic pathogens from community veterinary hospitals. Environmental samples were collected from 101 veterinary hospitals. The proportion of hospitals with positive environmental swabs were: E. coli-92%, Clostridium difficile (C. difficile)-5S%, methicillin-resistant Staphylococcus aureus (MRSA) -9%, blacMY-2 producing E. coli-
9%, methicillin-resistant Staphylococcus pseudintermedius-1%, Salmonella-2%.
Vancomycin-resistant Enterococcus spp., canine parvovirus and feline calicivirus were not isolated. Antimicrobial resistance prevalence in E. coli isolates was low. Important potential veterinary and human pathogens were recovered including Canadian epidemic strains MRSA-2 and MRSA-5, and C. difficile ribotype 027. There is an environmental reservoir of pathogens in veterinary hospitals. Additional studies are required to characterize risk factors associated with HAI in companion animals, including the role of the environment.
49 1.0 Introduction
Hospital-acquired infections are an important cause of morbidity and mortality in
both human and veterinary patients. Hospital-acquired infections are associated with
multiple factors, for example, patient susceptibility and sources of exposure (Griffin,
2007). In companion animals, HAI by antimicrobial resistant E. coli (Sanchez et al.,
2002), Clostridium difficile (Weese and Armstrong, 2003) and Acinetobacter baumanii
(Boerlin et al., 2001) with an associated environmental reservoir have been reported.
Environmental sources associated with hospital-acquired salmonellosis in horses have
also been documented (Schott et al., 2001, Ward et al., 2005), however, the role and
contribution of environmental pathogens in the epidemiology of HAI is not fully
understood.
In human medicine, environmental sites are not typically considered to be an
important source of exposure to pathogens and routine surveillance of environmental
sites is not recommended (CDC 2003). This may not apply to companion animal veterinary medicine, however, because the behaviours, housing and management of human patients are quite different than those of companion animals. For example, unlike humans, companion animals have close contact with floors during clinical examination, venipuncture and recovery from anesthesia. Moreover, floors of veterinary hospitals are more likely to be contaminated with infectious material (e.g., feces) and animal exploratory behaviour may place animals' noses and mouths in contact with these areas.
Therefore, sites such as floors and perhaps other sites may be of greater importance as environmental reservoirs of pathogens in companion animal medicine than human medicine.
50 Veterinary hospitals are an intersection of human and animal interaction. Thus, when investigating agents associated with hospital-acquired infections in veterinary medicine, different types of agents need to be considered: animal pathogens, antimicrobial-resistant bacteria that may be potential pathogens to humans or animals, zoonotic pathogens and microorganisms that are relatively resistant to environmental disinfection.
The objectives of this study were to determine: 1) the prevalence of environmental E. coli, Salmonella enterica, extended spectrum beta-lactamase (ESBL) E. coli, CMY-2 producing E. coli (blacwf-2 producing E. coli), C. difficile, vancomycin- resistant Enterococcus spp. (VRE) methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus pseudintermedius (MRSP), canine parvovirus (CPV) and feline calicivirus (FCV); 2) the antimicrobial susceptibility of environmental E. coli and Salmonella isolates; 3) the distribution of molecular types of C. difficile and MRSA; and 4) the associations between specific infection control practices and environmental recovery of E. coli, ampicillin-resistant E. coli (AMP-R E. coli), C. difficile and MRSA.
2.0 Materials and methods
2.1 Sample size calculations
We sought to enrol 100 veterinary hospitals to enable detection of an individual organism at a prevalence of 10% among hospitals with a precision of 6%, 80% of the time (Dohoo et al., 2003).
2.2 Veterinary hospital selection
Southern Ontario companion animal hospitals or offices, including those with additional licensures for food animal or equine facilities (mixed animal hospitals),
51 licensed by the College of Veterinarians of Ontario in 2005, were eligible for recruitment.
Eligible veterinary hospitals (n=766) were contacted by mail and invited to participate.
Participants were then asked to respond by mail, fax, or telephone with a completed
hospital-demographic survey (Appendix A.2.1).
2.3 Sampling
Environmental sampling in the hospitals was conducted in two phases. During
Phase One, individual sites were sampled throughout the hospital without pooling of any
sites. Sampling was performed in reception, treatment areas, examination areas, hospitalization areas, isolation areas, runs, boarding and grooming areas. The sites sampled were table surfaces (examination rooms, treatment, surgery, grooming and boarding areas), floor surfaces (reception, treatment, kennel rooms, surgery, isolation, grooming and boarding areas), equipment (stethoscopes, thermometers, otoscopes/ophthalmoscopes and otoscope tips), and areas of high human hand contact
(telephone, computer keyboards, taps, and doorknobs). The median number of sites sampled in each hospital was 23 (range 17-27). Based on the results of Phase One, the sites sampled in Phase Two were pooled: 1) kennel areas and runs; 2) examination and treatment tables; 3) floors; 4) isolation areas; 5) kitchen and bathroom taps, keyboards and telephones; 6) exam and treatment room sink taps; 7) otoscope tips, 8) otoscopes, ophthalmoscopes and stethoscopes; and 9) thermometers. Sites sampled for bacteriology were sampled using sterile, divided electrostatic cloths (Swiffer®, Proctor and Gamble,
Toronto, Ontario) and gloved hands. Gloves were changed between sampling of different sites. Samples for CPV and FCV were collected from kennel and isolation areas only and
52 sampling was done using a sterile cotton swab (Becton, Dickinson and Co., Franklin
Lakes, NJ, USA).
Sampling was performed between May 2005 and August 2005. Samples were
collected Monday through Friday, between 9am and 4pm. Sample collection was not
targeted to sites recently disinfected or sites that had been used, yet not cleaned or
disinfected. Samples were taken irrespective of environmental disinfection within the
practice.
2.4. Microbiology
2.4.1. Escherichia coli
Half of the electrostatic cloth was placed in 50 ml of buffered peptone water
(BPW) (Becton, Dickinson) and incubated at 37°C for 18-24 h. Then, 25 ml of BPW was
added to 25 ml of double strength E. coli medium (EC) broth (Becton, Dickinson) and incubated at 42°C for 18-24 h. Next, loops of broth were streaked onto Eosin Methylene
Blue agar (Becton, Dickinson) and incubated at 37°C for 18-24 h, then six presumptive
E. coli colonies were plated onto MacConkey agar (Becton, Dickinson) and incubated at
37°C for 18-24 h. Then, six presumptive E. coli colonies were plated onto tryptic soy agar (Becton, Dickinson) and incubated at 37°C for 18-24 h. Escherichia coli confirmation was conducted using Kovac's indole reagent (PML Microbiologicals Inc.,
Mississauga, Ontario) and Simmons citrate agar (Becton, Dickinson) for production of indole and no utilization of citrate as a sole carbon source, respectively
2.4.2. Extended spectrum (3-lactamase Escherichia coli
The initial preparation of the electrostatic cloth was the same as described above, except that the EC broth was streaked for isolated colonies onto MacConkey agar with 2
53 ug of cefpodoxime (Oxoid Company, Nepean, Ontario) and incubated at 37°C for 48 h in
accordance with the Clinical Laboratory Standards Institute (CLSI) guidelines (NCCLS,
2002). Escherichia coli were confirmed as described above.
2.4.3.blacMY-2 Escherichia coli
Escherichia coli isolates with the following antimicrobial resistance phenotype were selected for amplification, sequencing and hybridization using polymerase chain reaction (PCR) for the blacm-2 gene (M'Zali et al. 1997, Allen and Poppe, 2002): ampicillin (minimum inhibitory concentration (MIC) >32 ug/ml) together with amoxicillin-clavulanic acid (MIC >32 ug/ml), and either cefoxitin (MIC >8 fag/ml) or ceftriaxone (MIC >8 ug/ml) or ceftiofur (MIC >2 ug/ml) (See Susceptibility Testing below).
2.4.4. Salmonella
Salmonella was isolated using two methods. In the first method, 1 ml of BPW pre-enrichment was added to 9 ml Rappaport Vassiliadis (RV) broth (Becton, Dickinson) and incubated at 42°C for 24 h. Then, 1 ml of the RV broth was added to 9 ml
Tetrathionate broth (Becton, Dickinson) and incubated at 37°C for 18-24 h. Tetrathionate broth was streaked for isolated colonies onto a Xylose-lysine Tergitol 4 (XLT4) agar plate and incubated at 37°C for 18-24 h. Two colonies with morphologies typical of
Salmonella were streaked on to MacConkey agar and were incubated at 37°C for 18-24 h.
Presumptive Salmonella isolates were plated onto nutrient agar and incubated at 37°C for
18-24 h after which biochemical and serological testing was conducted on the isolates obtained.
54 In the second method, 0.1ml of the initial BPW pre-enrichment was inoculated onto Modified Semisolid Rappaport Vassiliadis (MRSV) agar (Becton, Dickinson) and incubated at 42°C for 24-72 h. Plates were examined for a Salmonella migratory pattern at 24,48 and 72 h of incubation. Colonies with typical Salmonella migration were streaked onto MacConkey agar and incubated at 37°C for 18-24 h. Two presumptive colonies were plated onto nutrient agar and incubated at 37°C for 18-24 h.
Salmonella confirmation was conducted using triple sugar iron agar slants
(Becton, Dickinson), Christenssen's urea agar (Becton, Dickinson) slants and by slide agglutination using Salmonella O antiserum polyvalent A-I and Vi. Salmonella isolates were serotyped at the Laboratory for Foodborne Zoonoses Salmonella reference laboratory using standard methods (Government of Canada 2006).
2.4.5. Vancomycin-resistant Enterococcus spp.
One quarter of the electrostatic cloth was placed in 50 ml BPW and incubated at
35°C for 24 h. Then, 1 ml of BPW was added to 9 ml VRE enrichment broth and incubated at 35°C for 24 h. This was plated onto selective agar plates (Oxoid) and incubated at 35°C for 48 h. Next, brown and black colonies were plated onto Columbia blood agar and incubated at 35°C for 24 h. Catalase-negative, Gram-positive cocci were confirmed as enterococci using the API Strep 20 biochemical identification system
(Oxoid).
2.4.6. Clostridium difficile
One quarter of the electrostatic cloth was placed in 50 ml of Clostridium difficile
Moxalactam Norfloxacin broth (CDMN) and was incubated in an anaerobic chamber at
37°C for 7 d. Then, 2 ml of the CDMN broth were added to 2 ml of 95% ethanol, which
55 was incubated at room temperature for 1 h, then centrifuged at 4400 g and the pellet was
streaked onto blood agar and incubated in an anaerobic chamber at 37°C for 48 h.
Clostridium difficile identification was confirmed by colony morphology, Gram stain
appearance, characteristic odour and 1-proline aminopeptidase activity (PRO Disc assay,
Carr-Scarborough Microbiologicals, Inc., Decatur, Georgia, USA). Isolates were
characterized for ability to produce toxins A, B and CDT binary by PCR (Kato et al.,
1998, Rupnik et al., 2003). Ribotyping was performed to further identify the strains
(Bidet et al., 1999) and patterns were compared with a collection of human and animal
isolates archived by the investigators (Martin et al., 2008).
2.4.7. Methicillin-resistant Staphylococcus aureus and Staphylococcuspseudintermedius
One quarter of the electrostatic cloth was placed in BPW and incubated at 35°C
for 24 h. Then, 1 ml of the BPW rinse was added to 9 ml of MRS A enrichment broth
(7.5% NaCl, 2.5 g/L yeast extract, 10 g/L tryptone and 10 g/L mannitol) and was
incubated at 35°C for 24 h. The broth was plated onto mannitol salt agar with 2 jig/ml
oxacillin (Oxoid) and was incubated at 35°C for 48 h. Coagulase and catalase positive,
Gram-positive cocci were identified as Staphylococcus species. Methicillin resistance was confirmed using the PBP2 assay (Oxoid). Identification to the species level was done using the API STAPH system (BioMerieux Canada Inc., St Laurent, Quebec) and polymyxin B susceptibility. MRS A isolates were typed by Smal PFGE and categorized described by Mulvey et al. (Mulvey et al., 2001), with two modifications: 1) the lysostaphin and lysozyme was added to buffer, and 2) the switch times were 0.5-90 s.
2.4.8. Virus isolation
56 Swabs were placed in cooled viral transport media and stored (2 to 6 h) in a
portable cooler with an ice pack. Next, the swab and media were vortexed and 1.25 ml of
the mixed media was placed in to cryovials and frozen at -86°C. Before inoculation, the
samples were centrifuged at 2100 - 2200 g for 10 min.
2.4.8.1. Canine parvovirus
Cell monolayers were prepared by seeding Lab-Tek 2-well Permanox slides
(Nalge Nunc International, Rochester, NY, USA) with 1:4 — 1:5 dilutions of Crandall feline kidney cells (1.5 ml per chamber). Sample supernatant was added to the seeded chamber slides (50 ul per well) and incubated for 5 d at 37°C with 5% CO2. A positive control using canine parvovirus stock virus and a negative control using an uninoculated chamber was included in each run. After 5 d, media were aspirated and monolayers were air-dried and then heat-fixed at 60°C on a slide warmer for 30 min. Following, slides were fixed in 3:2 acetone and phosphate buffered saline mixture (BPS) for 15 min and then dipped in methanol prior to fan-drying.
Fixed monolayers were stained with canine parvovirus monoclonal antibody
(TropBio Pty Ltd., Townsville, Queensland, Australia) and incubated in a moist chamber for 30 min at 37°C. Next, slides were rinsed 3-times for 4 min each time in PBS and then flooded with fluorescein isothiocyanate conjugated goat anti-mouse IgG (MP
Biomedicals, Solon, OH, USA) for 30 min at 37°C. After, the slides were rinsed in PBS
(3 x 4-min), followed by several rinses in reverse osmosis water. Slides were fan-dried and coverslipped with fluorescent antibody mounting medium before examining monolayers for viral-specific fluorescence.
2.4.8.2. Feline calicivirus
57 Cell monolayers were prepared as above except that 25 cm (5 ml) flasks were
seeded in place of chamber slides. Samples were added (100 JJ.1 per flask) and incubated
for 7 d with daily monitoring for cytopathic effects (CPE). A negative cell control was
included with each run. Samples were considered negative if no CPE was evident after 7
d post-inoculation.
2.5. Antimicrobial susceptibility testing
For each sample, susceptibility testing was performed on three E. coli isolates, all presumptive ESBL-ii. coli isolates, two confirmed Salmonella isolates from the first isolation method, and one confirmed isolate from the second isolation method. Minimum inhibitory concentrations were determined by broth microdilution methodology
(Sensititre®, Trek Diagnostic Systems Inc., Cleveland, OH, USA). The National
Antimicrobial Resistance Monitoring System (NARMS) microtitre plate configuration
(NARMS CMV1AGNF) was used with the following resistance breakpoints: amikacin
(>64 ug/ml), amoxicillin-clavulanic acid (>32 ug/mi), ampicillin (>32 ug/ml), cefoxitin
(>32 ug/ml), ceftiofur (>8 fig/ml), ceftriaxone (>64 ug/ml), chloramphenicol
(>32 ug/ml), ciprofloxacin (>4 ug/ml), gentamicin (>16 ug/ml), kanamycin (>64 ug/ml), nalidixic acid (>32 ug/ml), streptomycin (>64 ug/ml), sulfamethoxazole (>512 ug/ml), tetracycline (>16 ug/ml) and trimethoprim-sulfamethoxazole (>4 ug/ml). The Clinical
Laboratory Standards Institute breakpoints for resistance (NCCLS 2002) were used for all antimicrobials except streptomycin for which the NARMS 2001 susceptibility breakpoint was used (CDC 2003).
2.6. Generalized linear mixed models of factors potentially associated with environmental recovery of bacteria
58 The binary outcomes of interest were: recovery (or not) of E. coli, AMP-R E. coli,
C. difficile, and MRSA. The categorical independent variables were: the type of
disinfectant used for environmental disinfection, the type of hand cleansers used for hand
washing, an isolation area, in-hospital antimicrobials, and documented "Standard
Operating Procedures" (SOPs) for cleaning and disinfection of environmental areas, and
cleaning, disinfection and sterilization of equipment. Potential confounding variables
examined were type of hospital, number of staff in the veterinary hospital, number of
hospitalized patients per day, number of appointments seen per day and recovery of other
organisms. The data were captured through questionnaires administered to one veterinarian and one veterinary technician from each hospital (Appendix A.2.2. and
A.2.3.).
Unconditional associations between infection control practices and the outcomes of interest were evaluated using Fisher's exact test. Next, a single-level (hospital-level) multivariable logistic model was built for each outcome using a step-wise, forward selection process of predictors unconditionally associated with the outcome at p <0.2.
The p-value for entry into the model was 0.2 and for removal was 0.05 for all predictors including potential confounding variables. Further modelling was performed manually.
Potential confounding variables and other predictors that were removed during the step wise forward selection were reintroduced to the initial model to examine their effect on coefficients of the other factors.
Following the primary logistic model building procedure, a mixed multilevel
(level 1: site, level 2: hospital) modelling procedure was performed including site of bacterial recovery within the veterinary hospital as both random and fixed effects. The
59 model was built by manual backwards selection using significant variables (p ^0.05) in the primary logistic model and the potential confounders. Variables were retained in the final model if significantly associated with the outcome (p <0.05), significant by the likelihood ratio test (p <0.05), or their removal altered the coefficient of one or more of the other predictors by >10%. The distribution of the standardized residuals (hospital level) was assessed graphically with a normal (Q-Q) plot. Influential observations at the hospital level were assessed using Cook's distance. The intraclass correlation (ICC) for bacterial recovery was calculated using the variance provided from the model (Dohoo et al., 2003).
Descriptive statistics were performed using Micosoft Excel 2003 (Microsoft
Corporation, Redmond, Washington, USA) and Intercooled Stata 10.0 (StataCorp,
College Station, Texas, USA). Univariable and multivariable analyses were performed using Intercooled Stata (StataCorp). Mixed multi-level modelling was performed using
GLAAM (Rabe-Heskethe et al., 2002, Rabe-Heskethe and Skrondal 2008) within
Intercooled Stata (StataCorp).
The study procedures were approved by the Research Ethics Board at the
University of Guelph.
3.0 Results
One hundred and twenty-one hospitals responded with interest to the recruitment letter (response rate 16%) leaving a study population of 101 hospitals. Twelve could not be sampled because of time limitations and the other 8 were out of the geographic sampling region. Of the 101 hospitals, 90 were companion animal, ten were mixed animal, and one hospital treated primarily exotic animals. The median number (and
60 range) of full-time veterinarians per hospital was 2 (0-12), part-time veterinarians; 1 (0-
5), and other staff; 10 (3-45). The median number (and range) of appointments per day
was 10 (2-40), of dogs hospitalized each day; 3 (1-18) and cats; 3 (1-25).
In Phase One, E. coli was recovered from 23 (96%) of hospitals (n=24) and from
up to 13 different sites within individual hospitals (Figure 2.1). Clostridium difficile was
recovered from 83% of hospitals (n=27) from up to five different sites (Figure 2.1).
Combining data from Phases One and Two, E. coli and/or C. difficile were recovered
from at least one site in each veterinary hospital and each type of site sampled (Tables
2.1, 2.2). Overall, the hospital prevalence of recovery of E. coli and C. difficile (Phase
One and Phase Two) was 92% (n=93) and 58% (n=58), respectively (Table 2.1). Forty- seven percent (n=56) of the C. difficile ribotypes identified have been previously identified in humans, and 43% (n=52) have been previously identified in animals. The remaining 9% (n= 11) were newly identified ribotypes (Table 2.3). Among the 56 isolates of known human ribotypes, 36 (64%) produced Toxins A and B, 19 (34%) produced Toxins A, B and CDT, and 1 (2%) produced Toxin B. Ninety-four percent (n=
49) of the isolates from ribotypes only associated with animals were non-toxigenic. Four percent (n=2) of the isolates from known animal ribotypes had toxins A and B, and 2%
(n=l) had toxins A, B and CDT. Forty-five percent (n=5) of the newly identified types
(n=l 1) were toxigenic (Toxins A and B-80% (n=4), Toxins A, B and CDT-20% [n=l]).
Methicillin-resistant Staphylococcus aureus and MRSP were identified in 9% (n=9) and
7% (n=7) of the hospitals, respectively (Table 2.1). Thirty-eight percent (n=5) of the
MRSA isolates belonged to Canadian epidemic strain (cMRSA)-5 and 23% (n=3) were cMRSA-2. The remainder (n=5) were non-typeable. One isolate of Salmonella
61 Typhimurium isolated on XLT4 medium was recovered from an exam room floor of a different hospital. One isolate of Salmonella Mbandaka isolated on the MRSV medium was recovered from the run area of another hospital. The Salmonella isolates were susceptible to all antimicrobials tested. VRE, CPV and FCV were not recovered from any sampled sites.
The prevalence of antimicrobial resistance in E. coli isolates was low (0-13%), yet at least one resistant isolate was observed to all classes of antimicrobials tested (Table
2.4). Combined resistance to ampicillin and amoxicillin-clavulanic acid was only observed with resistance to a third-generation cephalosporin or to a cephamycin. Sixty- eight E. coli isolates were selected for testing for the WOCMY-2 gene including 47% (n=32) of the E. coli isolates obtained using ESBL selection methodology. Sixty percent (n=41) of the tested isolates were PCR positive for the blacMY-2 gene. These isolates were recovered from 9% (n=9) of the hospitals (Table 2.1). Ninety-five percent (n=39) of the blacMY-2 positive E. coli isolates were resistant to ampicillin, amoxicillin-clavulanic acid and cefoxitin. Although only 5% (n=2) isolates were resistant to ampicillin, amoxicillin- clavulanic acid, cefoxitin and ceftriaxone, isolates positive for the Z>/«CMY-2 gene had significantly more isolates (p<0.0l) with a MIC >8 jag/ml for ceftriaxone.
3.1. Generalized linear mixed models of potential factors associated with environmental recovery of bacteria
Models of potential factors associated with the environmental recovery of MRS A and AMP-R E. coli could not be generated because of insufficient power. A model could not be generated for potential factors associated with the environmental recovery of E. coli because of insufficient heterogeneity in the outcome. Using a multi-level logistic
62 modeling procedure, the use of in-hospital parenteral trimethoprim-sulfonamide combinations was positively associated with the recovery of environmental C. difficile
(odds ratio (OR) =2.73 /?=0.008,95% confidence interval (CI) (1.30,5.77)) (Table 2.5).
The use of in-hospital oral enrofloxacin was negatively associated with the recovery of C. difficile (OR = 0.51,/?=0.036, 95% CI (0.27, 0.96)). There were significantly higher odds of C. difficile recovery from kennel areas (OR=2.17/>=0.051,95% CI (1.01,4.74) and borderline significant lower odds of recovery from table surfaces (OR=0.37 ^=0.057,
95% CI (0.14,1.03) when compared to floors. Hospital demographic data, disinfectant use, hand cleansers, availability of SOPs for environmental cleaning and disinfection use or presence of an isolation unit were not significantly associated with environmental recovery of C. difficile. The ICChospitai of C. difficile recovery was 0.25 indicating that there was low to moderate within-hospital clustering of environmental C. difficile. The distribution of residuals was bounded between -3.4 and 1.4. The upper tail of the normal plot was skewed since few hospitals had strongly positive residuals when compared to the tail of the distribution. However, Cook's distance revealed no observations that were influential to the model.
4.0 Discussion
The recovered bacteria from environmental surfaces in this study are potential pathogens (e.g., E. coli, C. difficile) and also represent a pool of antimicrobial resistance genes (e.g., MRSA, MRSP, blacMY-2 positive E. coli) that could place human and animal health at risk. The recovered organisms may contribute to occurrence of hospital-acquired infections and possibly to the overall epidemiology of opportunist infections in the hospital and community including zoonoses. These bacteria can colonize or infect
63 companion animals and people, and this could lead to unwitting dissemination of bacteria
in the environment. In the absence of clinical signs, the identification of potentially
infectious patients or staff, for practical purposes, is difficult. Therefore, implementation
of appropriate infection measures to control the spread of these organisms in the
veterinary hospital environment is important.
Methicillin-resistant Staphylococcus aureus is an important antimicrobial resistant
zoonosis. The cMRSA-2 clone is a common North American human clone associated
with human hospital and community MRS A infections and colonization (Christianson et
al., 2007), companion animal clinical infections (Weese et al., 2006, Christianson et al.,
2007, Weese et al., 2007), colonization human contacts of animal patients (Weese et al.,
2006) and has been recovered from companion animal veterinarians (Hanselman et al.,
2006). The cMRS A-5 clone is the predominant one associated with equine colonization
and infections in North America (Weese et al., 2006) and can also colonize veterinary
personnel (Hanselman et al., 2006, Anderson et al., 2008). With both human and animal
reservoirs, there is an opportunity for MRSA transmission in veterinary hospitals through
contact between individuals. However, as demonstrated in this study an environmental
reservoir of MRSA could be an additional source for colonization or infection of both
humans and animals.
Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an emerging
antimicrobial resistant opportunist pathogen of companion animals. The relatively high prevalence of MRSP fromenvironmenta l sites within hospitals observed in this study
(hospital prevalence 7%) is quite concerning especially given the low frequency of colonization and infection observed in dogs (Griffeth et al., 2008, Hanselman et al., 2008,
64 Ruscher et al., 2008). Although it is a recognized zoonosis, no large human reservoir is known, yet it has been identified in veterinary personnel (Sasaki et al., 2007, van
Duijkeren et al., 2008). The observed high frequency of recovery may result from poor environmental disinfection within hospitals. We were unable to test this hypothesis because of limited statistical power. However, given the recent emergence of highly multi-drug resistant MRSP (Loeffier et al., 2007, Ruscher et al., 2008,Wettstein et al.,
2008) and the high frequency of recovery from veterinary hospitals, there is a need for further work describing the epidemiology of MRSP.
In addition, blacMY-2 positive E. coli was also recovered from environmental sites. This organism has been described in humans (Pitout et al., 2007) and many veterinary species (Allen and Poppe, 2002, Blanc et al., 2006, Singer et al., 2008, Smet et al., 2008) including dogs (Sanchez et al., 2002, Carattoli et al., 2005, Murphy 2009a), therefore, the recovery of WOCMY-2 positive E. coli was not surprizing. There are previous reports of HAI associated with this organism in dogs where a corresponding environmental reservoir was documented (Sanchez et al., 2002). Additionally, addressing many aspects of infection control, including environmental disinfection, aided in control of outbreaks of HAI associated with this pathogen.
Salmonella was recovered infrequently from hospitals. This could be reflective of the low level prevalence of carriage in healthy companion animals (Van Immerseel et al.,
2004, Wright et al., 2005, Lefebvre et al., 2006b, Murphy et al., 2009a). Comparable studies in healthy people have not been performed, however S. Typhimurium was the second most common serovar reported from clinical human isolates in Canada
(Government of Canada 2007). S. Mbandaka infections in humans (Whichard et al.,
65 2007) and food animals (Liebana et al., 2001, Guerin et al., 2005) have been described
(Liebana et al., 2001), yet not in companion animals. Outbreaks of salmonellosis associated with S. Typhimurium in people and companion animals linked with veterinary hospitals or animal shelters have been reported (Cherry et al., 2004, Wright et al., 2005).
In two outbreaks, environmental samples yielded the outbreak strain /phage type of S.
Typhimurium identified in the outbreak (Wright et al., 2005).
The high prevalence of C. difficile recovery, including human and animal associated ribotypes, is noteworthy; especially the identification of ribotype 027; an epidemiologically important ribotype, which has been associated with outbreaks of C. c//$?c//e-associated disease in people in North America and Europe (Kuijper et al., 2006) and has been recovered from a healthy hospital visitation dog in southern Ontario
(Lefebvre et al., 2006a). The recovery of C. difficile is not unexpected since humans and animals can be asymptomatically colonized, and as spore-forming bacteria, it is resistant to disinfectants. We were not able to demonstrate significant statistical associations between the recovery of C. difficile and the use of specific disinfectants, hand-cleansers and the presence of an isolation area. However, given the data in the study, the statistical power to determine any association was <10%.
We attempted to determine factors that may have been associated with the environmental recovery of bacterial. Because of statistical limitations, we were only able to generate a model for the recovery C. difficile. We were not able to demonstrate significant statistical associations between the recovery of C. difficile and the use of specific disinfectants, hand-cleansers and the presence of an isolation area; however we were hampered by low statistical power. Furthermore, the format of capturing data on the
66 hypothesized risk factors for bacterial recovery using a questionnaire may have been
inadequate. This method relied on veterinarians and veterinary technicians to accurately
and precisely describe environmental disinfection (e.g., contact time for disinfectants)
and other infection control practices (e.g., duration of handwashing). There are no
publications evaluating the accuracy of data on the evaluated infection control practices
gathered in this manner. Inaccuracies or imprecision of the responses provided by the
veterinarians and veterinary technicians may resulted in the misclassification of
hypothesized risk factors leading to an inability to determine associations between the
recovered organisms and the hypothesized infection control practices.
The recovery of C. difficile was significantly higher from kennels and tables, than
from floors. Kennels are frequently contaminated with fecal material, including diarrhea.
Tables in veterinary practices are high contact areas by animals and humans and can
easily be contaminated with fecal material. Quaternary ammonium compounds (QAC)
were the most frequently reported disinfectants used on table and kennels by
veterinarians and veterinary technicians from the investigated practices (Murphy et al.,
2009b). C. difficile is resistant to disinfection with QACs. The data from these studies
suggest that veterinary practices may need to consider intermediate-level disinfectants for routine disinfection of kennels and tables.
The observed associations between C. difficile recovery and the use of in-hospital parenteral trimethoprim-sulfonamide combinations (positive association) and oral enrofloxacin (negative association) were difficult to interpret. In human medicine, use of many antimicrobials have been identified as risk factors for C. difficile infection, including fluoroquinolones, clindamycin and cephalosporins (Kuijper et al., 2006,
67 Blossom and McDonald 2007, Elliott et al., 2007, Deshpande et al., 2008) however their
role as risk factors for human hospital environmental contamination with C. difficile have
not been published. Alternatively, the observed associations may be driven by other
unmeasured factors that were highly correlated with antimicrobial use, such as other
infection control practices, patient population characteristics or patient care and
management factors. Further studies are required to clarify the role, if any, of
antimicrobial use on environmental contamination with C. difficile.
Given the high prevalence of C. difficile, it is perhaps surprizing that CPV and
FCV were not recovered from any hospitals, since they all require high level disinfection
for removal from the environment. However, CPV and FCV are limited to dogs and cats, respectively, and shedding may have been uncommon in the study population.
Companion animals and people can be asymptomatically colonized with C. difficile which may lead to a greater opportunity for dissemination of C. difficile in the veterinary hospital environment. The differences in apparent prevalence could also be due to infection control practices used in the management of known or suspected cases of CPV, or cases and carriers of FCV. Veterinarians from the investigated hospitals reported that animals with known CPV or FCV associated disease, animals with clinical signs of disease associated with the gastrointestinal or respiratory tract and animals without vaccinations would require specific infection control measures (Murphy et al., 2009b).
Therefore, adequate infection control practices and low frequency of shedding could account for the lack of recovery of CPV and FCV. However it is possible that sensitivity of the sampling methodology was too low to detect CPV and FCV.
68 This study demonstrated that recovery of environmental bacteria was possible using sterile electrostatic cloths. These cloths are inexpensive, easily accessible, simple to use and sterilize. Other authors have had similar experiences using electrostatic cloths
(Burgess et al., 2004). Standardized sampling and microbiological methods for environmental organisms from the hospital environment have not been established. In addition, the sensitivity of recovery using different tools (e.g., cotton swabs, electrostatic cloths, contact plates) is unknown. Determining the sensitivity of recovery and development of sampling and microbiological standards will improve interpretation of results and reproducibility of study design. Another challenge in determining the role of environmental pathogens in the epidemiology of HAI, is that environmental contamination may be a consequence of infection, rather than a source of infection.
Selecting the appropriate study group, such as secondary cases rather than primary cases in an epidemic, may assist in determining real associations between an environmental reservoir of pathogens and HAI.
The issue of infection control in companion animal veterinary medicine is in the early stages. This study demonstrated that E. coli, blacMY-2 positive E. coli, C. difficile,
MRS A and MRSP were present in environmental sites within community veterinary hospitals. Although this study did not attempt to correlate HAI with an environmental reservoir of organisms, the high frequency of recovery of potential pathogens combined with inadequate infection control policies provides an opportunity for an excess of HAI in community companion animal veterinary hospitals. This preliminary study points to the need for research in infection control in community companion animal veterinary medicine. This includes studies that quantify the frequency of HAI, enhance
69 understanding endemic and epidemic HAI, describe the scope of conditions and
associated organisms contributing to HAI, describe factors associated with HAI,
including the contribution of an environmental reservoir, and factors associated with reducing the environmental burden.
Acknowledgements
We would like to acknowledge the following for their contributions to this study:
Alyssa Calder, Karlee Thomas and Adriana Sage for clinic sampling; staff of the
Canadian Research Institute for Food Safety, especially Gabriel Jantzi and Virginia
Young for E. coli and Salmonella isolation; Meredith Craig for data entry, Joyce
Rousseau for C. difficile, MRSA and MRSP isolation and typing; Rebeccah Travis and
Gerry Lazarek for PCR of the blacMY-2 gene; staff of the Laboratory for Foodborne
Zoonoses for susceptibility testing and typing of Salmonella isolates; staff at the Prairie
Diagnostics Services for virus isolation and the veterinary practices for participating in this project. This study was funded by the Ontario Veterinary College Pet Trust Fund and the Public Health Agency of Canada. Colleen Murphy was a recipient of the Ontario
Veterinary College Graduate Student Fellowship.
70 References
Allen, KJ, C Poppe. 2002. Occurrence and characterization of resistance to extended-
spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from
food-producing animals in Canada. Can J Vet Res, v. 66, p. 137-44.
Anderson, ME, S L Lefebvre, J S Weese. 2008. Evaluation of prevalence and risk factors
for methicillin-resistant Staphylococcus aureus colonization in veterinary personnel
attending an international equine veterinary conference. Vet Microbiol, v. 129, p. 410-7.
Bidet, P, F Barbut, V Lalande, B Burghoffer, J C Petit. 1999. Development of a new
PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett, v. 175, p. 261-6.
Blanc, V, R Mesa, M Saco, S Lavilla, G Prats, E Miro, F Navarro, P Cortes, M
Llagostera. 2006. ESBL- and plasmidic class C beta-lactamase-producing E. coli strains isolated from poultry, pig and rabbit farms. Vet Microbiol, v. 118, p. 299-304.
Blossom, DB, L C McDonald. 2007. The challenges posed by reemerging Clostridium difficile infection: Clin Infect Dis, v. 45, p. 222-7.
Boerlin, P, S Eugster, F Gaschen, R Straub, P Schawalder. 2001. Transmission of opportunistic pathogens in a veterinary teaching hospital. Vet Microbiol, v. 82, p. 347-59.
Burgess, BA, P S Morley, D R Hyatt. 2004. Environmental surveillance for Salmonella
71 enterica in a veterinary teaching hospital. J Am Vet Med Assoc, v. 225, p. 1344-8.
Carattoli, A, S Lovari, A Franco, G Cordaro, P Di Matteo, A Battisti. 2005. Extended-
spectrum beta-lactamases in Escherichia coli isolated from dogs and cats in Rome, Italy,
from 2001 to 2003. Antimicrob Agents Chemother, v. 49, p. 833-5.
Centres for Disease Control and Prevention (CDC). 2003. Recommendations of the CDC
and the Healthcare Infection Control Practices Advisory Committee (HICPAC). http://www.cdc.gov./ncidodVdhqp/gl_envfroninfection.html. Accessed February, 2009.
Cherry, B, A Burns, G S Johnson, H Pfeiffer, N Dumas, D Barrett, P L McDonough, M
Eidson. 2004. Salmonella Typhimurium outbreak associated with veterinary clinic.
Emerg Infect Dis, v. 10, p. 2249-51.
Christianson, S, G R Golding, J Campbell, M R Mulvey. 2007. Comparative genomics of
Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus. J Clin
Microbiol, v. 45, p. 1904-11.
Dohoo, I, W Martin, H Stryhn. 2003. Veterinary epidemiologic research. AVC Inc.
Charlottetown, Prince Edward Island.
Deshpande, A, C Pant, A Jain, T G Fraser, D D Rolston. 2008. Do fluoroquinolones predispose patients to Clostridium difficile associated disease? A review of the evidence.
Curr Med Res Opin, v. 24, p. 329-33.
72 Elliott, B, B J Chang, C L Golledge, T V Riley. 2007. Clostridium d$?c//e-associated
diarrhoea. Intern Med J, v. 37, p. 561-8.
Government of Canada. 2006. Canadian integrated program for antimicrobial resistance
surveillance (CIPARS) 2004. Public Health Agency of Canada.
Government of Canada. 2007. Canadian Integrated Program for Antimicrobial Resistance
Surveillance (CIPARS) 2005. Public Health Agency of Canada.
Griffeth, GC, D O Morris, J L Abraham, F S Shofer, S C Rankin. 2008. Screening for
skin carriage of methicillin-resistant coagulase-positive staphylococci and
Staphylococcus schleiferi in dogs with healthy and inflamed skin. Vet Dermatol, v. 19, p.
142-9.
Griffin, FA. 2007. 5 Million Lives Campaign. Reducing methicillin-resistant
Staphylococcus aureus (MRSA) infections. Jt Comm J Qual Patient Saf, v. 33, p. 726-31.
Guerin, MT, S W Martin, G A Darlington, A Rajic. 2005. A temporal study of
Salmonella serovars in animals in Alberta between 1990 and 2001. Can J Vet Res, v. 69, p. 88-99.
Hanselman, BA, S Kruth, J S Weese. 2008. Methicillin-resistant staphylococcal colonization in dogs entering a veterinary teaching hospital. Vet Microbiol, v. 126, p.
277-81.
73 Hanselman, BA, S A Kruth, J Rousseau, D E Low, B M Willey, A McGeer, J S Weese.
2006. Methicillin-resistant Staphylococcus aureus colonization in veterinary personnel.
Emerg Infect Dis, v. 12, p. 1933-8.
Kato, H, N Kato, K Watanabe, N Iwai, H Nakamura, T Yamamoto, K Suzuki, S M Kim,
Y Chong, E B Wasito. 1998. Identification of toxin A-negative, toxin B-positive
Clostridium difficile by PCR. J Clin Microbiol, v. 36, p. 2178-82.
Kuijper, EJ, B Coignard, P lull. 2006. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect, v. 12 Suppl 6, p. 2-18.
Lefebvre, SL, L G Arroyo, J S Weese. 2006a. Epidemic Clostridium difficile strain in hospital visitation dog. Emerg Infect Dis, v. 12, p. 1036-7.
Lefebvre, SL, D Waltner-Toews, A S Peregrine, R Reid-Smith, L Hodge, L G Arroyo, J
S Weese. 2006b. Prevalence of zoonotic agents in dogs visiting hospitalized people in
Ontario: implications for infection control. J Hosp Infect, v. 62, p. 458-66.
Liebana, E, D Guns, L Garcia-Migura, M J Woodward, F A Clifton-Hadley, R H Davies.
2001. Molecular typing of Salmonella serotypes prevalent in animals in England: assessment of methodology. J Clin Microbiol, v. 39, p. 3609-16.
Loeffler, A, M Linek, A Moodley, L Guardabassi, J M Sung, M Winkler, R Weiss, D H
Lloyd. 2007. First report of multiresistant, mecA-positive Staphylococcus intermedius in
74 Europe: 12 cases from a veterinary dermatology referral clinic in Germany. Vet
Dermatol, v. 18, p. 412-21.
M'Zali, FH, J Heritage, D M Gascoyne-Binzi, M Denton, N J Todd, P M Hawkey. 1997.
Transcontinental importation into the UK of Escherichia coli expressing a plasmid- mediated AmpC-type beta-lactamase exposed during an outbreak of SHV-5 extended- spectrum beta-lactamase in a Leeds hospital. J Antimicrob Chemother, v. 40, p. 823-31.
Martin, H, B Willey, D E Low, H R Staempfli, A McGeer, P Boerlin, M Mulvey, J S
Weese. 2008. Characterization of Clostridium difficile strains isolated from patients in
Ontario, Canada, from 2004 to 2006. J Clin Microbiol, v. 46, p. 2999-3004.
Mulvey, MR, L Chui, J Ismail, L Louie, C Murphy, N Chang, M Alfa. 2001.
Development of a Canadian standardized protocol for subtyping methicillin-resistant
Staphylococcus aureus using pulsed-field gel electrophoresis. J Clin Microbiol, v. 39, p.
3481-5.
Murphy, CP, R J Reid-Smith, J F Prescott, B N Bonnett, C Poppe, P Boerlin, J S Weese,
N Janecko, S A McEwen. 2009a. Occurence of antimicrobial resistance in selected bacteria in healthy dogs and cats presented to private veterinary clinics in Southern
Ontario. Can Vet J, v. 50, p. 1047-53.
75 Murphy, CP, R J Reid-Smith, J S Weese, S A McEwen. 2009b. Evaluation of specific infection control practices used by companion animal veterinarians in community veterinary practices in southern Ontario. Zoonoses Public Health. Epub ahead of print.
NCCLS. 2002. Performance standards for antimicrobial susceptibility testing. Approved standard M100-S15 M7-A-MIC Testing Section. National Committee for Clinical
Laboratory Standards, Wayne PA.
Pitout, JD, D B Gregson, D L Church, K B Laupland. 2007. Population-based laboratory surveillance for AmpC beta-lactamase-producing Escherichia coli, Calgary. Emerg Infect
Dis, v. 13, p. 443-8.
Rabe-Hesketh, S, A Skrondal. 2008. Multilevel and longitudinal modeling using Stata
(Second Edition). College Station, TX, Stata Press.
Rabe-Heskethe, S, A Skrondal, A Pickles. 2002. Reliable estimation of generalized linear mixed models using adaptive quadrature. The Stata Journal, v. 2, p. 1-21.
Rupnik, M, M Grabnar, B Geric. 2003. Binary toxin producing Clostridium difficile strains. Anaerobe, v. 9, p. 289-94.
Ruscher, C, A Lubke-Becker, C G Wleklinski, A Soba, L H Wieler, B Walther. 2008.
Prevalence of methicillin-resistant Staphylococcus pseudintermedius isolated from clinical samples of companion animals and equidaes. Vet Microbiol, v. 136, p. 197-201.
76 Sanchez, S, M A McCrackin Stevenson, C R Hudson, M Maier, T Buffington, Q Dam, J
J Maurer. 2002. Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs. J Clin Microbiol, v. 40, p. 3586-95.
Sasaki, T, K Kikuchi, Y Tanaka, N Takahashi, S Kamata, K Hiramatsu. 2007.
Methicillin-resistant Staphylococcus pseudintermedius in a veterinary teaching hospital. J
Clin Microbiol, v. 45, p. 1118-25.
Schott, HC, S L Ewart, R D Walker, R M Dwyer, S Dietrich, S W Eberhart, J Kusey, J A
Stick, F J Derksen. 2001. An outbreak of salmonellosis among horses at a veterinary teaching hospital. J Am Vet Med Assoc, v. 218, p. 1152-9.
Singer, RS, S K Patterson, R L Wallace. 2008. Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract. Appl
Environ Microbiol, v. 74, p. 6956-62.
Smet, A, A Martel, D Persoons, J Dewulf, M Heyndrickx, B Catry, L Herman, F
Haesebrouck, P Butaye. 2008. Diversity of extended-spectrum beta-lactamases and class
C beta-lactamases among cloacal Escherichia coli Isolates in Belgian broiler farms.
Antimicrob Agents Chemother, v. 52, p. 1238-43.
van Duijkeren, E, D J Houwers, A Schoormans, M J Broekhuizen-Stins, R Dcawaty, A C
Fluit, J A Wagenaar. 2008. Transmission of methicillin-resistant Staphylococcus intermedius between humans and animals. Vet Microbiol, v. 128, p. 213-5.
77 Van Immerseel, F, F Pasmans, J De Buck, I Rychlik, H Hradecka, J M Collard, C
Wildemauwe, M Heyndrickx, R Ducatelle, F Haesebrouck. 2004. Cats as a risk for transmission of antimicrobial drug-resistant Salmonella. Emerg Infect Dis, v. 10, p. 2169-
74.
Ward, MP, T H Brady, L L Couetil, K Liljebjelke, J J Maurer, C C Wu. 2005.
Investigation and control of an outbreak of salmonellosis caused by multidrug-resistant
Salmonella Typhimurium in a population of hospitalized horses. Vet Microbiol, v. 107, p.
233-40.
Weese, JS, J Armstrong. 2003. Outbreak of Clostridium difficile-associated disease in a small animal veterinary teaching hospital: J Vet Intern Med, v. 17, p. 813-6.
Weese, JS, H Dick, B M Willey, A McGeer, B N Kreiswirth, B Innis, D E Low. 2006a.
Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol, v. 115, p. 148-
55.
Weese, JS, M Faires, J Rousseau, A M Bersenas, K A Mathews. 2007. Cluster of methicillin-resistant Staphylococcus aureus colonization in a small animal intensive care unit. J Am Vet Med Assoc, v. 231, p. 1361-4.
Weese, JS, J Rousseau, B M Willey, M Archambault, A McGeer, D E Low. 2006b.
Methicillin-resistant Staphylococcus aureus in horses at a veterinary teaching hospital:
78 frequency, characterization, and association with clinical disease. J Vet Intern Med, v. 20, p. 182-6.
Wettstein, K, S Descloux, A Rossano, V Perreten. 2008. Emergence of methicillin- resistant Staphylococcus pseudintermedius in Switzerland: three cases of urinary tract infections in cats. Schweiz Arch Tierheilkd, v. 150, p. 339-43.
Whichard, JM, K Gay, J E Stevenson, K J Joyce, K L Cooper, M Omondi, F Medalla, G
A Jacoby, T J Barrett. 2007. Human Salmonella and concurrent decreased susceptibility to quinolones and extended-spectrum cephalosporins. Emerg Infect Dis, v. 13, p. 1681-8.
Wright, JG, L A Tengelsen, K E Smith, J B Bender, R K Frank, J H Grendon, D H Rice,
A M Thiessen, C J Gilbertson, S Sivapalasingam, T J Barrett, T E Besser, D D Hancock,
F J Angulo. 2005. Multidrug-resistant Salmonella Typhimurium in four animal facilities:
Emerg Infect Dis, v. 11, p. 1235-41.
79 Figure 2.1. Number of sites in the Phase One component of the study where environmental E.coli (n=24 hospitals) and C. difficile (n=27 hospitals) were recovered from companion animal veterinary hospitals.
Percentage of 20% Veterinary Hospitals l C. (Male 15% 5 E caff
Number of environmental sites with the veterinary hospitals
80 Table 2.1. Prevalence (%) of bacterial recovery from environmental sites within companion animals veterinary hospitals.
Organism Prevalence (%)
(95% Confidence Interval)
Hospitals Kennels Isolation*1 Runs Floors Tables
E. coif 92 48 66 27 72 26
(85, 96) (38, 59) (48,81) (18,36) (62,81) (18,35)
C. difficile 58 17 29 11 16 7
(48, 69) (10, 26) (15,46) (6,19) (9,25) (3,14)
Salmonella11 2 0d 0 1 1 0
(0.2, 7) (0,4)d (0,10)e (0.02, 5) (0.02, 5) (0,4)e
MRSAb 9 1 3 0 3 4
(4, 16) (0.02, 5) (0.07, 15) (0,4)e (0.6, 8) (1, 10)
MRSPC 7 1 3 0 3 2
(3,14) (0.02, 5) (0.07,15) (0,4)e (0.6,9) (0.2,7) blacMY-2 9 2 3 0 5 0
E.colf (4,16) (0.2,7) (0.07,15) (0,4)e (0,4)e aHospital n=101; n=100; cn=99; n=35. Isolation was not present every hospital. All other sites were present in each of the sampled hospitals; eOne-sided 97.5% confidence interval.
81 Table 2.2. Prevalence (%) of bacterial recovery from equipment within companion animal veterinary hospitals.
Organism Prevalence (95% Confidence Interval)
Telephone, Otoscope Stethoscopes, Thermometers
keyboards tips Oto/Ophthalmoscopes
and taps
E.colt 34 (25,44) 7d(2,16) 22(14,31) 28c(20,38)
C. difficile 15(9,24) 8e(2, 17) 10(5,18) llc(6, 19)
MRSAb 2 (0.2, 7) 2f (0.04, 9) 1 (0.02, 5) 0c(0,4)h
MRSPC 1 (0.03,5) 08(0,6) 1 (0.03, 5) 0(0,4)
blacMY-2 0(0,4) 0(0,4) 0(0,4) 2 (0.2, 7)
E.colt an=101 (unless noted); "n=100 (unless noted); cn=99 (unless noted); dn=55; en=66; *n=61
^1=56; hOne-sided, 97.5% confidence interval.
82 Table 2.3. Ribotypes and toxin profile of environmental Clostridium difficile isolates
(n=l 19) from companion animal veterinary hospitals.
Ribotype Species3 Toxin A Toxin B CDT Number of Percentage
isolates of isolates6
V Human + + - 25 21
Y Human + + + 8 7
W Human + + - 7 6
027 Human + + + 6 5
C Human + + + 2 2
K Human + + + 2 2
N Human + + + 0.8
L Human + + - 0.8
MSE Human + + - 0.8
MSI Human + + - 0.8
AK Human + + - 0.8
AA Human - + - 0.8
OVCB Canine - - - 27 23
OVCC Canine - - - 21 18
OVCCc Canine + + - 1 0.8
OVCH Canine - - - 1 0.8
SL3 Canine + + + 1 0.8
SL4 Canine + + - 1 0.8
CM 65 Not - - - 3 3
83 Ribotype Species" Toxin A Toxin B CDT Number of Percentage
isolates of iso!atesb
Described
(ND)d
CM 53 NDd - - - 3 3
CM 57 NDd + + - 2 2
CM 76 NDd + + + 1 0.8
CM 103 NDd + + - 1 0.8
CM 92 NDd + + - 1 0.8
"Known animal host where the ribotype has been identified; Sum does not equal 100 due to rounding; "Ribotype had two different toxin gene pattern; dNewly identified ribotype that has not been described in humans or an animal species.
84 Table 2.4. Distribution (%) of antimicrobial minimum inhibitory concentrations of environmental Escherichia coli isolates (n=1554) from veterinary hospitals.
Antimicrobial3 Distribution of minimum inhibitory concentrations b,c (ug/ml) Total Resistantd
0.015 0.03 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256
AMC 2 19 60 15 0.9 3 3(2,4)
AMK 2 28 59 9 0.8 0.06f 0 (0,0.2)e
AMP 7 50 25 4 0.1 13 13(11,14)
CHL 7 63 28 0.1 1 1(0.8,2)
CIP 97 0.2 1 0.4 1 1.7(1,2)
CRO 96 0.1 0.6 0.2 2 0.5 0.3* 0.3 (0.07, 0.66)
FOX 0.06 2 33 55 6 0.4 3 3.5 (3, 5)
GEN 11 72 16 1 0.06 0.06(0.001,0.4)
KAN 96 1 0.9 1.7(1,2)
NALg llf 76 10 0.1 0.06 3 3(2,4)
SOX8 90 3 0.06 0 (0,0.2)e
85 Antimicrobial3 Distribution of minimum inhibitory concentrations b'c (ug/ml) Total Resistant
0.015 0.03 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256
STR8 93 7(6,9)
SXT8 84 13 0.06 0.1 0.2 3 (2,4)
TIO 77 11 0.3 0.3 3 (2,4)
TCY 92 0.6 7(6,8)
"Antimicrobial Abbreviations: AMC- amoxicillin-clavulanic acid, AMK- amikacin, AMP-ampicillin, CHL-chloramphenicol, CIP- ciprofloxacin, CRO-ceftriaxone, FOX-cefoxitin, GEN-gentamicin, KAN-kanamycin, NAL-nalidixic acid, SOX-sulfizoxazole, STR- streptomycin, SXT-trimethoprim-sulphamethoxazole, TlO-ceftiofur, TCY-tetracycline. ''Minimum Inhibitory Concentration distribution: The unshaded fields indicate the MIC range tested for each antimicrobial in the plate configuration. The MICs at the upper or lower bound of the distribution are censored. The values at the upper bound are > to the value presented and the values at the lower bound are < the value presented. cDouble bar represents the resistant breakpoint. Single bar represents the susceptible breakpoint. dValues in brackets are 95% confidence intervals. Total resistant may not equal values presented in table due to rounding error. eOne sided 97.5% confidence interval. fExact MIC. BSusceptible breakpoint-NAL: <16 |jg/ml, Susceptible breakpoint-SOX:
<256 ug/ml, Resistant breakpoint-SOX: >512 ug/ml, Susceptible breakpoint-STR: <32 ug/ml, Susceptible breakpoint-SXT: <2 ug/ml.
86 Table 2.5. Results from a generalized linear mixed model3 of factors potentially associated with the environmental recovery of Clostridium difficile from veterinary hospitals.
Variable Odds Standard z-value p-value 95% Confidence
Ratio Error Interval
Use of in-hospital enrofloxacin 0.51 0.16 -2.10 0.036 0.27,0.96
Use of in-hospital parenteral 2.73 1.04 2.65 0.008 1.30,5.77 trimethoprim-sulfonamide combinations
Sitesb
Tables 0.37 0.19 -1.90 0.057 0.14,1.03
Kennels 2.17 0.87 1.93 0.051 1.01,4.74
Isolation 0.58 0.27 -1.14 0.255 0.23,1.47
Telephones, computer 1.06 0.46 0.14 0.891 0.45,2.47
keyboards, door knobs, and
taps on kitchen and bathroom
sinks
Taps on exam and treatment 1.11 0.54 0.21 0.832 0.43,2.87
room sinks
Otoscopes, Ophthalmoscopes 0.58 0.27 -1.15 0.250 0.23,1.46
and Stethoscopes
Otoscope tips 0.45 0.27 -1.35 0.177 0.14,1.43
Thermometers 0.59 0.28 -1.10 0.269 0.24,1.50
"Number of observations: Level 1 (sites within veterinary hospitals) n=807; Level 2
(veterinary hospitals) n=92. Number of iterations: 4. Log likelihood= -295.025. LR chi2=
41.88 (p-value=0.0000). Pseudo R2=0.0657. Level 2 variance= 1.045 deferent: Floors.
87 Chapter 3
A prospective cohort study of the effects of antimicrobial treatment on the incidence of antimicrobial resistance in generic fecal Escherichia coli isolates and isolation of Clostridium difficile. Salmonella enterica, blaCMY-
2 positive E. coli, methicillin-resistant Staphylococcus aureus,
Staphylococcus pseudintermedius, and vancomycin-resistant
Enterococcus spp. from dogs treated in community companion animal practices in southern Ontario.
Abstract
This study aimed to 1) compare the incidence of antimicrobial resistance in generic fecal E. coli isolates and the isolation of Clostridium difficile, Salmonella enterica, blacMY-2 positive E. coli, vancomycin-resistant Enterococcus spp. (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus pseudintermedius (MRSP) from dogs treated with antimicrobials to untreated, healthy dogs, and 2) determine the associations between antimicrobial treatment and the risk of antimicrobial resistance in fecal E. coli isolates, and the isolation of fecal C. difficile,
Salmonella enterica, blacw-2 positive E. coli, VRE, MRSA and MRSP. Thirty-two veterinary hospitals in southern Ontario volunteered to participate and recruit dogs treated with antimicrobials, and untreated dogs. Dogs were enrolled into five different cohorts: treatment with oral amoxicillin-clavulanic acid (n=12), oral cephalexin (n=33), oral fluoroquinolones (n=12), parenteral penicillin (n=9) and an untreated cohort (n=8).
The first fecal sample (day 0) was collected prior to antimicrobial treatment for the
88 treated dogs and on the day of enrolment for the untreated dogs. Follow-up fecal samples
were collected on days 7,14,28,90 and 180. The incidence rates of resistance to
amoxicillin-clavulanic acid (incidence rate ratio (IRR) 3.38p=0.03), cefoxitin (IRR 3.22
/?=0.04), ceftiofur (IRR 8.21 /K0.01) and ceftriaxone (IRR 6.70p=0.02) were significantly higher in dogs treated with cephalexin than untreated dogs. Treatment with cephalexin was strongly associated with resistance to amoxicillin-clavulanic acid (Hazard ratio (HR) 3.92/7=0.03), cefoxitin (HR 3.70p=0.03), ceftiofur (HR 10.10^=0.03) and ceftriaxone (HR 8.61 p=0.04). The incidence rate for isolation of C. difficile was significantly higher in dogs treated with amoxicillin-clavulanic acid (IRR 16.2/K0.01).
Antimicrobial treatment was not significantly associated with the recovery of Salmonella enterica, blacwY-2 positive E. coli, MRSA or MRSP. The associations between antimicrobial treatment with cephalexin and resistance in fecal E. coli isolates to amoxicillin-clavulanic acid, cefoxitin, ceftriaxone and ceftiofur are important findings that may have implications for animal health, human health and public health.
1.0. Introduction
Antimicrobials have a positive impact on animal and human health through the successful treatment and prevention of bacterial infections. Although potentially life- saving, their use is not without adverse side effects including antimicrobial-associated diarrhea, selection of antimicrobial resistance and facilitation of enteric colonization with opportunistic pathogens (Prescott et al., 2002). The potential adverse effects of antimicrobial use in companion animals may also have public health implications, given the close contact between companion animals and humans, and opportunities for spread of resistant zoonotic pathogens and commensals.
89 In companion animals, epidemiological studies have demonstrated that prior antimicrobial exposure was associated with antimicrobial resistance in commensal E. coli isolates from healthy (Murphy et al., 2009) and hospitalized dogs (Ogeer-Gyles et al.,
2006), in opportunistic pathogens (Medleau et al., 1986, Rantala et al., 2004), including methicillin-resistant Staphylococcus aureus (MRSA) (Faires, 2008), and colonization with C. difficile (Clooten et al., 2008, Lefebvre et al., 2009). An experimental study demonstrated that dogs treated with enrofloxacin were more effectively colonized with multidrug-resistant E. coli (Trott et al., 2004). However, some caution is required when drawing inferences from cross-sectional studies because they can be subject to reverse- causation bias for factors that are time-variant such as antimicrobial exposure. Moreover, case-control studies usually assess only associations between independent variables and a single outcome (e.g., either antimicrobial resistance or colonization). In contrast, the prospective cohort study design is not subject to reverse causation and it enables the assessment of risk factors for multiple outcomes associated with specific antimicrobial use. It also permits the calculation of incident (new) occurrences of antimicrobial resistance or colonization with specific organisms associated with antimicrobial treatment. Furthermore, data collected through a prospective study designed to address a specific objective are often of better quality than those collected for other purposes.
This study used a prospective cohort design to measure associations between antimicrobial treatment of dogs with specific antimicrobials and the incidence of antimicrobial resistance in fecal E. coli isolates, and fecal colonization of dogs with specific opportunistic pathogens. The antimicrobials selected for study were oral amoxicillin-clavulanic acid, cephalexin, a fluoroquinolone and parenteral penicillin,
90 based on findings of a previous hypothesis-generating study (Murphy et al., 2009), frequency of use in Southern Ontario, or importance to human health (Government of
Canada, 2006). We compared, through the use of Kaplan-Meier survival functions and
Cox proportional hazard modelling, antimicrobial treated dogs and healthy, untreated dogs with respect to: 1) time to isolation of antimicrobial resistant fecal E. coli isolates; and 2) time to isolation of fecal C. difficile, Salmonella enterica and blacuY-2 positive E. coli. We also describe the recovery of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus spp. from dogs treated with antimicrobials and healthy dogs.
2.0. Materials and methods
2.1. Veterinary hospital and dog recruitment
All veterinary hospitals eligible for recruitment were located in Southern Ontario and licensed by the College of Veterinarians of Ontario in 2005 as companion animal hospitals or offices, including those with additional licenses for food animal or equine hospital or mobile (mixed-animal practices). A recruitment letter was mailed to the veterinarian listed as the director of the practice (n=766) describing the study objectives.
Practices willing to participate were asked to respond by mail, fax, or telephone with a completed practice-demographic survey (Appendix A.2.1).
Dogs were enrolled into one of five cohorts: treatment with one of oral cephalexin, amoxicillin-clavulanic acid, a fluoroquinolone, or injectable penicillin and a non-antimicrobial treated (untreated) cohort. Dogs eligible for any cohort had no episodes of vomiting or diarrhea in the seven days before enrolment and no exposure to antimicrobials in the six weeks before enrolment.
91 Dogs were eligble for enrolment into one of the oral antimicrobial cohorts if they were prescribed mono-antimicrobial therapy with one of: cephalexin, amoxicillin- clavulanic acid or fluoroquinolone, and were managed as out-patients (i.e., not hospitalized). Eligible dogs were treated for disease conditions diagnosed by veterinarians at participating veterinary hospitals. The care of study dogs was entirely at the discretion of the veterinarian and not influenced by the research team. Dogs were eligible for enrolment into the injectable penicillin cohort if they received injectable penicillin for elective sterilization surgery. Dogs that were presented through front office appointments (e.g., wellness appointments, vaccination appointments) that did not require antimicrobial therapy were enrolled in the untreated group. Attempts were made, though periodic contact with veterinary clinics, to achieve similar age, sex and weight distributions in the untreated cohort as the antimicrobial treated cohorts.
Fecal samples were collected from all dogs on day 0 (antimicrobial treatment started within 12 h following fecal collection for treated dogs; the day of enrolment for the untreated dogs) and days 7,14,28,90 and 180. The individuals collecting the fecal samples (veterinarians, veterinary technicians or pet owners for day 0, pet owner for subsequent samples) were instructed to place fecal samples into the provided sterile containers, store under refrigeration and mail them as soon as possible to the laboratory.
A questionnaire (Appendix A.3.1) was completed by the pet owners at the time of enrolment. It elicited information on the pet's diet, types of treats consumed, types of water consumed, coprophagia, contact with livestock and participation in therapy-dog programs.
92 Participating veterinary practices were visited in person at the beginning of the
study to discuss, in detail, the role of practices in the study and to provide training for the
individuals (veterinary technicians or veterinarians) who were the primary individual(s)
responsible for the execution of the study in that practice on study procedures (e.g.,
eligibility criteria, study materials and documents, sample submission). Participating
clinics were telephoned every 4-6 weeks to discuss any problems or concerns and
instructed on desired age, sex and weight distributions of recruited untreated dogs.
The study procedures were approved by the Research Ethics Board at the
University of Guelph.
2.2. Sample size
Sample size calculations were performed using Epi Info 2002 (Centers for
Disease Control and Prevention, Atlanta, Georgia) and were supported by antimicrobial
susceptibility estimates for fecal E. coli from a previous study (Murphy et al., 2009).
Sample sizes were derived using a mean estimated prevalence of resistance to an
antimicrobial in the unexposed group of 6% and an estimated odds ratio of 7.0 (Murphy
et al., 2009) for resistance to an antimicrobial in exposed relative to nonexposed animals.
Using these estimates, 45 dogs were required for each cohort.
2.3. Laboratory methods
2.3.1. Escherichia coli
The methods for the isolation and identification of E. coli are described in Chapter
2 with the following exceptions. Initially, the feces were homogenized with 0.85% sterile saline solution (Fisher Scientific, Whitby, ON, Canada) (Ratio 1:2 by weight). One-half ml of the fecal slurry was added to 4.5 ml buffered peptone water and 0.6ml of this
93 solution was added to Brucella broth (Difco Laboratories, Detroit, Michigan, USA) and
50% glycerol (Fisher Scientific). Next, a loopful of the fecal slurry was placed onto
MacConkey agar (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) and was incubated at 37°C for 18 to 24 hours. The remainder of the isolation protocol and identification of E. coli isolates was as described in Chapter 2.
2.3.2 Putative extended-spectrum p-lactamase Escherichia coli
A loopful of fecal slurry was plated onto MacConkey agar with 2 ug of cefpodoxime (Oxoid Company, Nepean, Ontario) and incubated at 37°C for 48 h in accordance with the Clinical Laboratory Standards Institute (CLSI) guidelines (NCCLS
2002). Escherichia coli identity was confirmed as described in Chapter 2.
2.3.3 Salmonella enterica
The methods for the isolation of Salmonella enterica are described in Chapter 2 with the following exceptions. Only one isolation method was employed using Modified
Semisolid Rappaport Vassiliadis agar (Becton, Dickinson) and initially 0.5 ml of fecal slurry was mixed with 4.5 ml buffered peptone water and incubated at 37°C for 18 to 24 hours.
2.3.4 blacMY-2 positive Escherichia coli and Salmonella enterica
Escherichia coli and Salmonella enterica isolates with the following antimicrobial resistance phenotypes were selected for amplification, and sequencing using polymerase chain reaction (PCR) for the blacMY-2 gene (Allen and Poppe 2002): ampicillin (minimum inhibitory concentration (MIC) >32 ug/ml) together with amoxicillin-clavulanic acid (MIC >32 ug/ml), and either cefoxitin (MIC >8 ug/ml) or
94 ceftriaxone (MIC >8 ng/ml) or ceftiofiir (MIC >2 ug/ml) (See Susceptibility Testing
below).
2.3.5. Clostridium difficile
The methods used for the isolation of C. difficile are described in Chapter 2 with
the following exception. Approximately 100 to 300 mg of feces was added to 9 ml of
Clostridium difficile Moxalactam Norfloxacin broth and incubated at 37°C in an aerobic
incubator for 7 d.
2.3.6 Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius
The methods used for the isolation of MRS A and MRSP are described in Chapter
2 with the following exception. Approximately 100 to 300 mg of feces were added to 9
ml of MRSA enrichment broth (7.5% NaCl, 2.5 g/L yeast extract, 10 g/L tryptone and 10
g/L mannitol) and incubated at 35°C for 24 h.
2.3.7. Vancomycin-resistant Enterococcus spp.
The methods used for the isolation of VRE are described in Chapter 2 with the
following exception. Initially, approximately 100 to 300 mg of feces were added to 9 ml
VRE enrichment broth and incubated at 35°C for 24 h.
2.3.8. Antimicrobial susceptibility testing
The methods used for antimicrobial susceptibility testing are described in Chapter
2.
2.4. Statistical Analysis
Median age and weight of antimicrobial treated and untreated dogs were compared using the Mann-Whitney U test with significance at/?<0.05. Sex distributions were compared using Fisher's exact test with significance at/?<0.05. The Kaplan Meier
95 estimator was used to describe the survival function for antimicrobial susceptibility, and the isolation of fecal C. difficile, Salmonella enterica and E. coli with a blacMY-2 phenotype (ampicillin MIC >32 ug/ml, together with amoxicillin-clavulanic acid MIC
>32 |wg/ml, and either cefoxitin MIC >8 ug/ml or ceftriaxone MIC >8 ug/ml or ceftiofur
MIC >2 ug/ml) for each cohort. (Since not all isolates were screened for the W«CMY-2 gene (see Section 2.3.4. blacwsY-2 positive Escherichia coli and Salmonella enterica), we could not use isolation of blacMY-2 positive E. coli as an outcome measure.) The "failure events" in the survival functions were the first occurrence of resistance to a specific antimicrobial or the isolation of fecal C. difficile, Salmonella enterica and E. coli with a blacMY-2 phenotype. Differences in the survival functions were assessed using the Log rank, Wilcoxon and Peto-Prentice tests with significance at/? <0.05. The Fleming-
Harrington test was used to compare the survival functions that crossed, with greater weights given to earlier failure times (p>l, q=0).
Cox proportional hazards models were used to measure the association between antimicrobial treatment and the time-to-outcome; the main effects tested were associations between antimicrobial treatment (cephalexin, amoxicillin-clavulanic acid, fluoroquinolones or penicillin) and antimicrobial resistance (each antimicrobial as an individual outcome), or isolation of C. difficile, orE. coli with a WATCMY-2 phenotype. The effect of antimicrobial treatment was modelled separately for each cohort (e.g., cephalexin treatment) versus untreated when there was a significant difference at/? <0.05 in the survival function or incidence rate. The other explanatory or confounding variables evaluated for some or all cohorts were age, weight (continuous), sex, consumption of pig ear treats, rawhide treats, raw meat, raw bones, coprophagia, drinks water from a toilet,
96 contact with livestock and participation as a therapy dog (categorical). Interaction terms were not assessed because of limitations associated with the small sample size.
Cox proportional hazard models at the dog level using an Efron approximation for tied survival data were generated for each outcome, beginning with the main binary treatment variable, then other possible explanatory or confounding variables one at a time. Results were reported when the main treatment effect was significant atp <0.05.
Other explanatory variables were included in the model if they were significant atp
<0.05, or they affected the coefficient of other variables in the model by >10% (i.e., were confounders) or were significant in the model atp <0.2 using a Wald test.
The assumption of proportional hazards was tested with significance atp <0.05 for each of the explanatory variables and a global test for the entire model. Sensitivity analysis examining complete positive and negative correlations between censoring and the outcome of interest was performed to evaluate the assumption of independent censoring. Complete positive correlation was performed by refitting the model after recoding all the censored observations as failing at the time of censoring and complete negative correlation was performed by refitting the model with an additional day of observation at day 365. The assumption of independent censoring was assumed to be violated if coefficients for the predictors in the refitted models changed by 10% or were no longer significant atp <0.05. Overall model fit was assessed by graphically assessing the distribution of the modified Cox-Snell residuals for a unit exponential distribution.
The residuals were assessed to have a unit exponential distribution when the cumulative hazard was a straight line with an intercept of 0 and a slope near 1. Analogous measures ofR2 were calculated as measures of the models' performance (Hosmer et al., 2008). The
97 distribution of the Deviance and Score residuals relative to time was assessed graphically for any possible outliers. The distribution of scaled Score residuals relative to time was assessed graphically for influential observations.
3.0. Results
Fifty-three veterinary hospitals responded with initial interest (7% response rate) and 32 were recruited to the study. Twenty-one of 32 (66%) of the enrolled veterinary hospitals recruited dogs (n=74) during the 24 month study period. The median number of dogs enrolled per hospital was 2 (range 1-10). Thirty-three dogs were enrolled into the cephalexin cohort, 12 into each of the amoxicillin-clavulanic acid and fluoroquinolone cohorts, 9 into the penicillin cohort and 8 into untreated cohort (Tables A.3.1 and A.3.2).
Twelve dogs were later removed from the study; 6 from the cephalexin cohort, four from the amoxicillin-clavulanic acid cohort and two from the penicillin cohort. Eight of these were removed because they required treatment with another antimicrobial, three were euthanized or died, and one at the request of the owner. The frequency of responses to the questionnaire and numbers of dogs for which questionnaires were completed are shown in Table A.3.3. The overall response rate for all questions was 98%.
Three hundred and forty eight fecal samples of the 417 expected (with complete compliance and adjusting for censoring) were submitted (fecal submission rate: 83%) and the median number of samples per dog was 5 (range 1 to 6) and 87% of the samples were collected within 2 days (range 0-4 days) of the expected date of collection. The overall prevalence (95% confidence interval (CI)) of isolation of bacteria from the fecal samples was: E. coli, 84% (79%, 87%); E .coli with Z>/aCMY-2phenotype, 13% (9%, 17%); C.
98 difficile, 9% (6%, 12%); Salmonella enterica, 9% (6%, 12%); MRSA, 1% (0.2%, 2%);
and VRE, 0% (97.5% one-sided CI (0,1%).
Thirty isolates of 13 different ribotypes of C. difficile were recovered from 20
dogs (Table 3.1), and 6 (46%) of the ribotypes recovered were previously reported from humans. Overall, 18 (60%) of the isolates were toxigenic, including all (n=16) of the ribotypes originally identified from human clinical samples, one isolate (ribotype CM 76) first identified from the environment of a veterinary hospital and one isolate (ribotype SL
3) initially recovered from the feces of a dog.
One isolate of MRSA was recovered from each of three dogs from different veterinary practices over the study period; the incidence rate for MRSA isolation was 0.4 dogs/1000 dog days. Two dogs were in the untreated cohort and the other was in the penicillin cohort. The two isolates from the dogs in the untreated cohort were negative for the Panton Valentine leukocidin (PVL) toxin and belonged to the Canadian epidemic strain MRSA (C-MRSA)-2 (USA 100). The third isolate was PVL-positive and belonged to C-MRSA-10 (USA300).
Thirty isolates of Salmonella enterica were recovered from 11 dogs from 14 fecal samples. Salmonella Typhimurium (n=13,43%) and Salmonella Heidelberg (n=13,43%) were the most frequently recovered serotypes. Three isolates (10%) of Salmonella
Ouakam and one isolate (3%) of Salmonella Indiana were also recovered. Where more than one isolate of Salmonella enterica was isolated from a fecal sample (median 2 isolates, range 1-3), they were always the same serovar. Eight dogs had Salmonella enterica isolated from one fecal sample and three dogs had Salmonella enterica recovered from two fecal samples. Two of the three dogs had the same serotype
99 recovered from each fecal sample (Heidelberg at days 90 and 180 or Typhimurium at days 28 and 90) and one dog had different serotypes recovered (Typhimurium at day 0 and Indiana at day 14). The Salmonella enterica isolates were susceptible to all the tested antimicrobials, with the exception of two isolates of Salmonella Typhimurium from one dog (Cohort: cephalexin; Day 0 fecal sample) that were resistant to amoxicillin- clavulanic acid (MIC >32 ug/ml), ampicillin (MIC >32 ug/ml), cefoxitin (MIC >8 ug/ml), ceftiofur (MIC >8 ug/ml) and had intermediate susceptibility to ceftriaxone (MIC
16 ug/ml) and one of these isolates was PCR-positive for the blacMY-2 gene.
3.1. Kaplan Meier survival functions
3.1.1 Antimicrobial susceptibility of Escherichia coli isolates
One thousand and two isolates of E. coli were submitted for susceptibility testing and all tested isolates were susceptible to amikacin and at least one isolate was resistant to each of the other antimicrobials. In the amoxicillin-clavulanic acid cohort, no isolates were resistant to chloramphenicol and gentamicin. In the cephalexin cohort, none was resistant to ciprofloxacin; in the penicillin cohort none was resistant to amoxicillin- clavulanic acid, ciprofloxacin, ceftriaxone, cefoxitin, ceftiofur, gentamicin, kanamycin and nalidixic acid, and in the untreated cohort, none was resistant to ciprofloxacin, kanamycin and nalidixic acid (Figures 3.1 and A.3.1).
One hundred and ninety five (19%) E. coli isolates were resistant to ampicillin
(MIC >32 ug/ml), amoxicillin-clavulanic acid (MIC >32 ug/ml) and either cefoxitin
(MIC >8 ug/ml), ceftriaxone (MIC >8 ug/ml) or ceftiofur (MIC >2 ug/ml). Seventy-one percent (138/195) of these isolates were PCR-positive for the WOCMY-2 gene.
100 In general, the incidence rate of isolation of resistant E. coli was low for all
cohorts (Figures 3.1 and A.3.1, Table 3.2) and the estimated survival probability of
antimicrobial susceptibility did not fall below the 50th percentile for any of the tested antimicrobials (Figures 3.1 and A.3.1). The estimated survival probabilities of susceptibility to amoxicillin-clavulanic acid (Log rank/?=0.04, Wilcoxon test^=0.05,
Peto-Prentice j?=0.04) (Figure 3.1b), ceftiofur (Log rank/?=0.01, Wilcoxon test/?=0.01,
Peto-Prentice/?=0.01) (Figure 3.1c) and ceftriaxone (Log rank/?=0.04, Wilcoxon test
/?=0.03, Peto-Prentice/?=0.03) (Figure 3.1d) were significantly lower for E. coli isolates from dogs treated with cephalexin than untreated dogs. In dogs treated with cephalexin, the highest rates of antimicrobial resistance in E. coli to amoxicillin-clavulanic acid
(Figure 3.1b), ceftiofur (Figure 3.1c) and ceftriaxone (Figure 3.Id) were between day 0 and day 14 when compared to untreated dogs.
The estimated survival probability of susceptibility to chloramphenicol was greater in E. coli isolates from dogs treated with cephalexin (Log rank/?=0.05, Wilcoxon test/?=0.10, Peto-Prentice/?=0.05) (Figure 3.1e) or amoxicillin-clavulanic acid (Log rank
/?=0.05, Wilcoxon test/?=0.06, Peto-Prentice/?=0.05) (Figure 3.1e) than untreated dogs.
In the untreated dogs, the rate of antimicrobial resistance to chloramphenicol was similar between each time interval. The estimated survival probability for susceptibility to ampicillin (Log rankp=0.04, Wilcoxon test/?=0.05, Peto-Prentice/?=0.04) was greater for E. coli isolates from dogs treated with penicillin than untreated dogs (Figure 3.1a) and the rate of antimicrobial resistance to ampicillin from untreated dogs was similar between each time interval.
101 The incidence rate of resistance among E. coli isolates from dogs treated with
cephalexin was significantly higher for amoxicillin-clavulanic acid (incidence rate ratio
(IRR) 3.38, 95 % confidence interval (CI) (1.01,17.7)^=0.03), ceftriaxone (IRR 6.7,
95% CI (1.03,286.07) j!7=0.02), ceftiofur (IRR 8.21, 95% CI (1.29, 34.33)/K0.01), and
cefoxitin (IRR 3.22,95% CI (0.96,17)/?=0.04) than untreated dogs (Table 3.2). There
were, however, no significant differences in the incidence rates of resistance in E. coli from dogs treated with amoxicillin-clavulanic acid, fluoroquinolones or penicillin and untreated dogs (Table 3.2).
3.1.2 Escherichia coli with a blacuY-i phenotype
There were no significant differences in the survival functions for not isolating E. coli with a blacMY-2 phenotype between antimicrobial treated and untreated dogs (Figure
A.3.2); however, the incidence rate for the isolation of fecal E. coli with a blacMY-2 phenotype from dogs treated with cephalexin was significantly higher than untreated dogs
(Table 3.3).
3.1.3. Clostridium difficile
The estimated survival probability of not isolating C. difficile from dogs treated with amoxicillin-clavulanic acid was significantly lower than for untreated dogs (Figure
3.2) and the incidence rate for the isolation of C. difficile from dogs treated with amoxicillin-clavulanic acid was significantly higher than for dogs in the untreatedcohort
(Table 3.3). The rate of isolation of C. difficile in dogs treated with amoxicillin- clavulanic acid was highest between days 7 to 90 and was comparatively lower from day
0 to day 7.
3.1.4 Salmonella enterica
102 There were no significant differences in the estimated survival probabilities of not
isolating Salmonella enterica (Figure A.3.3) or the incidence rates of isolation of
Salmonella enterica among dogs treated with any antimicrobial or untreated dogs (Table
3.3).
3.2. Cox proportional hazard models
Using Cox proportional hazard modelling, there was no significant association between treatment with amoxicillin-clavulanic acid and isolation of C. difficile (Hazard
Ratio (HR) 2.1, 95% CI (0.03,120) p=0.26), nor between treatment with cephalexin and
the recovery of 6/«CMY-2 E. coli phenotype (HR 2.84, 95% CI (0.84,9.62) p=0.09). The addition of other variables to these models did not change the above estimates.
3.2.1 Antimicrobial susceptibility of Escherichia coli isolates
Resistance to amoxicillin-clavulanic acid, ceftriaxone, ceftiofur and cefoxitin was strongly associated with treatment with cephalexin, and coprophagia was negatively associated with resistance to amoxicillin-clavulanic acid, ceftriaxone, ceftiofur and cefoxitin (Table 3.4). The distribution of the modified Cox-Snell residuals indicated that each of the models fit the data poorly. The range of values for the R2 analogs from the models for the association between cephalexin treatment and the hazard of antimicrobial resistance to amoxicillin-clavulanic acid was 13-41%; ceftriaxone 14-57%; ceftiofur 19-
65% and cefoxitin 12-40%.
Resistance to ampicillin was negatively associated with treatment with injectable penicillin (HR 0.09,95% CI (0.01,0.75)p=0.03) and positively associated with the consumption of pig ears (HR 5.00,95% CI (1.21,20.58) /?=0.03). However, the
103 distribution of the modified Cox-Snell residuals indicated that the models fit the data
poorly. The range of values for the R2 analogs from this model was 18-71%.
There were no significant associations between antimicrobial resistance to
chloramphenicol and treatment with cephalexin (HR 0.15,95% CI (0.01,1.40) p=0.10) or
amoxicillin-clavulanic acid (HR 0.25, 95% CI (0.001,14.6) p=0.15), nor did the addition
of other variables to these models alter the main effects.
4.0. Discussion
We observed that treatment of dogs with cephalexin, a first generation cephalosporin, was strongly associated with fecal E. coli resistance to amoxicillin- clavulanic acid (pMactam inhibitor combination), cefoxitin (cephamycin), and ceftriaxone and ceftiofur (third-generation cephalosporins). This is an important finding because a recent study showed that cephalexin was the most frequently prescribed non-topical (oral and parenteral) antimicrobial for non-hospitalized dogs treated in practices in Southern
Ontario, representing 33% of non-topical antimicrobial prescriptions (Chapter 4).
These observations indicate that substantial selection pressure from cephalexin use in the dog population of Ontario is reflected in greater risk of resistance in E. coli from treated dogs. This is important because the Veterinary Drugs Directorate of Health
Canada (following the lead of the World Health Organization) has classified third and fourth generation cephalosporins, cephamycins and P-lactam inhibitor combinations as of
"Very High Importance" to human health because they are regarded as essential for treatment of serious bacterial infections of humans, with few (or none in some circumstances) available alternative antimicrobials for effective treatment in the case of emergence of resistance to these agents (Government of Canada 2006). Moreover, there
104 is potential for sharing of E. coli clones between dogs and humans (Johnson and Clabots
2006, Johnson et al., 2008) and also for transmission of antimicrobial resistance determinants within the intestine to other bacteria (e.g., Salmonella enterica.) (Poppe et al., 2005, Jiang et al., 2006). In the case of Salmonella this is also worrisome since there can be zoonotic transmission of Salmonella enterica between dogs and humans and the
Salmonella serotypes recovered from dogs have also been identified in human clinical infections (Government of Canada 2008).
The observed associations between cephalexin treatment and antimicrobial resistance to a (3-lactam inhibitor combination, cephamycin and third-generation cephalosporins suggested that cephalexin treatment could also be associated with a
WacMY-2 phenotype since bacteria expressing the blacuY-2 gene are resistant to these antimicrobials. However, no significant differences were observed in the survival functions for E. coli with a WOCMY-2 phenotype in dogs treated with cephalexin and untreated dogs (Log rank test p=0.07, Wilcoxin testp=0.07, Peto-Prentice test/?=0.07), nor in the Cox proportional hazard model (p=0.09), although the incidence rate of isolation E. coli with a blacMY-2 phenotype was significantly higher from dogs treated with cephalexin than from the untreated dogs (p=0.05). In each of these evaluations, the p-values were close to the cut-off for significance, suggesting that there may have been insufficient power in this dataset to consistently demonstrate a true association. If such an association actually does exist, it could be important for the reasons described above.
Coprophagia had an apparent protective effect on E. coli resistance to amoxicillin- clavulanic acid, cefoxitin, ceftiofur and ceftriaxone in models of treatment with cephalexin. Other studies have reported protective associations between coprophagia and
105 isolation of C. difficile from the feces of dogs (Lefebvre et al., 2006, Lefebvre et al.,
2009). Perhaps coprophagia facilitates establishment of intestinal flora that provides
some resistance to colonization with certain bacteria.
The apparent protective association between treatment with injectable penicillin
and antimicrobial resistance to ampicillin is difficult to interpret because E. coli are
typically intrinsically resistant to the narrow spectrum penicillins, therefore this may be a
biased or spurious finding. If real, the association may reflect penicillin-induced
alterations in susceptible Gram-positive fecal flora with secondary changes in the E. coli
population.
There was also a strong association between the consumption of pig ear treats and
isolation of antimicrobial resistant E. coli from dogs treated with injectable penicillin. A
previous cross-sectional study demonstrated a positive association between the
consumption of pig ear treats and antimicrobial resistance to trimethoprim-
sulfamethoxazole in fecal E. coli isolates from healthy dogs (Murphy et al., 2009). The consumption of pig ear treats was strongly associated with the isolation of fecal
Salmonella enterica from dogs in another study (Lefebvre et al., 2008). In Canada, pig ear treats have been contaminated with Salmonella enterica, including antimicrobial resistant Salmonella enterica (Finley et al., 2008) and were associated with an outbreak of Salmonella Mantis in humans (Clark et al., 2001). It is possible that pig ear treats may be contaminated (at least occasionally) with bacteria other than Salmonella enterica, including antimicrobial resistant E. coli that may colonize or infect animals and humans, and that treatment with penicillin disturbed the intestinal microflora to improve colonization by these resistant bacteria.
106 The slopes of the survival functions for susceptibility to amoxicillin-clavulanic acid, cefoxitin, ceftiofur and ceftriaxone in E. coli isolates from dogs treated with cephalexin suggest a treatment effect. These dogs were treated with cephalexin for a range of 5 to 14 days, with 51% (17/33) treated for 14 days, and after treatment, the slopes of the survival functions from treated dogs were similar to those from untreated dogs. The differences in the survival functions for susceptibility to chloramphenicol in E. coli isolates between untreated dogs and dogs treated with amoxicillin-clavulanic acid or cephalexin are likely spurious results since the/?-values were at the cut-off for significance for the Log rank and Peto-Prentice tests and above the cut-off for significance for the Wilcoxin test. This could be as a consequence of the small sample size and a low frequency of antimicrobial resistance to chloramphenicol.
Previous research showed that prior antimicrobial administration was associated with hospital-associated colonization of dogs with C. difficile (Clooten et al., 2008), and antimicrobial therapy is a well-established risk factor for C. difficile-associated infection in humans (CDAI) (Elliott et al., 2007, Blondeau, 2009); normal enteric flora is believed to protect individuals from colonization with C. difficile (Elliott et al., 2007). In our study, the slope of the survival function for not isolating C. difficile is also suggestive of a treatment effect on colonization with C. difficile. The greatest rate of incident isolation of
C. difficile was between days 7 and 28, with another relatively large drop in the survival function between days 28 and 90, and a very small change in the survival function between day 0 and 7. These data suggest that the risk period for colonization with C. difficile did not occur immediately after initiation of treatment with amoxicillin- clavulanic acid, but was delayed for 1-2 weeks, and continued for months following the
107 discontinuation of treatment. Similarity, the risk period for C. difficile infection or C. d^cj'/e-associated-diarrhea in humans ranges from 4 weeks to 6 months following antimicrobial exposure (Dupont et al., 2008). Therefore, when investigating the association between antimicrobial exposure and C. difficile colonization, infection or associated-diarrhea, it is necessary to consider antimicrobial exposure that occurred months prior to the event.
The diversity of C. difficile ribotypes recovered from study dogs is notable. The most frequently identified ribotype, MOH V, was first ^identified from human fecal samples, albeit at a low frequency (Martin et al., 2008). Furthermore, MOH V and another ribotype, OVC B, were the most common ribotypes recovered from environmental samples in a recent study of veterinary clinics in Southern Ontario
(Chapter 2). These findings suggest that while particular C. difficile ribotypes may be more strongly associated with either humans or dogs, there is some sharing of ribotypes, perhaps from exposure to common sources, such as food (Rodriguez-Palacios et al.,
2007, Rodriguez-Palacios et al., 2009) or through inter-species transmission of C. difficile
(Arroyo et al., 2005).
Isolation of MRS A strains (CMRSA-2 and CMRSA-10) in this study was not unexpected. CMRSA-2 and CMRSA-10 are common human strains and previous studies of MRS A infection and colonization in dogs demonstrated that strains from dogs were often the same as those isolated from the human population of the same region (Weese et al, 2006, Christianson et al, 2007, Weese et al., 2007, Lefebvre and Weese, 2009). We found no evidence in this study that fecal shedding of MRS A by dogs was associated with antimicrobial therapy. Similarly, the incidence rate of Salmonella enterica shedding
108 by dogs in this study was not associated with antimicrobial therapy. Salmonella
Typhimurium and Salmonella Heildelberg were the most frequently recovered serotypes,
as they were in a study of therapy dogs in Ontario (Lefebvre et al., 2008). These
serotypes are also among the top three recovered from human clinical specimens in
Canada (Government of Canada 2008) and outbreaks of Salmonella Typhimurium
affecting both companion animals and humans have been reported (Wright et al., 2005).
The commonality of canine and human Salmonella serotypes may reflect a common source of exposure. Dogs and humans may have a tendency towards colonization with similar Salmonella enterica.; however, dogs are potentially an important source of
Salmonella enterica for human infections, including antimicrobial resistant infections with blacMY-2 postive Salmonella Typhimurium.
The small number of dogs enrolled into this study was a limitation. Participation by veterinarians may have been limited by the labour intensity of the study, the time required to recruit study dogs, and pet owner willingness to participate. Attempts were made to minimize and simplify the work required of the study liaisons, and each clinic did not need to enrol many dogs (n=8) to achieve the initial sample size targets
(ntotai=270). Although consultation with the participating veterinary practices at the beginning of the study suggested that these sample size targets were achievable, the failure to do so necessitated some modifications to the methodology and subsequent data analysis. The original study design included two untreated groups; one for the oral- antimicrobial cohort and another for the injectable penicillin cohort, to account for the differences in the source populations of the antimicrobial-treated dogs. Dogs presented for elective spay or neuter and not treated with injectable penicillin were to be enrolled
109 for untreated cohort for the injectable penicillin cohort. However, no clinics were able to
enrol any dogs into this cohort, and, thus, only the one untreated cohort was available for
analysis. Nevertheless, compliance by pet owners in submission of fecal samples was
very good, perhaps due to high motivation, selection of likely compliant pet owners by
the study liaisons for participation in the study, tools provided to assist the study liaison
and pet owner with sampling dates, and reminders to the pet owners to provide samples.
All the models in this study had relatively poor fit to the data, and the R2 analogs
suggested that the explanatory variables in the models accounted for little of the variation
or randomness in the data. This may be a result of variables not included in the models,
such as previous antimicrobial therapy, or the small sample size of the study. We
attempted to collect data on previous antimicrobial therapy by having the veterinary
practices submit the medical records for the enrolled dogs; however, there was poor
submission compliance for medical records of the untreated cohort and we were unable to
examine this association. Due to this lack of fit, the estimates for the hazard ratios reflect
direction of the association, but are probably not good estimates of the strength of the
associations. Additionally, the results should be considered relevant mainly to the
sampled population and are not widely generalizable. Nevertheless, they are useful, particularly when developing further studies examining the association of antimicrobial treatment on the occurrence of resistance.
In conclusion, the most important findings of the study were the associations between antimicrobial treatment with cephalexin and the increased incidence of antimicrobial resistance in fecal E. coli to amoxicillin-clavulanic acid, cefoxitin, ceftriaxone and ceftiofur. These associations have animal and public health implications.
110 The prospective cohort design of the study permits these associations to be components of causal inferences in the epidemiology of antimicrobial resistance. Other notable findings were the higher incidence rate of the isolation of E. coli isolates with a blacMY-2 phenotype in dogs treated with cephalexin, and the commonality of Salmonella serovars and C. difficile ribotypes isolated from study dogs and those previously recovered from humans.
Acknowledgements
We would like to thank Nicol Janecko and Virginia Young for their work in coordinating and managing this project; the Laboratory for Foodborne Zoonoses students at the
Canadian Research Institute for Food Safety, for E. coli and Salmonella isolation;
Meredith Craig and other students for data entry, Joyce Rousseau for C. difficile, MRS A and MRSP isolation and typing,- Rebeccah Travis and Gerry Lazarek for PCR of the
WacMY-2 gene; and staff of the Laboratory for Foodborne Zoonoses for susceptibility testing and typing of Salmonella isolates. We would like to also thank the veterinary practices, veterinarians, veterinary technicians, other clinic staff, pet owners and dog for participating in this study. This study was funded by the Ontario Veterinary College Pet
Trust Fund and the Public Health Agency of Canada. Colleen Murphy was a recipient of the Ontario Veterinary College Graduate Student Fellowship.
Ill References
Allen, KJ, C Poppe. 2002. Occurrence and characterization of resistance to extended-
spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from
food-producing animals in Canada. Can J Vet Res, v. 66, p. 137-44.
Arroyo, LG, S A Kruth, B M Willey, H R Staempfli, D E Low, J S Weese. 2005. PCR
ribotyping of Clostridium difficile isolates originating from human and animal sources. J
Med Microbiol, v. 54, p. 163-6.
Blondeau, JM. 2009. What have we learned about antimicrobial use and the risks for
Clostridium difficile-associated diarrhoea? J Antimicrob Chemother, v. 63, p. 238-42.
Christianson, S, G R Golding, J Campbell, M R Mulvey. 2007. Comparative genomics of
Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus. J Clin
Microbiol, v. 45, p. 1904-11.
Clark, C, J Cunningham, R Ahmed, D Woodward, K Fonseca, S Isaacs, A Ellis, C
Anand, K Ziebell, A Muckle, P Sockett, F Rodgers. 2001. Characterization of Salmonella
associated with pig ear dog treats in Canada. J Clin Microbiol, v. 39, p. 3962-8.
Clooten, J, S Kruth, L Arroyo, J S Weese. 2008. Prevalence and risk factors for
Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit.
Vet Microbiol, v. 129, p. 209-14.
112 Dupont, HL, K Garey, J P Caeiro, Z D Jiang. 2008. New advances in Clostidium difficile
infection: changing epidemiology, diagnosis, treatment and control. Curr Opin Infec Dis,
v. 21, p. 500-7.
Elliott, B, B J Chang, C L Golledge, T V Riley. 2007. Clostridium difficile-associated
diarrhoea. Intern Med J, v. 37, p. 561-8.
Faires, MC. 2008. Evaluation of methicillin-resistant Staphylococcus aureus and
characterization of S. pseudintermedius in dogs and cats (A thesis presented to the
Faculty of Graduate Studies of the University of Guelph in partial fulfilment of
requirements for the degree of Master of Science) University of Guelph, Guelph Ontario.
Finley, R, R Reid-Smith, C Ribble, M Popa, M Vandermeer, J Aramini. 2008. The
occurrence and anti-microbial susceptibility of Salmonellae isolated from commercially
available pig ear pet treats. Zoonoses Public Health, v. 55, p. 455-61.
Government of Canada. 2008. Canadian Integrated Program for Antimicrobial Resistance
Surveillance (CIPARS) 2007- Preliminary Results. Guelph (Ontario). Public Health
Agency of Canada.
Government of Canada. 2006. Categorization of antimicrobial drugs based on importance in human medicine, http://www.hc-sc.gc.ca/dhp- mps/consultation/vet/consultations/amrramhum-med-eng.php. Accessed June 2009.
113 Hosmer, DW, S Lemeshow, S May. 2008. Applied survival analysis: Regression
modeling of time-to-event data. p. 194-5. John Wiley & Sons, Hoboken, New Jersey.
Jiang, X, H Yang, B Dettman, M P Doyle. 2006. Analysis of fecal microbial flora for
antibiotic resistance in ceftiofur-treated calves. Foodborne Pathog Dis, v. 3, p. 355-65.
Johnson, JR, C Clabots. 2006. Sharing of virulent Escherichia coli clones among household members of a woman with acute cystitis. Clin Infect Dis, v. 43, p. el01-8.
Johnson, JR, K Owens, A Gajewski, C Clabots. 2008. Escherichia coli colonization patterns among human household members and pets, with attention to acute urinary tract infection. J Infect Dis, v. 197, p. 218-24.
Lefebvre, SL, R Reid-Smith, P Boerlin, J S Weese. 2008. Evaluation of the risks of shedding Salmonellae and other potential pathogens by therapy dogs fed raw diets in
Ontario and Alberta. Zoonoses Public Health, v. 55, p. 470-80.
Lefebvre, SL, R J Reid-Smith, D Waltner-Toews, J S Weese. 2009. Incidence of acquisition of methicillin-resistant Staphylococcus aureus, Clostridium difficile, and other health-care-associated pathogens by dogs that participate in animal-assisted interventions.
J Am Vet Med Assoc, v. 234, p. 1404-17.
Lefebvre, SL, D Waltner-Toews, A S Peregrine, R Reid-Smith, L Hodge, L G Arroyo, J
114 S Weese. 2006. Prevalence of zoonotic agents in dogs visiting hospitalized people in
Ontario: implications for infection control. J Hosp Infect, v. 62, p. 458-66.
Lefebvre, SL, J S Weese. 2009. Contamination of pet therapy dogs with MRS A and
Clostridium difficile. J Hosp Infect. E-pub ahead of press.
Martin, H, B Willey, D E Low, H R Staempfli, A McGeer, P Boerlin, M Mulvey, J S
Weese. 2008. Characterization of Clostridium difficile strains isolated from patients in
Ontario, Canada, from 2004 to 2006. J Clin Microbiol, v. 46, p. 2999-3004.
Medleau, L, R E Long, J Brown, W H Miller. 1986. Frequency and antimicrobial
susceptibility of Staphylococcus species isolated from canine pyodermas. Am J Vet Res, v. 47, p. 229-31.
Murphy, CP, R J Reid-Smith, J F Prescott, B N Bonnett, C Poppe, P Boerlin, J S Weese,
N Janecko, S A McEwen. 2009 Occurence of antimicrobial resistance in selected bacteria in healthy dogs and cats presented to private veterinary clinics in Southern Ontario. Can
Vet J, v. 50, p. 1047-53.
NCCLS. 2002. Performance standards for antimicrobial susceptibility testing. Approved standards M100-S15 M7-A-MIC testing section. National Committee for Clinical
Laboratory Standards, Wayne PA.
115 Ogeer-Gyles, J, K A Mathews, W Sears, J F Prescott, J S Weese, P Boerlin. 2006.
Development of antimicrobial drug resistance in rectal Escherichia coli isolates
from dogs hospitalized in an intensive care unit. JAVMA, v. 229, p.694-9.
Poppe, C, L C Martin, C L Gyles, R Reid-Smith, P Boerlin, S A McEwen, J F Prescott, K
R Forward. 2005. Acquisition of resistance to extended-spectrum cephalosporins by
Salmonella enterica subsp. enterica serovar Newport and Escherichia coli in the turkey
poult intestinal tract. Appl Environ Microbiol, v. 71, p. 1184-92.
Prescott, JF, W J Hanna, R Reid-Smith, K Drost. 2002. Antimicrobial drug use and
resistance in dogs. Can Vet J, v. 43, p. 107-16.
Rantala, M, E Lahti, J Kuhalampil, S Pesonen, A K Jarvinen, Saijonmaa-Koulumies, T
Honkanen-Buzalski. 2004. Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs. Acta Vet Scand, v. 45, p. 37-45.
Rodriguez-Palacios, A, R J Reid-Smith, H R Staempfli, D Daignault, N Janecko, B P
Avery, H Martin, A D Thomspon, L C McDonald, B Limbago, J S Weese. 2009. Possible seasonality of Clostridium difficile in retail meat, Canada. Emerg Infect Dis, v. 15, p.
802-5.
Rodriguez-Palacios, A, H R Staempfli, T Duffield, J S Weese. 2007. Clostridium difficile in retail ground meat, Canada. Emerg Infect Dis, v. 13, p. 485-7.
116 Trott, DJ, L J Filippich, J C Bensink, M T Downs, S E McKenzie, K M Townsend, S M
Moss, J J Chin. 2004. Canine model for investigating the impact of oral enrofloxacin on
commensal coliforms and colonization with multidrug-resistant Escherichia coll J Med
Microbiol, v. 53, p. 439-43.
Weese, JS, H Dick, B M Willey, A McGeer, B N Kreiswirth, B Innis, D E Low. 2006.
Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic
pets and humans in veterinary clinics and in the household. Vet Microbiol, v. 115, p. 148-
55.
Weese, JS, M Faires, J Rousseau, A M Bersenas, K A Mathews. 2007. Cluster of methicillin-resistant Staphylococcus aureus colonization in a small animal intensive care unit. J Am Vet Med Assoc, v. 231, p. 1361-4.
Wright, JG, L A Tengelsen, K E Smith, J B Bender, R K Frank, J H Grendon, D H Rice,
A M Thiessen, C J Gilbertson, S Sivapalasingam, T J Barrett, T E Besser, D D Hancock,
F J Angulo. 2005. Multidrug-resistant Salmonella Typhimurium in four animal facilities.
Emerg Infect Dis, v. 11, p. 1235-41.
117 Figure 3.1. Kaplan-Meier estimator of the survival probability of antimicrobial susceptibility of
E. coli isolates from dogs treated with antimicrobials and untreated dogsa, where at least two of the estimated survival probabilities are significantly different.
Susceptibility to ampicillin 3.1b Susceptibility to amoxicillin-clavulanic acid 1.00- IE iu/b- l_. I 0.75-1 I , •a
3 i 1 0.25- 1
o.oo- 0 7 14 28 90 T f I""11 • . i \ Days after enrolment in the study 0 7 14 28 90 Days after enrolment in the study
3.1c Susceptibility to ceftiofur 3.1d Susceptibility to ceftriaxone 1.00 1.00- TP T^ it •f 1 0.75 S 0.75^ !
0.50
I 0.25
T—i—i—i i 0 7 14 28 90 0 7 14 28 90 Days after enrolment in the study Days after enrolment in the study
-J • 16 Susceptibility to chloramphenicol i.ooH "^ £ t I ? 0.50-^
0.25
0 7 14 28 90 Days after enrolment in the study Legend
"Amoxicillin-clavulanic acid Cephalexin — Fluoroquinolones Penicillin — Control "For dogs treated with antimicrobials, the day 0 sample was collected prior to antimicrobial therapy and antimicrobial treatment started within 12 hours of collection of the day 0 sample. For the untreated control dogs, the day 0 sample was collected on the day of enrolment.
118 Figure 3.2. Kaplan-Meier estimator of the survival probability of not isolating
Clostridium difficile from dogs treated with antimicrobials and untreated dogsa'b
1.00H—i
I 0.75 2 2 Q. 1 •£ 0.50-
CO I 025 at
0.00H T 1— 7 14 28 90 180 Days after enrolment in the study
Legend
—Amoxicillin-clavulanicacid — Cephalexin — Fluoroquinolones • Penicillin — Control
aFor dogs treated with antimicrobials, the day 0 sample was collected prior to antimicrobial therapy and antimicrobial treatment started within 12 hours of collection of the day 0 sample. For the untreated control dogs, the day 0 sample was collected on the day of enrolment. bCephalexin treatment compared to controls: Log rank test p=0A2, Wilcoxin test/>=0.21, Peto-Prentice test/?=0.13; Amoxicillin-clavulanic acid treatment compared to controls: Log rank testp=0.01, Wilcoxin test p=0.0S, Peto- Prentice test/?=0.02; Fluoroquinolone treatment compared to controls: Log rank test p=0.59, Wilcoxin test/?=0.89, Peto-Prentice test/7=0.61; Penicillin treatment compared to controls: Log rank test^=0.40, Wilcoxin test/?=0.68, Peto-Prentice test/>=0.84.
119 Table 3.1. The frequency of Clostridium difficile ribotypes recovered from dogs treated with
antimicrobials and untreated dogs.
Ribotype8 Number of isolates Frequency Toxin A Toxin B CDT
MOHV 10 33% Positive Positive Negative
OVCB 7 23% Negative Negative Negative
MOHAI 2 7% Positive Positive Negative
OVCC 2 7% Negative Negative Negative
MOHAA 3% Negative Positive Negative
MOHAL 3% Positive Positive Positive
MOHAN 3% Positive Positive Negative
MOHQ 3% Positive Positive Negative
CM 173b 3% Negative Negative Negative
CM53C 3% Negative Negative Negative
CM76C 3% Positive Positive Negative
OVCH 3% Negative Negative Negative
SL3 3% Positive Positive Negative
The ribotypes with the "MOH" designation were originally identified from human fecal samples.
The ribotypes with "OVC" and "SL" were originally identified in dogs. "This is a newly identified ribotype. "These ribotypes were originally identified from environmental sites within veterinary hospitals.
120 Table 3.2. The incidence rate (per 1000 dog days) of resistance to tested antimicrobials in fecal E.
coli from dogs treated with antimicrobials and untreated dogs.
Tested Antimicrobial cohort Antimicrobial
Amoxicillin- Cephalexin Fluoroquinolones Penicillin Untreated
clavulanic
acid
Amoxicillin- 5.1 9.1" 2.5 2.7
clavulanic acid
Ampicillin 5.8 9.6 5.8 1.1 6.3
Chloramphenicol 0 0.4 1.7 1.1 2.7
Ciprofloxacin 0.73 0 0.8 0 0
Ceftriaxone 2.2 6.18 0.8 0 0.9
Ceftiofur 2.2 7.4a 0.8 0 0.9
Cefoxitin 5.1 8.7" 0.8 0 2.7
Gentamicin 0 0.87 0.8 0 0.9
Kanamycin 0.7 0.4 0.8 0 0
Nalidixic-acid 1.5 0.4 0.2 0 0
Streptomycin 1.5 1.7 3.3 2.2 0.9
Sulfisoxazole 2.2 1.3 4.2 2.2 3.6
Tetracycline 0.7 4.3 2.5 1.1 1.8 methoprim- 0.7 0.9 3.3 1.1 2.7 sulfamethoxazole
The incidence rates are significantly different than the untreated cohort.
121 Table 3.3. The incidence rate (dogs per 1000 dog days) of isolation of E. coli isolates with a
W«CMY-2phenotype, Clostridium difficile and Salmonella enterica from dogs treated with
antimicrobials and untreated dogs.
Antimicrobial Cohort
Bacteria of Interest Control Amoxicillin- Cephalexin Fluoroquinolone Penicillin
clavulanic
acid
E. coli isolates with a blacMY-2 phenotype
Incidence Rate 2.7 5.1 8.2 0.8 0
IRR Referent 1.9 3.1 0.31 0
(95% CI)8 (0.43, 11.4) (0.99, 16) (0.005,3.84) (0, 2.93)
/7-value 0.40 0.05 0.16
Clostridium difficile
Incidence Rate 0.7 6.3 2.9 1.3 0.8
IRR Referent 8.8 4.1 (0.62, 1.8 1.17(0.01,
(95% CI) (1.29,380) 172) (0.09, 106) 92)
p-value 0.01 0.14 0.68 0.92
Salmonella enterica
Incidence Rate 2.6 0.7 0.1 2.2 1.8
IRR Referent 0.28 0.5 (0.07, 0.83 0.68
(95% CI) (0.005,3.5) 3.7) (0.11,6.1) (0.06, 6.0)
/»-value 0.29 0.41 0.82 0.71
"IRR (95% CI): Incidence Rate Ratio (95% Confidence Interval).
122 Table 3.4. Cox proportional hazard models for resistance to amoxicillin-clavulanic acid,
ceftriaxone, ceftiofur and cefoxitin in fecal E. coli isolates from dogs.
Outcome Explanatory Variables
Treatment with Cephalexin8 Coprophagiab
Resistance to amoxicillin-clavulanic acid
Hazard Ratio 3.92 0.28
(95% confidence interval) (1.16, 13.2) (0.08, 0.96)
/7-value 0.03 0.04
Resistance to ceftriaxone
Hazard Ratio 8.61 0.13
(95% confidence interval) (1.07, 62.2) (0.02, 0.99)
p-value 0.04 0.05
Resistance to ceftiofur
Hazard Ratio 10.1 0.11
(95% confidence interval) (1.35, 76.7) (0.01, 0.82)
p-value 0.03 0.03
Resistance to cefoxitin
Hazard Ratio 3.70 0.30
(95% confidence interval) (1.09, 12.56) (0.09, 0.98)
/7-value 0.03 0.05
"Referent: untreated healthy dogs "Referent: dogs that did not participate in coprophagia
123 Chapter 4
Out-patient antimicrobial drug use in dogs and cats for new disease
events from community companion animal practices in Ontario. Abstract
The objectives of this study were to describe non-topical (oral and parenteral)
antimicrobial use in dogs and cats; to compare the dose (mg/kg) and frequency of
administration prescribed to those recommended in a pocket-type formulary reference;
and to evaluate antimicrobial use in feline upper respiratory tract disease, feline lower
urinary tract disease and canine infectious tracheobronchitis. Eighty-four veterinarians
submitted 1,807 records over a 12 month study period describing incident (new) disease
events and associated prescription treatments (if required) in dogs and cats. Fifty-one
percent (n= 1,009) of the recorded prescription events were for antimicrobials, and 70%
(n=705) of the antimicrobial treatments were non-topical, pMactams were the most
frequently prescribed antimicrobial class for both dogs and cats, representing 65% and
67%, respectively, of all non-topical antimicrobial prescription events, and were the
predominant class for treatment in most body sites. The most frequently prescribed
antimicrobials in dogs were cephalexin (33% of recorded antimicrobial prescription
events) and amoxicillin-clavulanic acid (16%), and in cats were amoxicillin-clavulanic
acid (40%) and cefovecin (17%). In dogs and cats, fluoroquinolones represented 7% and
12%, respectively, of the recorded antimicrobial prescription events. In dogs, 18% (n=70)
of the antimicrobial prescription events were dosed below and 8% (n=28) above the referenced range. In cats, 30% (n=55) were dosed below and 19% (n=35) above the referenced range. In dogs, 67% of the disease events associated with canine infectious
124 tracheobronchitis were treated with antimicrobials. In cats, 70% and 74% of the disease events associated with feline upper respiratory tract and lower urinary tract disease, respectively, were treated with antimicrobials. Of the diagnostic test events recorded,
0.8% (n= 40) were for bacterial culture and antimicrobial susceptibility testing, which were performed for 3% and 8% of the disease events treated with antimicrobials in dogs and cats, respectively. Data from this study suggest that cefovecin and fluoroquinolones may be over-used in this study population and antimicrobial use for the treatment of feline upper respiratory tract disease, feline lower urinary tract disease and canine kennel cough could probably be reduced. Additionally, the use of bacterial culture and antimicrobial susceptibility by veterinarians as a diagnostic tool to support diagnoses and antimicrobial therapy could be improved.
1.0. Introduction
The discovery of antimicrobials for the treatment or prevention of bacterial infections is among the most important advances in modern medicine. However, shortly after their discovery, acquired antimicrobial resistance was observed to adversely affect clinical outcomes (Forbes, 1949). Antimicrobial use, both in humans and in animals is an important contributor to antimicrobial resistance is antimicrobial use, few published data are available regarding antimicrobial use in companion animal veterinary practice.
Previous studies generally reported frequencies (Odensvik et al., 2001, Holso et al., 2005,
Weese, 2006) or quantities of antimicrobial use (DANMAP, 2007). The few studies that investigated appropriate antimicrobial selection (Watson and Maddison, 2001, Rantala et al., 2004) were performed in study populations from teaching veterinary hospitals
(Rantala et al., 2004, Holso et al., 2005, Weese, 2006) that may not be representative of
125 community veterinary practices, or were based on surveys which are subject to recall bias
(Watson and Maddison, 2001). Previous studies have not reported a systematic evaluation of antimicrobial use that includes quantifying antimicrobial use, investigation of antimicrobial use in specific disease conditions, and evaluation of appropriate dosing of antimicrobials.
Documenting how, why and which antimicrobial drugs are used in general veterinary practice, and the circumstances of their use, is necessary in order to determine whether improvements are needed to reduce resistance. Therefore, the objectives of this study were to 1) describe non-topical antimicrobial use in dogs and cats in Ontario,
Canada, and 2) to investigate adherence to formulary referenced doses (mg/kg) and frequencies of administration (Allen et al., 2004), and 3) to evaluate antimicrobial use in feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis.
2.0. Materials and methods
2.1. Veterinarian recruitment
Letters describing the study were mailed to all general practice companion animal veterinarians located in Ontario (Appendix A.4.1) identified using the College of
Veterinarians professional classification codes (College of Veterinarians of Ontario).
Veterinarians certified by the American Board of Veterinary Practitioners (canine or feline) were included; however, all other specialities were excluded from recruitment.
Veterinarians were asked to respond to the initial recruitment letter whether they were interested or not in the project; those who responded with interest were contacted by telephone to discuss the project prior to committing to participation.
126 2.2. Data-entry journal pre-test
Ten randomly-selected respondents agreed to pre-test a study journal using the instructions for case eligibility (see Section "2.4. Data Collection" for description of study journal). They completed a feed-back questionnaire addressing the inclusion and exclusion criteria, difficulties in understanding or completing the journal, length of time required to complete the journal and the number of potential eligible patients presented to their practice on the day that they completed the journal (Appendix A.4.2.). Additionally, the pre-test veterinarians were contacted by telephone to further discuss their experience with the study or journal. The completed pre-test journals were evaluated for completeness and clarity of the data provided, which were then entered into the study database to assess its suitability. Following the pre-test, the journal was modified and the definition of case eligibility was refined. The pre-test data were excluded from the study.
2.3. Eligibility criteria
Veterinarians were asked to complete journals for incident disease events diagnosed in dogs and cats managed as outpatients through front office, house-call or after-hours/emergency appointments. These included incident disease events that were treated with prescription or non-prescription medications where treatment was deferred pending the results of diagnostic testing, or where treatment was not required or declined by the client. Additionally, data were collected on incident disease events that would have been managed on an out-patient basis, but where the animal was euthanized.
An incident disease event was defined as any new abnormality consistent with a disease or pathological process in dogs or cats worthy of notation in the medical record, whether or not it required treatment at the time of presentation. This definition included
127 disease events that recurred after a period of resolution (as assessed by the attending veterinarian, e.g., skin infections), or other disease events, such as weight changes or dental disease.
2.4. Sample size estimation
Data from the pre-test determined that the median number of eligible patients on a given day was 3 (range 1-10). Previous experience in community-based research in a similar veterinary population suggested that the positive response rate of veterinarians to the recruitment letter would range from 6-24%. We estimated that up to five eligible patients per veterinarian per journal day would provide us with data from 2000-6000 patients (with 100% compliance). Previous experience suggested that compliance by the participating veterinarians would range from 30-50%, providing data from 1000-3000 patients, sufficient to achieve the study objectives.
2.5. Data collection
Veterinarians were asked to participate for a 12 month period and were assigned one of four days in a month (Day 3,11,19, or 27 of the month), through a formal randomization process, to complete their journal for up to a maximum of five eligible patients that were presented on each of their journal days. The information gathered in the journal included signalment of the animal, incident disease event name (up to three per animal), duration (acute, chronic or recurrent), severity (mild, moderate, severe), pre existing disease conditions, diagnostic tests performed (e.g., history, physical exam, biochemistry, bacterial culture and antimicrobial susceptibility), type of patient contact
(e.g., scheduled office appointment, emergency), and treatment administered or prescribed (e.g., no treatment, treatment with prescription medications, owner declined
128 therapy). Veterinarians were given the choice to complete the journal in a hard (paper) copy or identical electronic format (Appendix A.4.3.). Completed journals were returned to the research team by fax, mail or email. Veterinarians indicated on a form when no eligible cases were available for a particular month. If a veterinarian was not working on their assigned day in a particular month, they were instructed to complete the journals on their next working day.
2.6. Statistical analysis
2.6. J General descriptive statistics
All data were entered into a project specific database1 and data analysis was performed using commercial software2. Descriptive statistics were performed by describing categorical variables as proportions and continuous data as medians with ranges. The analysis was limited to disease events that were treated with non-topical antimicrobials administered orally or parenterally. Frequencies of use of antimicrobials were described according to the first-line, second-line or third-line classification of therapy as described by Weese (2006), and to the Category I, II or HI classification for importance to human health, as defined by the Government of Canada (2006).
1 Created in Microsoft Access 2003. Microsoft Corporation, Redmond, Washington,
USA.
2 Intercooled STATA 10. StataCorp, College Station Texas, USA; Microsoft Excel.
Microsoft Corporation, Redmond, Washington, USA.
129 The disease event descriptions were classified by specific body sites or disease conditions (Tables A.4.1 and 2). The frequency of antimicrobial treatments in specific body sites or disease conditions was calculated by the following formula:
Number of prescription events with non-topical antimicrobial "X" in body site "Y"
Total number of non-topical antimicrobial prescription events in body site "Y"
Associations between the duration of oral antimicrobial therapy and severity (mild versus moderate or severe) or duration (acute versus chronic or recurrent) of disease were determined using the Mann Whitney U test with significance at/? <0.05. Associations among the severity or duration of the disease event, animal species and use of bacterial culture and antimicrobial susceptibility testing were determined using Fisher's exact test with significance at <0.05 where the 95 % confidence interval (95% CI) did not include the null.
2.6.2. Comparison to formulary referenced dose range (mg/kg) and frequency of administration
Recommended dose ranges (mg/kg) and frequencies of administration were obtained from Allen et al. (2005); a readily available, pocket-type, Canadian formulary designed as a practical aid to veterinarians. The median (range) dose (mg/kg) used in study animals was determined for each non-topical antimicrobial, along with the distribution of the frequency of administration. The frequency of antimicrobial prescription events that were above and below the recommended dose range was also determined. For non-topical antimicrobials that were administered in capsule, pill or
130 tablet form, the recommended doses were adjusted by ± 10% to allow for limitations of formulation.
2.6.3. Non-topical antimicrobial use in specific diseases
Non-topical antimicrobial use was described specifically for feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis (Table A.4.3). In addition, associations between non-topical antimicrobial treatment and severity of disease (mild versus moderate/severe), chronicity of the disease (acute versus chronic/recurrent) and sex were determined using the Fisher's exact test with significance at/?<0.05 where the 95% CI did not include the null.
Associations between non-topical antimicrobial treatment and age or weight were determined using the Mann Whitney U test with significance at/?<0.05. 3.0.
3.0 Results
3. J. Demographics
Recruitment letters were mailed to 2,406 veterinarians in Ontario and 218 responded (overall response rate: 9%), with 178 expressing interest in participating in the study (positive response rate of respondents: 82%). Positive respondents were contacted by telephone to further discuss the project and 109 agreed to participate in the project.
Eighty-two percent of the study-participants were graduates from the Ontario Veterinary
College and the median year of graduation was 1991 (range 1956- 2006). Female veterinarians were overrepresented (p<0.01) in the study population (67%) when compared to the proportion of female companion animal veterinarians in Ontario (54%).
Ninety-three percent of the participants described themselves as companion animal veterinarians and 7% as mixed animal veterinarians.
131 Over the study period, 1,807 journals were submitted by 84 veterinarians (total number enrolled n=109; participation rate: 77%). The median number of months of participation was 9 (range 0-13). Twelve veterinarians formally withdrew from the study over the study period (no reason reported n= 9, parenteral leave n=2, changed type of practice n=l).
3.2. Journals
Of the 1,807 journals submitted, 70% (n=l, 256) involved dogs of 107 breeds and mixed breeds; 2.5% (31/1,256) did not list breed. Mixed-breeds were most frequently reported (25%), followed by Labrador Retriever (10%), Golden Retriever (7%) and Shih
Tzu (6%); other breeds were each represented at a frequency of less than 3%. Thirty per cent (n=551) of journals involved cats, comprising 20 breeds; 73% (n=392) were domestic short hair, 13% (n=71) were domestic long hair, 4% (n=24) were domestic medium hair, 3% (n=14) were Siamese and no breed was indicated for 2% (n=12). All other breeds were represented at a frequency of less than 2%. The reported age, sex and weight distributions of the dogs and cats treated with non-topical antimicrobials are presented in Table A.4.4.
There were 2,125 disease events reported with 550 unique disease event descriptions, and for 213 (11%) events no disease name was reported. Sixty-nine percent
(n=l,311) and 31% (n=601) of the disease events were reported in dogs and cats, respectively. Table A.4.5 shows the distribution of the disease conditions (e.g., acute, chronic, and recurrent) and the severity (e.g., mild, moderate and severe) of the disease events treated with non-topical antimicrobials reported by the veterinarians.
132 There were 5,188 diagnostic-test events recorded for 1,789 animals and of these,
0.8% (n=40) were bacterial culture and antimicrobial susceptibility. In dogs and cats,
bacterial culture and susceptibility was performed in 3% and 8%, respectively, of the
disease events treated with antimicrobials; and, in all cases, antimicrobial treatment was
prescribed. In dogs and cats, of the bacterial culture and susceptibility testing performed,
59% (n=10) and 67% (n=10) respectively, were performed for disease events associated
with the urinary tract. Disease events that were listed as chronic or recurrent were
significantly (OR 2.5, 95% CI (1.1, 5)/?=0.02) more likely to have a bacterial culture and
susceptibility performed than disease conditions listed as acute, and culture and
sensitivity were performed more often in cats (OR 2, 95% (1.1, 5)p=0.04) than dogs.
There was no significant association between the severity of disease and having a
bacterial culture and susceptibility performed (OR 2,95% CI (0.9,4A)p=0.07)
3.3. Antimicrobial prescription events
There were 1,984 precription events recorded and 51% (n= 1,009) were for
antimicrobials; 70% (n=705) were non-topical (oral and parenteral) antimicrobials, 17%
(n=171) were otic preparations, 10% (n=100) were ophthalmic and 3% (n=33) were skin
preparations. Fifty-five percent and 49% of the prescription events in dogs and cats
respectively, were antimicrobials. The incidence rate of antimicrobial prescription events
were 23 events per 100 veterinarian-hours worked, 14 events per 100 patients examined
and 52 events per 100 patients diagnosed with a new disease event. There were 111 prescription events where a corresponding disease event was not recorded and 89%
(n=99) of these were prescription events with non-topical antimicrobials.
133 The most frequently prescribed antimicrobial class was P-lactams for both dogs
and cats, representing 65% and 67% of all non-topical antimicrobial prescription events, respectively (Tables 4.1, 4.2). Cephalosporins (predominantly cephalexin; 33% (n=180) of antimicrobial prescription events) were the most commonly prescribed antimicrobial in dogs, followed by amoxicillin-clavulanic acid (n=78,16% of antimicrobial prescription events) and metronidazole (n=78 each, 16% of antimicrobial prescription events). The next most frequently prescribed antimicrobials in dogs were fluoroquinolones (n=34,
7%). In cats, amoxicillin-clavulanic acid (n=88, 40%) and cefovecin (n=38,17%) were the most commonly prescribed antimicrobials. Fluoroquinolones (n=26,12%) and lincosamides (clindamycin) (n= 16, 7%) were the third and fourth most frequently prescribed antimicrobials. In both dogs and cats, all other antimicrobials were prescribed at frequencies less than 3%.
In dogs and cats, P-lactams were used to treat disease events in every body site reported by veterinarians (Tables 4.1,4.2, A.4.6, A.4.7) and were the predominant antimicrobial class used in each body site, with the exception of the gastrointestinal tract, for which disease events were most commonly treated with metronidazole (Tables 4.1,
4.2). When prescribed in dogs, fluoroquinolones were mostly used to treat diseases affecting the urinary tract or ears, but they were also to treat almost every other body site reported by veterinarians (Table 4.1). In cats, fluoroquinolones were commonly used to treat disease events associated with the urinary tract and respiratory tract (Table 4.2).
Clindamycin was mostly used to treat disease events associated with the dentition/oral cavity, abscesses and wounds (including bite wounds) (Table 4.2).
134 Using the criteria defined by Weese (2006), first-line antimicrobials were the most frequently prescribed in both cats and dogs. Antimicrobials in category I of importance to human health (Government of Canada 2006) were the most frequently prescribed in cats; in dogs, prescribed antimicrobials were predominately within
Category I and II (Table 4.3).
For antimicrobials that were administered orally, the median (range) duration of therapy was 10 days in both dogs and cats (range 1 to 35 days and 4 to 42 days, respectively) (Tables 4.4,4.5). Frequently, therapy was prescribed for 7 days (dogs 23%, cats 33%), 10 days (dogs 24%, cats 33%) or 14 days (dogs 28%, cats 20%). Dogs with disease events classified as chronic or recurrent were treated significantly longer (median duration: 10 days (1-28 days)) than dogs with acute disease (median duration: 9 days (2-
21 days) (p<0.01). Cats with disease events classified as moderate or severe had a longer duration of therapy (median duration: 10 days (4-28 days)) than those with mild disease
(median duration: 7 days (5-14 days)) (p=0.04). There was no significant association between the duration of therapy and the severity of the disease event in dogs (p=0.34) or the chronicity of the disease event in cats (p=0.08)
In dogs, some or all prescription events involving ampicillin, cefazolin, cefovecin, ceftiofur, metronidazole, procaine penicillin, procaine/benzathine penicillin, trimethoprim/sulfadiazine and tylosin were administered parenterally by single injection.
Excluding treatments with cefovecin (n=14; for which single injection has a duration of action of 14 days), five of these treatments (ampicillin, n=l, procaine penicillin, n=2, procaine/benzathine penicillin, n=l, tylosin n=l) were the only antimicrobial therapy prescribed to the animal. In cats, some or all prescription events with ampicillin,
135 cefovecin, enrofloxacin, procaine penicillin, procaine/benzathine penicillin, and tylosin
were administered parenterally, all by a single injection. Excluding treatments with
cefovecin (n=9), three of these treatments (procaine/benzathine penicillin, n=2, tylosin,
n=l) were the only antimicrobial therapy prescribed to the animal.
3.4. Comparison to a formulary referenced dose range (mg/kg) and frequency of
administration
In dogs, the median dose (mg/kg) was outside the referenced range for
chloramphenicol, clindamycin, doxycycline, metronidazole, tetracycline and tylosin and
the dosing frequency was outside the referenced range for amoxicillin-clavulanic acid,
azithromycin, chloramphenicol, enrofloxacin, metronidazole, and tetracycline
hydrochloride/novobiocin sodium/prednisolone combination (Delta albaplex®)(Table
4.6). In dogs, 26% (n=98) of the recorded prescriptions (n=378) overall were dosed
(mg/kg) outside of the referenced range; 18% (n=70) below and 8% (n=28) above the
referenced range.
In cats, the median doses (mg/kg) reported for most antimicrobials were within
the referenced dose range, with the exception of procaine penicillin and
procaine/benzathine penicillin (Table 4.7). In cats, 49% (n=90) of the recorded
prescriptions (n=183) were dosed outside of the referenced range, with 30% (n=55)
below and 19% (n=35) dosed above the referenced range.
In general, the frequencies of administration reported by veterinarians followed
those of the referenced formulary (Tables 4.6,4.7). However, for enrofloxacin in cats, the most commonly reported frequency of administration was once daily or every 24 hours
(Table 4.6, 4.7), whereas the referenced frequency is twice daily or every 12 hours
136 administration. Nine percent (n=l) of the prescription events exceeded 5 milligram (mg) total daily dose; however, in 73% (n=8) of the prescription events, the total daily dose was less than 4.5 mg.
3.5. Specific disease conditions
3.5.1. Feline upper respiratory tract disease
Incident disease event descriptions that were consistent with feline upper respiratory tract disease represented 6% (n=37) of all disease event descriptions in cats, and 70% (n=28) were treated with antimicrobials (Table 4.8). These represented 13% of all non-topical antimicrobial prescription events in cats. Amoxicillin-clavulanic acid was the most frequently prescribed antimicrobial to treat this condition (33%), followed by doxycycline (15%) and fluoroquinolones (14%). Cats with moderate to severe feline upper respiratory tract disease were significantly more likely to be treated with antimicrobials (OR=7, 95% CI (1.1,48)/>=0.02) than cats with mild disease. There was no association between antimicrobial treatment and chronicity (acute versus chronic/recurrent) of disease (OR=2,95% CI (0.3, 23)/?=0.7), age (p=0.7), weight
0?=0.9) or sex (OR=1.4,95% CI (0.2,10)/?=1.00) of the cat.
3.5.2. Feline lower urinary tract disease
Six percent (n=39) of the incident disease event descriptions in cats were consistent with feline lower urinary tract disease, of which 74% (n=29) were treated with antimicrobials (Table 4.8). Prescription events associated with this condition represented
13% of all non-topical antimicrobial prescription events in cats. Amoxicillin-clavulanic acid was the most frequently prescribed antimicrobial (55%), followed by fluoroquinolones (27%). There were no significant associations between antimicrobial
137 treatment and the severity of disease (OR=1.4, 95% CI (0.2, S)p=0J), chronicity of
disease (OR=0.4, 95% CI (0.1,2)^=0.25), weight (p=0.18), age (p=0.97) or sex
(OR=0.67, 95% CI (0.1, 4)/?=0.7).
3.5.3. Canine infectious tracheobronchitis
Incident disease event descriptions by veterinarians that were consistent with
canine infectious tracheobronchitis represented 2% (n=31) of all disease event
descriptions in dogs and 67% (n=21) were treated with antimicrobials (Table 4.8) and
represented 4% of all antimicrobial prescription events in dogs. The most frequently
prescribed antimicrobials were amoxicillin, amoxicillin-clavulanic acid and
chloramphenicol. Dogs with moderate or severe disease were more likely to be treated
with antimicrobials (OR=29,95% CI (2,1337)/><0.01) than dogs with mild disease.
There was also a significant difference (p=0.002) in the ages of the dogs that were treated
with antimicrobials. The median (range) age of dogs with infectious tracheobronchitis
that were treated with antimicrobials was 6 months (8 weeks to 11 years) and the median
age of dogs not treated with antimicrobials was 7 years (8 months to 14 years). There
were no significant associations between antimicrobial treatment and chronicity (OR=0.9,
95% CI (0.1, 7)^=1.00), weight (p=0.2) or sex (OR=0, 95% CI (0.0,1.2)p=0.14).
4.0. Discussion
Our findings that antimicrobials were the most commonly prescribed
medications, with p-lactams as the most frequently prescribed class, are similar those of previous studies investigating antimicrobial use in a tertiary-care teaching hospital in
southern Ontario (Weese 2006), and in studies from other countries, including those
involving tertiary care veterinary hospitals (Rantala et al., 2004), questionnaires
138 completed by veterinarians describing antimicrobial use (Watson and Maddison 2001), prescription data from university pharmacies (Holso et al., 2005), antimicrobial dispensing data collected from veterinary drug wholesalers (Odensvik et al., 2001) and a national monitoring system (DANMAP, 2007). Using the criteria for first, second and third-line antimicrobial therapy described by Weese (2006), study veterinarians prescribed predominately first-line antimicrobials and did not report use of any third-line antimicrobials (e.g., carbapenems, vancomycin). However, a high proportion of prescriptions were second-line antimicrobials, especially in cats (29%); comprising mainly of the relatively high frequencies of cefovecin and fluoroquinolone use.
Cefovecin is a subcutaneously administered long-acting third-generation cephalosporin, with duration of action of 14 days. This mode of administration is attractive in cats, since oral administration can be difficult or in some cases impossible. Third-generation cephalosporins are classified by Health Canada as of very high importance in human medicine (Category I antimicrobials) as they meet the criteria of being essential for the treatment of serious bacterial infections of humans with limited or no availability of alternative antimicrobials for effective treatment in case of emergence of resistance to these agents (Government of Canada 2006). It has been suggested that third-generation cephalosporins be limited to second-line antimicrobial therapy, when supported by bacterial culture and susceptibility data indicating a lack of appropriate first-line options
(Weese 2006). Bacterial culture and susceptibility testing was, however, not recorded in any of the prescription events involving use of cefovecin in dogs or cats in this study.
It has also been suggested that fluoroquinolones should be limited to second-line therapy (Weese 2006) and, like cefovecin, fluoroquinolones have been classified as very
139 high importance in human medicine (Category I antimicrobials) (Government of Canada
2006). The frequency of antimicrobial prescription events with fluoroquinolones reported in this study (overall 9%) is higher than reported in other studies (Odensvik et al., 2001,
Rantala et al., 2004, Holso et al., 2005, Weese, 2006, DANMAP 2007). For example, in
Denmark, fluoroquinolones represented 1% (by weight) of all antimicrobials used in companion animals (DANMAP 2007) and in a tertiary-care teaching hospital in southern
Ontario, fluoroquinolones comprised 5% of overall antimicrobial prescriptions. This suggests that fluoroquinolone use by veterinarians in Ontario could probably be reduced.
Twenty-five percent of the non-topical antimicrobial prescription events in this study were not consistent with the referenced formulary (Allen et al., 2004) with respect to dosing (mg/kg) (under or over dosed) or frequency of administration. Conventional wisdom suggests that under-dosing and reduced duration of treatment can lead to therapeutic failure (Pea et al., 2008), perhaps increasing the need for use of second-line antimicrobials or antimicrobials with greater importance to human medicine (Category I or II) and consequently further increases in Category I antimicrobial selection pressure.
Administering antimicrobials at higher than the recommended dose, frequency or duration of administration may also have adverse impact on patients, including toxicity and disruption of commensal flora (Pea et al., 2008).
In veterinary medicine, adequate oral dosing with capsules, pills or tablets can be challenging. The variation in normal adult size of companion animals is greater than 45 kilograms; it would be better to ensure appropriate dosing and compliance by owners if manufacturers would formulate the capsules, pills or tablets in concentrations to accommodate this variation. For some medications, such as marbofloxacin, convenient
140 dosing for animals with body weight less than 4.5 kg is difficult given the available tablet concentrations (25 mg, 50 mg, 100 mg, 200 mg). Similarly, for cephalexin (tablet concentrations: 250 mg, 500 mg) dosing at the high end of the recommended dose range
(40 mg/kg) is inconvenient for body weights greater than 30 kg. However, difficulties in dosing are rarely encountered with suspensions or injections, therefore other factors (e.g., dose approximation by veterinarians or use of other references for treatment guideines) may explain the observed high frequency (53%) of treatments dosed outside the referenced range. We did not seek to determine these other factors, but research in this area would be useful.
We also observed use of antimicrobials for feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis, including treatment with cefovecin and fluoroquinolones. Antimicrobial use is normally not required for these conditions, as they are typically associated with viruses (e.g., feline herpes virus, feline calicivirus, canine parainfluenza virus, canine adenovirus, canine influenza), may be self limiting even when associated with other micro-organisms (e.g.,
Bordetella bronchiseptica, Chlamydophila felis) or not associated with a bacterial infection (e.g., feline lower urinary tract disease) (Kruger et al., 2009, Greene 2006),
Antimicrobial use in cats in the study population could possibly be decreased by up to
25% if routine use of antimicrobials for the management of feline upper respiratory tract disease or feline lower urinary tract disease was discontinued.
Many veterinary professional organisations have published prudent or judicious antimicrobial use guidelines (CVMA 2000, AAFP 2001, Morley et al., 2005,
AAFP/AAHA 2006, AVMA 2006). All advocate use of bacterial culture and
141 susceptibility as tools to support diagnosis and as guides to therapy. However, in this study population the frequency of bacterial culture and susceptibility testing was extremely low (overall 4%). Laboratory tests increase the financial cost to the client; however, there are other types of costs associated with inappropriate or improper therapy at the individual level and population level. These may include prolonged treatment, increased morbidity, need for more expensive, second or third-line antimicrobials or those with higher importance in the treatment of human infections.
In summary, antimicrobials are critical for the management of bacterial infections and veterinarians accept a responsibility for stewardship of these drugs. The findings in this study population suggest that antimicrobial use could be improved by reducing the frequency of use third-generation cephalosporins (e.g., cefovecin) and fluoroquinolones, and reducing the use of antimicrobials in the treatment of feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis.
Additionally, veterinarians could make greater use of bacterial culture and susceptibility to support their diagnoses and therapeutic choices. Following antimicrobial selection, veterinarians also need to ensure that the medications are prescribed following the recommended dose (mg/kg) and frequency of administration.
Acknowledgements
We thank Nicol Janecko for managing the project, the Laboratory for Foodborne
Zoonoses co-op and summer students for data management and entry, and the veterinarians for participating the study. This study was funded by the Ontario Veterinary College Pet
Trust Fund and the Public Health Agency of Canada. Colleen Murphy was a recipient of the Ontario Veterinary College Graduate Student Fellowship.
142 References
Allen D.G, Pringle J.K, Smith D.A., Pasloske K., and Dayger K. 2005. Handbook of
Veterinary Drugs. Baltimore, Maryland. Lippincott Williams and Wilkins.
American Association of Feline Practitioners (AAFP). 2001. Basic guidelines of judicious therapeutic use of antimicrobials in cats. http://www.catvets.com/uploads/PDF/antimicrobials.pdf. Accessed September, 2009.
American Association of Feline Practitioners/American Animal Hospital Association
(AAFP/AAHA). 2006. Basic guidelines ofjudiciou s therapeutic use of antimicrobials. http://www.aahanet.org/PublicDocuments/AAFP_AAHA_AntimicrobialGuidelines.pdf.
Accessed September, 2009.
American Veterinary Medical Association (AVMA). 2006. American association of feline practitioners/American animal hospital association basic guidelines of judicious therapeutic use of antimicrobials. Revised 2009. http://www.avma.org.subzero.lib.uoguelph.ca/issues/policy/jtua_aafp_aaha.asp.Accessed
September, 2009.
Canadian Veterinary Medical Association (CVMA). 2000. Guidelines on the prudent use of antimicrobial drugs in animals. http://canadianveterinarians.net/Documents/Resources/Files/85_Resources_Prudent-Use- of-Antimicrobial-Drugs-in-Animals.pdf. Accessed September, 2009.
143 College of Veterinarians of Ontario (CVO). Professional classification codes. http://cvo.org/pdfs/directory_codes.pdf. Accessed September, 2009.
DANMAP. 2007. Use of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, foods and humans in Denmark. http://www.danmap.org/pdfFiles/Danmap_2007.pdf. Accessed September, 2009.
Forbes, GB. 1949. Infection with penicillin-resistant staphylococci in hospital and general practice. Br Med J, v. 2, p. 569-71.
Government of Canada. 2006. Categorization of Antimicrobial Drugs Based on
Importance in Human Medicine http://www.hc-sc.gc.ca/dhp- mps/consultation/vet/consultations/amr_ram_hum-med-eng.php. Accessed September,
2009.
Greene, C. 2006. Infectious diseases of the dog and cat, St. Louis, Missouri, Saunders
Elsevier.
Holso, K, M Rantala, A Lillas, S Eerikainen, P Huovinen, L Kaartinen. 2005. Prescribing antimicrobial agents for dogs and cats via university pharmacies in Finland—patterns and quality of information. Acta Vet Scand, v. 46, p. 87-93.
144 Kruger, JM, C A Osborne, J P Lulich. 2009. Changing paradigms of feline idiopathic
cystitis. Vet Clin North Am Small Anim Pract, v. 39, p. 15-40.
Morley, PS, M D Apley, T E Besser, D P Burney, P J Fedorka-Cray, M G Papich, J L
Traub-Dargatz, J S Weese. 2005. Antimicrobial drug use in veterinary medicine. J Vet
Intern Med, v. 19, p. 617-29.
Odensvik, K, K Grave, C Greko. 2001. Antibacterial drugs prescribed for dogs and cats in Sweden and Norway 1990-1998. Acta Vet Scand, v. 42, p. 189-98.
Pea, F, F Pavan, M Furlanut. 2008. Clinical relevance of pharmacokinetics and pharmacodynamics in cardiac critical care patients. Clin Pharmacokinet. v 47, p. 449-62.
Rantala, M, K Holso, A Lillas, P Huovinen, L Kaartinen. 2004. Survey of condition- based prescribing of antimicrobial drugs for dogs at a veterinary teaching hospital. Vet
Rec,v. 155, p. 259-62.
Watson, AD, J E Maddison. 2001. Systemic antibacterial drug use in dogs in Australia.
Aust Vet J, v. 79, p. 740-6.
Weese, JS. 2006. Investigation of antimicrobial use and the impact of antimicrobial use guidelines in a small animal veterinary teaching hospital. 1995-2004. J Am Vet Med
Assoc, v. 228, p. 553-8.
145 Table 4.1. The frequencies of non-topical antimicrobial prescription events for incident disease events8 in dogs reported by veterinarians in the submitted journals.
Antimicrobial Overall Skin GF Urinary Respiratory Anal Dental/ Ears Digits Genitalia Eyes
(N=486) (N=153) (N=90) (N=32) (N=28) Gland Oral (N=14) (N=9) (N=7) (N=5)
nr/o n(% n (%) n(% n(%) (N=22) (N=23) n(%) n(%) n(%) n(%) n(%) n(%) p-lactams
Amoxicillin 44(9) 6(4) 8(25) 2(7) 1(5) 3(14) 1(7) 3 (33) 2 (29) 1(20)
Amoxicillin-clavulanic 78 (16) 21 (14) 12 (38) 5(18) 12 (55) 6(26) 1(7) 2(40)
acid
Ampicillin 5(1) 1 (0.6) 1(1) 2(7) 1(14)
Cefazolin 1 (0.2)
Ceftiofur 3 (0.6) 2(1)
Cefovecin 19(4) 9(6) 1(1) 2(6) 1(4) 1(4) 1(7)
Cephalexin 160 (33) 103 (67) 4(13) 2(7) 4(18) 2(9) 7(50) 5 (56) 2 (29) 2(40)
Procaine Penicillin 3 (0.6)
Procaine/ 3 (0.6) 2(2) 1(4) 1(14)
benzathine Penicillin
146 Antimicrobial Overall Skiii GF Urinary Respiratory Anal Dental/ Ears Digits Genitalia Eyes
(N=486) (N=153) (N=90) (N=32) (N=28) Gland Oral (N=14) (N=9) (N=7) (N=5)
n(%) n (%) n (%) n (%) n (%) (N=22) (N=23) n(%) n(%) n(%) n(%)
n (%) n (%)
Fluoroquinolones
Enrofloxacin 24 (5) 6(3) 6(19) 1 (4) 1 (5) 2(14) 1(14)
Marbofloxacin 5 (1) 1 (0.6) 1 (1) 2(7)
Orbifloxacin 5 (1) 1 (0.6) 1 (4) 1 (5) 1(7)
Lincosamides 1(11)
Clindamycin 15(3) 1 (0.6) 11(48) Tylosin 15 (3) 12(13)
Macrolides
Azithromycin 3 (0.6) 1 (0.6) 1(4) Erythromycin 1 (0.2) 1(1)
Tetracyclines
Doxycycline 4 (0.8) 3(H) Tetracycline 1 (0.2)
147 Antimicrobial Overall Skin GIb Urinary Respiratory Anal Dental/ Ears Digits Genitalia Eyes
(N=486) (N=153) (N=90) (N=32) (N=28) Gland Oral (N=14) (N=9) (N=7) (N=5)
n(% n(%) n (%) n (%) n(%) (N=22) (N=23) n(%) n(%) n(%) n %) n (%) n (%)
Tetracycline, 4 (0.8) 2(7)
novobiocin and
prednisolone
combination
Sulfonamides and
Combinations
Sulfadimethoxine 5 0) 1 (0.6) 4(4) 2(7)
Trimethoprim/ 5(1) 4(4)
Sulfadiazine
Other
Chloramphenicol 5(1) 4(14)
Metronidazole 78 (16) 64 (71) 1(4) 1(5)
"Veterinarians' disease event descriptions were aggregated into body sites or specific disease conditions. Antimicrobial prescriptions
for body sites, specific disease conditions or clinical signs where n is less than 5 are described in Appendix A.4.6.bGastrointestinal.
148 Table 4.2. The frequency of non-topical antimicrobial prescription events for incident disease events3 in cats reported by veterinarians in the submitted journals.
Antimicrobial Overall Respiratory Urinary GPAbscess Skin Dental/ Bite Digits Wound Ear Anal
(N=219) (N=35) (N=29) (N=24) (n=18) (N=18) Oral (N=12) (N=6) (N=4) (N=4) Gland
n(%) n(%) n(%) n(%) n(%) n(%) (N=13) n(%) n(%) n(%) n(%) (N=3)
n (%) n (%)
P-lactams
Amoxicillin 11(5) 3(9) 2(7) 1(6) 1(17) 1(25)
Amoxicillin-clavulanic 88(40) 13(37) 16(55) 3(12) 9(50) 12(67) 4(31) 6(50) 4(67) 1(25) 1(33)
acid
Ampicillin 4(2) 2(8) 1(8)
Cefovecin 38 (17) 4(11) 3(10) 1(4) 4(22) 4(22) 2(15) 4(33) 1(17) 2(50) 2(50) 1(33)
Procaine Penicillin 1 (0.5) 1(6)
Procaine/ 4(2) 1(4)
benzathine Penicillin
loroquinolones
Enrofloxacin 15(7) 5(14) 5(17) 1(33)
Marbofloxacin 4(2) 2(7) 1 (6) 1 (8)
149 Antimicrobial Overall Respiratory Urinary GIb Abscess Skin Dental/ Bite Digits Wound Ear Anal
(N=219) (N=35) (N=29) (N=24) (n=18) (N=18) Oral (N=12) (N=6) (N=4) (N=4) Gland
n (%) n (%) n (%) n(%) n(%) n(%) (N=13) n(%) n(%) n(%) n(%) (N=3)
n (%) n (%)
Orbifloxacin 7(3) 2(6) 1 (3) 1 (4) 1(6) 1(25)
Lincosamides
Clindamycin 16(7) 3(17) 6(46) 1(8) 1(25)
Tylosin 2(1)
Macrolides
Azithromycin 4(2) 3(9)
Erythromycin 2(1) 1(3) 1(4)
Tetracyclines
Doxycycline 4(2) 4(11)
Tetracycline 1 (0.5) 1(4)
150 Antimicrobial Overall Respiratory Urinary GI Abscess Skin Dental/ Bite Digits Wound Ear Anal
(N=219) (N=35) (N=29) (N=24) (n=18) (N=18) Oral (N=12) (N=6) (N=4) (N=4) Gland
n(%) n(%) n(%) n(%) n(%) n(%) (N=13) n(%) n(%) n(%) n(%) (N=3)
n (%) n (%)
Tetracycline, 1 (0.5)
novobiocin and
prednisolone
combination
Other
Metronidazole 18(8) 12(50)
"Veterinarians' disease event descriptions were aggregated into body sites or specific disease conditions. Antimicrobial prescriptions for other body sites, specific disease conditions or clinical signs are described in Appendix A.4.7. bGastrointestinal
151 Table 4.3. Percentages of prescription events with non-topical antimicrobials for incident
disease events in dogs and cats by recommended use as first, second or third line therapy
in veterinary medicine8 and importance for use to treat human infectionsb.
Category Percent of prescriptions in Percent of prescriptions
dogs (n=486) in cats (n=219)
Use in veterinary medicine*
First-line therapyb 88% 71%
Second-line therapy0 12% 29%
Importance for use to treat
human infections4
Classification I (Very 45% 78%
High Importance)6
Classification II (High 53% 20%
Importance/
Classification ffl 2% 2%
(Medium Importance)6
"Weese, 2006. "Penicillins (penicillin G, amoxicillin, or ampicillin), potentiated penicillins (amoxicillin- clavulanic acid), first-generation cephalosporins (cefazolin or cephalexin), second-generation cephalosporins (cefoxitin), trimethoprim-sulfonamide, tetracyclines (tetracycline or doxycycline), lincosamides (clindamycin, metronidazole), macrolides (erythromycin or tylosin). 'Fluoroquinolones, other cephalosporins. ""Government of Canada, 2006. Third-generation cephalosporins, fluoroquinolones, nitroimidazoles (metronidazole), penicillin-pMactamase inhibitor combinations. fFirst and second- generation cephalosporins (including cephamycins), lincosamides, macrolides, penicillins, trimethoprim/sulfamethoxazole.8Phenicols, sulphonamides, tetracyclines, trimethoprim.
152 Table 4.4. Body site-specific distributions of the duration of antimicrobial therapy for
incident disease events3 in dogs as reported by veterinarians in submitted journals.
Body site of Disease Median (days) Minimum (days) Maximum (days)
Event
Skin(n=130) 14 5 30
Gastrointestinal 7 2 21
(n=80)
Urinary (n=30) 10 6 35
Respiratory (n=23) 10 3 21
Anal Gland (n=21) 10 7 20
Dental/Oral (n=20) 10 1 28
Ears (n=12) 10 7 21
Digits (n=8) 7 7 14
Genitalia (n=4) 9 7 14
Eyes (n=5) 14 10 14
Veterinarians' disease event descriptions were aggregated into body sites or specific disease conditions.
153 Table 4.5. Body site-specific distributions of the duration of antimicrobial therapy for
incident disease events3 in cats as reported by veterinarians in the submitted journals.
Body Site of Disease Median (day) Minimum (day) Maximum (day)
Event
Respiratory (n=21) 10 4 42
Urinary (n=26) 10 7 21
Gastrointestinal (n=17) 7 4 28
Abscess (n=13) 10 7 14
Skin(n=13) 7 7 14
Dental/Oral (n=10) 10 7 14
Bite (n=8) 10 7 14
Digits (n=5) 7 7 7
Wound (n=2) NA 7 14
Ear (n=2) NA 10 30
Anal Gland (n=2) 10 10 10
Veterinarians' disease event descriptions were aggregated into body sites or specific disease conditions.
154 Table 4.6. The reported doses and frequencies of prescriptions with non-topical antimicrobials8 for incident disease events in dogs by veterinarians in the submitted journals.
Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range p-lactams
Amoxicillin (n=36) 10-22 mg/kg q8-l 2h 17 mg/kg 3(8) 3(8) 35 (97) 1 (3)
(9,27)
Amoxicillin- 13.75 mg/kg q 12h 14 mg/kg 38(58) 19(29) 19(29) 2(3) 64(97)
clavulanic acid (4,31)
(n=66)
Ampicillin (n=5)c 11 -22 mg/kg q6-8h 22 mg/kg 2 (40) 1 (20) 1 (20) Single injection
(10,28)
Cefazolind (n=l) 5-15 mg/kg q6-8h Not reported Single injection
155 Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range
Cefovecine(n=12) 8mg/kg 8mg/kg 8(67) 7(58) 1(8) Single injection
(1,10)
Cephalexin 20-40 mg/kgq8-12h 24mg/kg 9 (7) 8 (6) 1 (0.7) 130(96) 5(4)
(n=135) (9,47)
Ceftiofurf (n=2) 2.0 mg/kg q24h 2.1 mg/kg Single injection
(2.0,2.3)
Procaine Penicillin 20,000 IU/kg q 12- Not reported Not reported
(n=2) 24h
Procaine/ 15,000 IU/kg' 39,000 IU/kg 1(100) 1(100) Single injection
benzathine
penicillin (n=l)
Fluoroquinolones
156 Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range
Enrofloxacin 5-20 mg/kg per day 6 mg/kg 4(20) 3(15) 1(5) 16 (80) 4(20)
(n=20) (2.5,13)
Marbofloxacinh 2.75-5.55 mg/kg 3 mg/kg 4(80) 4(80) 5 (100)
(n=5) q24h (1.6,3.4)
Orbifloxacin (n=5) 2.5-7.5 mg/kg q24h 3 mg/kg 5 (100)
(2,4.5)
Lincosamides
Clindamycin 11 mg/kg ql2h or 6 mg/kg 9(69) 9(69) 1(8) 12 (92)
(n=13) 22 mg/kg q24hr (2,11)
Tylosin (n=8) 5-10 mg/kg q8-12h 13 mg/kg 6(71) 6(71) 7(88) 1(12)
20-40 mg/kg q!2h (11,17) 8(100) 8(100)
157 Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range
Macrolides
Azithromycin 3.3-10 mg/kg q24h 8mg/kg 1(50) 1 (50) 1 (50) 1 (50)
(n=2) (3,12)
Erythromycin 10-20 mg/kgq8h lSmg/kg11 1 (100)
(n=l)
Tetracyclines
Doxycycline8 (n=3) 5-20mg/kgql2h 2.8mg/kg 2 (67) 2 (67) 3(100)
(1.3,4.5)
Tetracycline (n=l) 25-50 mg/kg q6-8h 15 mg/kg 1 (100) 1 (100) 1 (!00)
158 Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range
Tetracycline, 4.3-30 mg/kg ql 2h 12 mg/kg 1 (50) 1 (50)
novobiocin,
prednisolone
combination1" (n=2)
Sulfonamides and
Combinations
Sulfadimethoxine 25 mg/kg ql2-24h 23 mg/kg 2(50) 1(25) 1(25) 4(100)
(n=4) (21,34)
Trimethoprim/ 15-30 mg/kg ql 2h 17 mg/kg 2(40%) 1(20) 1(20) 3(60) 2(40)
sulfadiazine (n=5) (15,42)
Other
159 Antimicrobial Referenced dose Doses reported Frequencies of prescription Frequencies of administration
and frequency in study events outside of referenced dose as reported in study journals
journals range n (%)
(Median (range)) n (%)
Outside Below Above q24h ql2h q8h
Range Range Range
Chloramphenicol SO mg/kg q8h 35 mg/kg 2(67) 2(67) 1 (33) 2 (67)
(n=3) (31,49)
Metronidazole 25-65 mg/kg q24h 14 mg/kg 16 (24) 16(24) 7(10) 59 (88) 1 (2)
(n=67) (6,35)
"AH medications were given orally unless otherwise noted. "Allen et al (200S). CA11 were given by intravenous or subcutaneous injection. "Recommended dose fromproduc t monograph. Route of administration not reported. "Recommended dose range obtained from product monograph, with a single subcutaneous injection having a duration of action of 14 days. 'Recommended dose from product monograph. Route of administration was subcutaneous injection. gThere are other recommended dosing regimes for doxycycline, however all reported prescription used a ql2h dosing frequency.b Recommended dose from product monograph.
160 Table 4.7. The reported doses and frequencies of prescriptions with non-topical antimicrobials3 for incident disease events in cats by veterinarians in the submitted journals.
Antimicrobial Referenced dose and Doses reported in Frequencies of prescription Frequency of administration
frequencyb study journals events outside of referenced as reported in study journals
(Median (range)) dose range n (%)
n(%)
Outside Below Above q24h ql2h q8hr
Range Range Range
p-lactams
Amoxicillin 10-22 mg/kgq8-12h 18mg/kg 1(10) 1 (10) 2 (20) 8 (80)
(n=10) (11,31)
Amoxicillin- 62.5 mg per cat ql2h See Results 4(6) 2(3) 2(3) 73 (100)
clavulanic acid
(n=73)
Ampicillin ll-22mg/kgq6-8h 19mg/kg 1(33) 1 (33) Single injection
(n=3)c (9,28)
Cefovecind 8mg/kg 8mg/kg 14(44) 6(19) 8(25) Single injection
(n=32) (5,12)
161 Antimicrobial Referenced dose and Doses reported in Frequencies of prescription Frequency of administration
frequency study journals events outside of referenced as reported in study journals
(Median (range)) dose range n(%
n(%)
Outside Below Above q24h ql2h q8hr
Range Range Range
Procaine 20,000 lU/kg ql2-24h 16,500' 1 (100) 1(100) Single injection
Penicillin (n=l)
Procaine/ 20,000 IU/kg 7,500f 3 (100) 3 (100) Single injection
benzathine
penicillin (n=3)
Fluoroquinolones
Enrofloxacin 2.5mg/kgql2h 3.6mg/kg 5(45) 4(36) 1(9) 10(91) 1(9)
(n=ll) (1.4,5)
Marbofloxacin6 2.75 mg per kg q24h 2.6 mg/kg 4(100) 2(50) 2(50) 4(100)
(n=4) (1.9,3.8)
Orbifloxacin 2.5-7.5 mg/kg q24h 2.9 mg/kg (1.7, 2 (33) 2 (33) 6(100)
(n=6) 3.8)
162 Antimicrobial Referenced dose and Doses reported in Frequencies of prescription Frequency of administration
frequency study journals events outside of referenced as reported in study journals
(Median (range)) dose range n(%)
n(%)
Outside Below Above q24h ql2b q8hr
Range Range Range
Lincosamides
Clindamycin 5.5 -11 mg/kgql2hor 11- 5mg/kg 6(50) 5(42) 1(8) 3(25) 9(75)
(n=l 2) 22 mg/kg q24hr (4,26)
Tylosin (n=2) 5-10 mg/kg q8-12h 7.6f mg/kg 1 (50) 1 (50)
20-40 mg/kg ql2h
Macrolides
Azithromycin 5-10 mg/kg q24h 8 mg/kg 2 (100)
(n=2) (7.5,8.5)
Erythromycin 10-20 mg/kg q8h Not reported 1 (50) 1 (50)
(n=2)
Tetracyclines
163 Antimicrobial Referenced dose and Doses reported in Frequencies of prescription Frequency of administration
frequency11 study journals events outside of referenced as reported in study journals
(Median (range)) dose range n (%)
n("/o Outside Below Above q24h ql2h q8hr
Range Range Range
Doxycycline1 5-20 mg/kg ql2h 5 mg/kg 4(100) 4 (100) 4 (100)
(n=4) (3,6)
Tetracycline, 1 tablet per cat ql2h Not reported
novobiocin,
prednisolone
combination6
(n=l)
Other
Metronidazole 25 mg/kg q24h 14 mg/kg 10(71) 4(29)
(n=14) Or (6,24)
10 mg/kg ql2h
164 aAH medication were given orally unless otherwise noted. bAllen et al. 2005. CA11 were given by intramuscular or subcutaneous injection. dRecommended dose range obtained from product monograph, with a single injection having a duration of action of 14 days. "Recommended dose from product monograph, there are other recommended dosing regimes for doxycycline, however all reported prescription used a ql2h dosing frequency.
165 Table 4.8. The percent of events of feline upper respiratory tract disease, feline lower
urinary tract disease and canine infectious tracheobronchitis treated with antimicrobials.
Percent Treated with Antimicrobials
Antimicrobial Feline Upper Feline Lower Urinary Canine Infectious
Respiratory Tract Disease Tracheobronchitis
Tract Disease
P-lactams
Amoxicillin 11% 7% 19%
Amoxicillin- 33% 55% 19%
clavulanic acid
Cephalexin 9%
Cefovecin 11% 10% 5%
Fluoroquinolones
Enrofloxacin 7% 17%
Marbofloxacin 7%
Orbifloxacin 7% 3% 5%
Macrolides
Azithromycin 11%
Erythromycin 4%
Tetracyclines
Doxycycline 15% 9%
Tetracycline, 9%
novobiocin and
prednisolone
combination
Other
Chloramphenicol 19%
Metronidazole 5%
166 Chapter 5
Evaluation of specific infection control practices used by companion
animal veterinarians in community veterinary practices in southern
Ontario.
As accepted for publication by Zoonoses and Public Health
Abstract
This study evaluated specific infection control practices in community veterinary practices in southern Ontario. Environmental disinfection, management of infectious patients and antimicrobial use in clean surgical procedures were investigated. Community companion animal veterinary practices (n=101) in Southern Ontario were recruited and a questionnaire was administered to one veterinarian and one veterinary technician from each practice. The veterinarian questionnaire gathered data on clinic demographics, management of infectious patients, infectious diseases of concern, environmental disinfection and antimicrobial use in surgical procedures. The veterinary technician questionnaire gathered data on environmental disinfection. None of the veterinary practices had a formal infection control program. Sixty-five percent (n=66) of the veterinary practices did not have an isolation area and 61% (n=40) of these practices did not employ any specific infection control measures for infectious cases. The products most frequently used for environmental disinfection were hydrogen peroxide based or quaternary ammonium compounds. Bleach was the agent most commonly used for environmental disinfection of infectious body fluids; however 60% of the veterinarians and 40% of the veterinary technicians did not identify a product for environmental disinfection of infectious body fluids. Twenty-four percent of the veterinarians reported
167 using antimicrobials for elective sterilization surgeries and 60% reported using
antimicrobials in other clean surgical procedures. There is a need for community
veterinary practices to develop infection control programs specific to their individual practice. In addition, veterinarians should discontinue the common use of antimicrobials
for clean elective sterilization surgical procedures.
1.0. Introduction
Even before widespread acceptance of the germ theory, isolation of infectious patients, hospital-hygiene, handwashing and asepsis were identified as important for prevention of hospital-associated infections (HAT) in humans (Larson, 1997). In the
1970s, the Centers for Disease Control and Prevention (CDC) established the National
Nosocomial Infection Surveillance system to integrate human hospital-surveillance activities and to further develop infection control programs. During this same time period, the CDC also initiated the Study on the Efficacy of Nosocomial Infection Control that was a national epidemiological study on the impact of hospital infection control and surveillance activities (Hughes, 1987). Hospital-based infection control in veterinary medicine has not had the same degree of research, and development and implementation of standards (Morley, 2004). Research studies on infection control in veterinary medicine are limited in number and scope (Benedict et al., 2008, Wright et al., 2008). Recently, a compendium of infection control practices for veterinary clinics was released (Elchos et al., 2008); however there is a dearth of objective data available for guideline development and assessment.
There are reports of infectious disease outbreaks associated with community veterinary practices including zoonotic pathogens such as Salmonella (Cherry et al.,
168 2004, Wright et al., 2005) and methicillin-resistant Staphylococcus aureus (MRSA)
(Weese et al., 2006), and animal-specific pathogens such as feline calicivirus (Schorr-
Evans et al., 2003). Despite these reports, the epidemiology of HAI in companion animal
veterinary medicine is unknown. It is likely that the occurrence of HAI in community
companion animal practice is under-recognised, under-investigated and under-reported.
While outbreaks receive the most attention, endemic infections, such as surgical site
infections, infectious diarrhea, upper respiratory tract disease and invasive device (i.e.
intravenous catheter site) infections may account for the majority of HAI. General
practice veterinarians often do not have adequate skills or resources to investigate
possible outbreaks or do surveillance to understand and address endemic HAI. While
absolute numbers of infections may be low, the public health consequences of HAIs by zoonotic pathogens could be substantial given the close interaction between people and their pets.
To our knowledge, no objective evaluations of the effect of infection control measures in veterinary medicine have been published. In order to begin to evaluate the value of specific infection control practices, it is necessary to understand which infection control practices are used and their frequency of use. The objective of this survey was to gather data on specific infection control practices in companion animal veterinary hospitals in southern Ontario, Canada. In particular the use of disinfectants in environmental disinfection, management of infectious patients and use of antimicrobials for clean surgical procedures were investigated.
2.0. Materials and methods
169 Veterinary hospitals in southern Ontario, Canada licensed as companion animal
hospitals or offices by the College of Veterinarians of Ontario in 2005 were eligible for recruitment. The additional licensures for food animal or equine facilities (mixed animal practice) were not excluded from the pool of eligibility. A recruitment letter was mailed to the veterinarian listed as the director of these practices (n=766) describing the study objectives. Practices willing to participate were asked to respond by mail, fax, or telephone with a completed practice-demographic survey (Appendix A.2.1).
Following recruitment, practices were visited by the researchers. Data were collected through questionnaires that were administered verbally to one veterinarian and one veterinary technician at each practice (Appendix A.2.2 and A.2.3). Prior to starting the study, the questionnaire was pre-tested by one veterinarian and one veterinary technician in each of four veterinary practices. Following the pre-test, the questionnaire was revised before the initiation of the full study. Data from the pre-test was not included in the study results.
Once the study was initiated, the questionnaire was verbally administered to the veterinarian and veterinary technician who were most involved with the practices* infection control program. In the absence of a person filling this role, then the questionnaire was administered to the veterinarian and/or veterinary technician involved with decision-making regarding hospital policy concerning the companion animal aspect of the practice. The questionnaire administered to the veterinarians gathered data on baseline clinic demographics and used open questions to capture data on the management
(from an infection control perspective) of possible or known infectious patients, infectious diseases, infectious agents or clinical signs of concern, disinfection practices
170 and antimicrobial use in surgical procedures. Veterinarians were also asked to rank the importance of appropriate cleaning and disinfection in their practice and to list reasons for appropriate cleaning and disinfection. Since "appropriate" cleaning and disinfection can vary under different clinical situations, the term "appropriate" was not defined and each veterinarian was allowed to make his or her own interpretation. In addition, in practices where an isolation unit was used as part of the management of known or suspect infectious cases, the veterinarians were not asked about other infection control measures such as barrier precautions. Since these infection control measure can vary depending on the infectious disease, we sought only to describe the practices used when an isolation area was not available. The questionnaire administered to technicians asked the same open questions on disinfection practices as were asked in the veterinarian questionnaire.
Veterinarians and veterinary technicians were interviewed separately and not informed of the other's responses.
The study procedures were approved by the Research Ethics Board at the
University of Guelph.
2.1. Statistical methods
Descriptive analysis was performed. Categorical responses were described as proportions. Median values and ranges were calculated for counts. Differences in proportions were tested using the Fisher's exact test. The Stuart-Maxwell test statistic and the kappa statistic tested symmetry and agreement, respectively, between veterinarian and veterinary technician responses to questions on disinfection practices. Pairs were tested where both the veterinarian and the veterinary technician knew information regarding disinfection practices. A/?-value of <0.05 was considered significant.
171 3.0. Results
One hundred and twenty one clinics responded with interest to the recruitment letter (response rate 16%). There was no significant difference (p=0.45) between the positive response rate of companion animal only practices and mixed animal practices.
Twenty of these clinics were not included because they were outside of the geographic sampling region or could not be contacted for follow-up. Of the 101 practices, 90 practices were companion animal only, 10 were mixed animal practices, and 1 practice treated primarily exotic animals (companion animals other than dogs and cats). The number of staff (including veterinarians and all support staff) in each practice ranged from 3-45 (median 10). The number of canine or feline appointments seen each day ranged from 2-40 (median= 10). The number of dogs and cats hospitalized each day ranged from 1-18 (median 3) and 1-25 (median 3), respectively. Eighty-eight of the clinics offered non-medical services such as boarding, grooming or adoption.
None of the participating veterinary clinics had an infection control program or an individual specifically involved with infection control. Consequently, in each practice, the questionnaire was completed by the veterinarian and veterinary technician who was involved with decision-making regarding hospital policy concerning the companion animal aspect of the practice.
A separate isolation unit for possible or known infectious cases was present in
35% (n=35) of the clinics. In practices with a separate isolation unit (n=35), 69% (n=24) of the interviewed veterinarians reported that this area was exclusively used for potentially or known infectious cases. Sixty-five percent (n=66) of the practices did not have an isolation area and in 61% (n=40) of the practices, the interviewed veterinarians
172 reported that no specific infection control measures were employed when hospitalizing a possible or known infectious case. Nevertheless, 39% (n=26) of the veterinarians from practices without an isolation unit (n=66) described infection control measures to manage known or suspected infectious patients. Each practice in this cohort attempted to physically separate the infectious patient from the other hospital population and for 35%
(n=9) of the clinics, this was the only infection control measure used. Forty-two percent
(n=l 1) described placing a known or suspected infectious patient in a kennel far from other patients and 27% (n=7) of the veterinarians described placing known or suspected infectious patients in a regular hospitalization kennel room that was emptied of other patients. Nineteen percent (n=5) of the veterinarians reported placing possible or known infectious cases in a portable kennel in an office and 11% (n=3) in a treatment area.
Following the physical separation of patients, the next frequently employed measure was the use of protective barriers. Fifty-seven percent (n=15) of the veterinarians in this cohort described using some type of protective barrier when handling a known or suspected infectious patient. Thirty-one percent (n=8) described using disposable gloves or booties when handing a patient and 27% (n=7) reported using a designated lab coat when handling a known or possible infectious patient. Other less frequently reported infection control strategies in this cohort of practices included the use of dedicated stethoscopes and thermometers which was reported by 15% (n=4) of the veterinarians, handling or cleaning infectious cases last (12%, n=3), closing off areas where the infectious patient had been (4%, n=l), disinfecting areas with infectious cases with bleach (15%, n=4) and increasing the frequency of cleaning (8%, n=2).
173 Among the 26 clinics that employed self-described infection control measures,
50% (n=13) of the veterinarians described two infection control strategies and 8% (n=2) described three. Thirty-one percent (n=8) of the veterinarians described a combination of physical separation and protective clothing barriers. Twelve percent (n=3) of the veterinarians reported combining physical separation with cleaning the area last and 8%
(n=2) reported physical separation combined with cleaning the area with bleach. Among the two clinics where the veterinarians described three infection control strategies, one used dedicated equipment and closed areas where the infectious patient had been. The other clinic cleaned the area with the infectious patient last and disinfected the area with bleach. These measures were used in addition to physical separation of patients.
The open question requesting veterinarians to list clinical signs, infectious agents or diseases that would require isolation or infectious disease precautions (hereafter referred to as infectious diseases) had a 99% (n=100) response rate by veterinarians and
638 individual responses were provided. The median number of distinct infectious diseases reported by veterinarians was 6 (range 1-10). Ninety-five percent (n=95) of the veterinarians reported using isolation or other infection control practices as part of the management of patients with infectious diseases associated with the gastrointestinal or respiratory systems and these patients were the largest group identified as requiring isolation or other infection control measures (Table 5.1) accounting for 71% (n=451) of the responses provided by the veterinarians. Additionally, veterinarians (n=100) reported these other conditions as warranting isolation or other infection control measures: leptospirosis (32%), unvaccinated animals (24%), feline immunodeficiency virus (14%), feline leukemia virus (13%), rabies (10%) ringworm (9%), fleas (6%) fever (5%), and
174 feline infectious peritonitis (5%). Less than 5% of the veterinarians interviewed listed the following as requiring isolation or other infection control measures: neurological signs, mange, any dermatological lesions, bloody urine, elevated kidney or liver parameters, and sick puppies or kittens. One percent of the veterinarians also reported using isolation areas to house scared animals or pocket pets.
Ninety-eight percent (n=99) of the veterinarians ranked the importance of appropriate cleaning and disinfection protocols in their practice as moderate to high. One veterinarian ranked this as not important and one veterinarian did not respond to this question. Ninety-nine percent of the veterinarians (n=100) were able to provide reasons for appropriate cleaning and disinfection in the practice and 279 responses were provided. The median number of reasons listed by veterinarians was 3 (range 0-4). The reasons given by veterinarians for using appropriate cleaning and disinfection are presented in Table 5.2. Ninety-five percent of the veterinarians reported using appropriate cleaning and disinfection to reduce disease transmission between patients or zoonotic disease transmission, and over 50% reported that using appropriate cleaning and disinfection practices were important to the esthetics of the practice (Table 5.2).
For most areas (in the clinic), hydrogen peroxide-based oxidizing agents or quaternary ammonium compound (QAC) products were the most frequently used products for environmental disinfection according to the veterinarians and veterinary technicians (Table 5.3). Bleach was more commonly used for disinfecting areas contaminated with potentially infectious body fluids. However, 60% (n=60) of veterinarians and 40% (n=40) of technicians did not know if any products were specifically used for potentially infectious body fluids. Overall, 82% (n=100) of the
175 veterinarians interviewed and 45% (n=45) of the veterinary technicians interviewed did not know what disinfectant was used in at least one area of the practice, hi situations where both the veterinarian and technician from the same clinic were able to identify the product that was used in a given area, the agreement between them was substantial. The value of kappa for the type of product used on tables was 0.9 (n= 80,/?=0.00), kennels 0.8
(n= 77,p=0.00), floors 0.8 (n= 5S,p=0.00), runs 0.6 (n= 58,/>=0.00) and infectious body fluids 0.6 (n=26,p=0.00).
In addition to the products listed in Table 5.3, other products or product combinations were reportedly used at a low frequency. One veterinarian reported that an ammonia and bleach combination was used to disinfect the kennels, tables and runs in the practice. One veterinary technician and one veterinarian from different practices reported that a chlorohexidine solution (Savlon®) was used to disinfect kennels, runs and table surfaces. One technician reported that a QAC and a formaldehyde-based product
(Formacide®) were used to disinfect the kennels and one veterinarian from a different clinic reported using Formacide® to disinfect tables and sites contaminated with infectious body fluids.
The reported methods of preparation or dilution of disinfectants is presented in
Table 5.4. Few veterinarians and technicians reported diluting the disinfectant as described by the manufacturer and most did not know how the products were prepared for use. Ninety-nine percent (n=99) of the veterinarians and 98% (n=99) of the technicians surveyed did not know the procedures used to prepare the products for use in one or more sites in the practice. Since there was so little knowledge on the preparation of disinfectants for use by veterinarians and veterinary technicians, there were few
176 practices where both the veterinarian and veterinary technician reported methods of preparation. In these practices, the agreement between the veterinarians and the veterinary technicians was poor. The value for kappa for the preparation of disinfectant for tables was: 0.03 (n=7,/>=0.58), kennels: 0.1 (n=13,/?=0.20), floors: 0.00 (n=9,p- value=incalculable due to zero cells in the table) and runs: 0.31 (n=8, p=0.02). Of note, there were only two clinics where a veterinarian and a technician each knew about the dilution used for infectious fluids and both pairs used their own clinic specific dilution.
The value of kappa for this pair was 1.0 (n=2,p=0.08).
Twenty-four percent (n=24) of the veterinarians reported using antimicrobials for clean elective sterilization surgeries and two veterinarians provided no response. Forty- two percent (n=10) of these veterinarians reported using procaine (short acting) penicillin, 42% (n=10) used benzethaine (long acting) penicillin and 8% (n=2) did not specify the type of penicillin used. One (4%) veterinarian solely used ampicillin- sulbactam and one used either penicillin or ampicillin-sulbactam for elective sterilization surgeries. Sixty percent (n=60) of the veterinarians used antimicrobials for clean, non- elective surgical procedures (Table 5.5). The median number of different antimicrobials used by veterinarians was 2 (range 1-4). pMactams were reportedly used by 97% (n=58) of the veterinarians, fifteen percent (n=9) used potentiated P-lactams and fluoroquinolones were used by 12% (n=7) of the veterinarians.
4.0. Discussion
Infection control in veterinary hospitals is an emerging discipline and formal infection control policies are uncommon in most veterinary practices (Wright et al., 2008) and tend to be limited to veterinary teaching hospitals (Benedict et al., 2008). A
177 universal goal of any infection control program is to reduce the occurrence of HAIs. In veterinary medicine, an effective hospital infection control program functions to protect animal and human health (Traub-Dargatz et al., 2004). None of the veterinarians or veterinary technicians interviewed for this study identified a formal infection control program in their practice. This is a potentially serious problem in the standard of care in community companion animal veterinary medicine that could have legal, ethical, animal health and occupational health ramifications. Without a recognised infection control program in a practice, there is no opportunity to assess the effectiveness of any infection control measure. Although veterinarians and veterinary technicians did not identify an infection control program in their practice, they were able to describe features of an infection control program: environmental disinfection, use of isolation and management of potentially or known infectious patients. However, these features were generally underutilized, or inadequately instituted. Thus, institution of a formal infection control program could help improve the potential efficacy of some infection control practices that are already in place.
The barrier components of an infection control program that were evaluated in this study were the use of isolation and use of barrier precautions when handling infectious or potentially infectious patients. These practices can reduce HAI by providing a barrier to transmission of an infectious disease. In human medicine, isolation of a patient in a private room is recommended when the patient has poor hygienic habits, contaminates the environment or cannot be expected to assist in maintaining infection control precautions to limit the transmission of disease (HIPAC 1996, Government of
Canada 1999). All hospitalized, infectious companion animals would fulfill these criteria
178 for private room isolation. However, the majority of veterinary clinics surveyed did not have an isolation unit and companion animal veterinary facilities in Ontario are not currently required to have an isolation area (CVO 2006). Given the absence of regulation for isolation of potentially infectious patients, the low frequency of isolation of infectious patients and use of protective barriers, an opportunity is present for an excess of hospital- associated infections to occur.
In contrast, the American Animal Hospital Association (AAHA), a non- regulatory, professional organization requires veterinary practices to have formal infection control policies including a designated, negative pressure isolation area for infectious or potentially infectious cases (AAHA 2007). Some veterinarians in this study reported compliance with AAHA standards as a reason for appropriate cleaning and disinfection practices. However, compliance with these standards was imperfect since no participating practice had a formal infection control policy.
It is concerning that many clinics without isolation facilities did not even report measures to separate potentially infectious cases from the general clinic population. In some clinics, potentially infectious animals were placed in areas such as treatment and office areas that may increase the risk of transmission to other patients and staff. This action places infectious or potentially infectious patients in areas of the hospital with high patient and staff traffic. This may actually increase the risk that individuals may be exposed to the infectious patient.
Most veterinarians in this study reported that patients exhibiting either clinical signs or known infections associated with the respiratory or gastrointestinal tract required isolation or other infection control measures. This is a very good infection control policy
179 for companion animals, however, there is evidence of inadequacies in the handling of other potentially or known infectious patients. For example, rabies is endemic in Ontario
(Government of Canada 2007) and is an important deadly zoonotic disease. However only 10% of the veterinarians listed rabies as a reason for use of isolation or other infection control measure and 3% of the veterinarians listed neurological signs as a reason for isolation or other infection control measure. These findings are in agreement with data reported by Wright et al. (2008). They reported limited use of personal protective equipment by veterinarians when examining a patient with neurological signs
(Wright et al., 2008). Vaccination of companion animals is required by law in Ontario
(Government of Ontario 2007) and vaccine protection is likely very high, yet not complete. Clinical signs of rabies can include a variety of neurological signs including behavioural changes, cranial and peripheral nerve deficits, seizures, and paralysis.
Rabies should be included as a differential diagnosis in an animal presenting with neurological clinical signs and appropriate infection control measures including transmission precautions to protect from inadvertent exposure to saliva should be employed.
Most veterinarians rated the use of appropriate cleaning and disinfection in their practice as important and recognize that their function is to reduce the transmission of disease. Nevertheless, there appears to be poor understanding of disinfection practices by veterinarians and technicians because most did not know what product was used for disinfection of areas contaminated with infectious body fluids and very few knew how disinfectants were prepared for use for these situations. This finding is not surprising since no veterinary clinic claimed to have an infection control policy. However,
180 environmental disinfection is an important element in hospital-based infection control.
Optimal choice of disinfectant depends on many factors including the microbial agent, detergent used, quantity of organic matter, water pH, the surface or equipment to be disinfected, cost, ease of use, and toxicity (Rutala 1996). Bleach was the most commonly reported disinfectant for infectious fluids. Bleach, as a hypochlorite solution, is an intermediate level disinfectant and is considered adequate for environmental disinfection of infectious fluids in companion animal veterinary practice, including areas that may be contaminated with bacterial spores (Dwyer 2004). However, hypochlorite solutions are not without drawbacks. When hypochlorite is combined with an acid or ammonia, toxic chlorine or chloramines gases, respectively, may be produced. One veterinarian reported use of this combination. An effective infection control plan would include safety precautions that would prevent the misuse of these chemicals that may place staff, the public and patients at risk.
Peroxiguard®™ is an accelerated hydrogen peroxide product reportedly used by veterinarians and veterinary technicians in this study to disinfect areas with infectious body fluids. It is available to human health care under the trade name Virox®™ and is an intermediate level disinfectant. The efficacy of Virox®™ against C. difficile is similar to hypochlorite solutions (Perez et al., 2005), however label claims are limited to bacterial and virucidal applications including canine parvovirus (Sattar 2004). Further research needs to be done regarding the spectrum of activity of Peroxiguard®™ and it may be a viable, less toxic, environmentally friendly alternative to hypochlorite solutions for environmental disinfection of areas potentially contaminated with bacterial spores.
181 Study veterinarians and veterinary technicians commonly listed QACs and peroxygen compounds (Virkon®™) as disinfectants used for environmental disinfection of areas with infectious body fluids. The QACs used in veterinary clinics in this study were low-level disinfectants and were commonly used among study practices for disinfection of areas with infectious body fluids. However, the use of these products should be limited to routine disinfection and only for disinfection of infectious fluids where the agent is known to be susceptible to QACs. Although some QACs have activity against non-enveloped viruses (Jimenez and Chiang, 2006), most do not. Some manufactures of QACs reportedly used in this study have virucidal claims against canine parvovirus, yet studies have indicated poor efficacy against these viruses (Scott, 1980,
Kennedy et al., 1995, Eleraky et al., 2002). Consequently, veterinarians may be misinformed about the spectrum of activity of QACs and use these products inappropriately. In addition to QACs, veterinarians may also be misinformed about the spectrum of activity of Virkon®™. Virkon®™ is marketed for human and veterinary medicine as bactericidal, fungical, sporicidal and virucidal including nonenveloped viruses. However, studies have indicated that Virkon®™ may have poor sporicidal activity (Coates, 1996, Hernndez et al., 2000). The information that veterinarians receive regarding the efficacy and spectrum of activity of environmental disinfectants is usually limited to data supplied by the manufacturer. There is a need for independent research on veterinary environmental disinfectants that is accessible to veterinarians. This could provide veterinarians with objective data that would assist them in the selection of environmental disinfectants to suit their infection control needs.
182 Judicious or prudent antimicrobial use is an important component of a hospital
infection control program, including antimicrobial use in surgical patients. Prudent use of antimicrobials may reduce the risk of adverse events including disruption to commensal flora, promotion of resistance, colonization or infection with opportunistic pathogens, patient toxicity, and drug reactions including anaphylaxis. The classification of surgical wounds on the basis of bacterial contamination was developed by the National Academy of Sciences-National Research council in 1964 (Berard and Gandon, 1964). In human and veterinary medicine, clean surgical wounds have been defined as non-traumatic wounds without infection or inflammation, where breaks in aseptic technique have not occurred and luminal organs are not entered (Berard and Gandon 1964, Fossum 1997).
Antimicrobial prophylaxis or therapy is not indicated for clean surgical wounds unless there has been a break in asepsis, the surgical procedure or anaesthetic period is prolonged, conducted by an inexperienced surgeon, involves placement of an implant or in an immunocompromised patient. (Fossum 1997). Notwithstanding these recommendations and those of expert veterinary panels (Morley et al., 2005), most veterinarians in this study commonly reported using antimicrobials for clean surgical procedures. However, we cannot assess whether antimicrobial use in me manner is prudent. In order to assess judiciousness of antimicrobial use in these surgical cases, randomized controlled clinical trials (RCCT) are required. The limited number of RCCTs in clean surgical procedures that have been performed in companion animal veterinary medicine have demonstrated that perioperative use of ampicillin offered no benefit to the surgical outcome (Vasseur et al., 1985) or that perioperative antimicrobial administration was only beneficial when the duration of surgery was greater than 90 minutes in length
183 (Whittem et al., 1999). These studies agree with the current recommendations, however
further studies are required to define the clinical situations where antimicrobial use is
prudent and beneficial. This is important since many veterinarians in the study reported using antimicrobials in clean surgical procedures and inappropriate antimicrobial use places the individual patient at risk of an avoidable adverse event and also increases the risk to populations by contributing to the occurrence of resistance.
A limitation of this study was the low response rate, however in the authors' experience this response rate is not unexpected from the target population from which the study population was derived. In other studies from the same target population, response rate ranged from 10% to 24% (Murphy et al. 2009a, Murphy et al. 2009b, Chapter 4).
This study did not attempt to gather other demographic data such as practice size from veterinary practices that did not respond. These data are not available through comprehensive sources such as regulatory or voluntary veterinary professional organizations. It is the authors' experience gathering this data through follow-up postal- mailings, phone-calls and emails from non-responding veterinarians from this target population are almost impossible (Murphy, unpublished observations). One factor which may have contributed to the low response rate was the decision to recruit practices through directing our mailing to the listed director of the veterinary practice, rather than targeting all veterinarians in our geographical sampling region. Participation in the study was then dependant upon the interest of the director of the practice, and not the other associate-veterinarians in the practice. The effect of this factor is unknown, and may not be strong since a study targeting the recruitment of veterinarians in a similar geographical region had a lower response rate than observed in this study (Chapter 4). Factors
184 associated with veterinary participation in research studies from this target population is
an area that requires investigation.
The goal of this study was to describe specific aspects of infection control in community veterinary hospitals, not to evaluate infection control practices as a whole in these hospitals. Infection control policies are specific to individual facilities addressing the patient population, facility design, staffing and level of risk aversion. However, the goals of an infection control program in any veterinary hospital should be the same; to limit the transmission of infection, reduce a patient's susceptibility to infection and increase individual's resistance to an infection. Identification of infectious or potentially infectious patients, reducing the risk of transmission of disease through the use of isolation and barriers, adequate environmental disinfection and appropriate antimicrobial use are some specific actions that are used to address these goals. These are all areas that require attention in community veterinary hospitals in southern Ontario and presumably in veterinary hospitals in most other regions. Yet, without understanding the epidemiology of HAIs including frequency of infections, agents associated with these infections, the types of infections seen and the determinants associated with these infections, the effect of these deficiencies on the occurrence of hospital-associated infections is unknown.
Acknowledgements
We would like to acknowledge and thank the following for their assistance with the study: Nicol Janecko, Alyssa Calder, Karlee Thomas, Andriana Sage, Gabriel Jantzi,
Virginia Young, Meredith Craig and the participating veterinary practices. This study was funded by the Ontario Veterinary College Pet Trust Fund and the Public Health
185 Agency of Canada. Colleen Murphy was a recipient of the Ontario Veterinary College
Graduate Student Fellowship.
186 References
American Animal Hospital Association (AAHA). http://www.aahanet.org/accreditation/index.aspx Accessed June 2007.
Benedict, KM, P S Morley, D C Van Metre. 2008. Characteristics of biosecurity and infection control programs at veterinary teaching hospitals. J Am Vet Med Assoc, v. 233, p. 767-73.
Berard, F, J Gandon. 1964. Postoperative wound infections: The influence of ultravioloet irradiation of the operating room and of various other factors. Ann Surg, v. 160(S), p.l-
192.
Cherry, B, A Burns, G S Johnson, H Pfeiffer, N Dumas, D Barrett, P L McDonough, M
Eidson. 2004. Salmonella Typhimurium outbreak associated with veterinary clinic.
Emerg Infect Dis, v. 10, p. 2249-51.
Coates, D. 1996. Sporicidal activity of sodium dichloroisocyanurate, peroxygen and glutaraldehyde disinfectants against Bacillus subtilis. J Hosp Infect, v. 32, p. 283-94.
College of Veterinarians of Ontario (CVO). 2006. Minimum standards for veterinary facilities in Ontario; Titles 1-11. http://www.cvo.org/uploadattachments/Minimumstandards2006.pdf. May 2007.
Dwyer, RM. 2004. Environmental disinfection to control equine infectious diseases. Vet
187 Clin North Am Equine Pract, v. 20, p. 531-42.
Elchos, BL, J M Scheftel, B Cherry, E E DeBess, S G Hopkins, J F Levine, C J Williams.
2008. Compendium of veterinary standard precautions for zoonotic disease prevention in
veterinary personnel. J Am Vet Med Assoc, v. 233, p. 415-32.
Eleraky, NZ, L N Potgieter, M A Kennedy. 2002. Virucidal efficacy of four new
disinfectants J Am Anim Hosp Assoc, v. 38, p. 231-4.
Fossum, T, C Hedlund, D Hulse, A Johnson, H Seim, M Willard, G Carroll. 1997. Small
Animal Surgery. Mosby-Year Book, Inc. St. Louis, Missouri.
Government of Ontario. Health Protection and Promotion Act-R.R.0 1990 Reg 567,
Rabies Immunization, http://www.search.e-laws.gov.on.ca/en/isysquery/b0cbc963-cc64-
4981-b95f-4eaee40c9451/l/frame/?search=browseStatutes&context= Accessed August
2007.
Government of Canada. 2007. Positive Rabies in Canada: Lab Submissions and Clinicals.
Canadian Food Inspection Agency. http://www.inspection.gc.ca/englisri/anima/heasan/disemala/rabrag/statse.shtml.
Accessed August 2007.
Government of Canada. 1999. Routine practices and additional precautions for preventing the transmission of infection in health care. CCDR, v. 25 S4.
188 Hernndez, A, E Martro, L Matas, M Martin, V Ausina. 2000. Assessment of in-vitro
efficacy of 1% Virkon against bacteria, fungi, viruses and spores by means of AFNOR
guidelines. J Hosp Infect, v. 46, p. 203-9.
Hospital Infection Control Practices Advisory Committee (HIPAC). 1996. Guideline for
isolation precautions in hospitals. Part II. Recommendations for isolation precautions in hospitals. Am J Infect Control, v. 24, p. 32-52.
Hughes, JM. 1987. Nosocomial infection surveillance in the United States: historical perspective. Infect Control, v. 8, p. 450-3.
Jimenez, L, M Chiang. 2006. Virucidal activity of a quaternary ammonium compound disinfectant against feline calicivirus: a surrogate for norovirus. Am J Infect Control, v.
34, p. 269-73.
Kennedy, MA, V S Mellon, G Caldwell, L N Potgieter. 1995. Virucidal efficacy of the newer quaternary ammonium compounds. J Am Anim Hosp Assoc, v. 31, p. 254-8.
Larson, E. 1997. A retrospective on infection control. Part 1: Nineteenth century- consumed by fire. Am J Infect Control, v. 25, p. 236-41.
Morley, PS. 2004. Surveillance for nosocomial infections in veterinary hospitals. Vet
Clin North Am Equine Pract, v. 20, p. 561-76.
189 Morley, PS, M D Apley, T E Besser, D P Burney, P J Fedorka-Cray, M G Papich, J L
Traub-Dargatz, J S Weese. 2005. Antimicrobial drug use in veterinary medicine. J Vet
Intern Med, v. 19, p. 617-29.
Murphy, CP, R J Reid-Smith, J F Prescott, B N Bonnett, C Poppe, P Boerlin, J S Weese,
N Janecko, S A McEwen. 2009a. Occurence of antimicrobial resistance in selected bacteria in healthy dogs and cats presented to private veterinary clinics in Southern
Ontario. Can Vet J. v.50, p.1047-53.
Murphy, CP, R J Reid-Smith, J S Weese, S A McEwen. 2009b. Evaluation of specific infection control practices used by companion animal veterinarians in community veterinary practices in southern Ontario. Zoonoses Public Health. Epub ahead of print.
Perez, J, V S Springthorpe, S A Sattar. 2005. Activity of selected oxidizing microbicides against the spores of Clostridium difficile: relevance to environmental control. Am J
Infect Control, v. 33, p. 320-5.
Rutala, WA. 1996. APIC guideline for selection and use of disinfectants. Am J Infect
Control, v. 24, p. 313-42.
Sattar, SA. 2004. Evaluation of the effectiveness of a 7% accelerated hydrogen peroxide- based formulation against canine parvovirus. Virox Technologies, Inc..
Schorr-Evans, EM, A Poland, W E Johnson, N C Pedersen. 2003. An epizootic of highly
190 virulent feline calicivirus disease in a hospital setting in New England. J Feline Med
Surg,v. 5, p. 217-26.
Scott, FW. 1980. Virucidal disinfectants and feline viruses. Am J Vet Res, v. 41, p. 410-
4.
Traub-Dargatz, JL, D A Dargatz, P S Morley, M Dunowska. 2004. An overview of infection control strategies for equine facilities, with an emphasis on veterinary hospitals.
Vet Clin North Am Equine Bract, v. 20, p. 507-20.
Vasseur, PB, H A Paul, L R Enos, D C Hirsh. 1985. Infection rates in clean surgical procedures: a comparison of ampicillin prophylaxis vs a placebo. J Am Vet Med Assoc, v. 187, p. 825-7.
Weese, JS, H Dick, B M Willey, A McGeer, B N Kreiswirth, B Innis, D E Low. 2006.
Suspected transmission of methicillin-resistant Staphylococcus aureus between domestic pets and humans in veterinary clinics and in the household. Vet Microbiol, v. 115, p. 148-
55.
Whittem, TL, A L Johnson, C W Smith, D J Schaeffer, B R Coolman, S M Averill, T K
Cooper, G R Merkin. 1999. Effect of perioperative prophylactic antimicrobial treatment in dogs undergoing elective orthopedic surgery. J Am Vet Med Assoc, v. 215, p. 212-6.
Wright, JG, S Jung, R C Holman, N N Marano, J H McQuiston. 2008. Infection control
191 practices and zoonotic disease risks among veterinarians in the United States. J Am Vet
Med Assoc, v. 232, p. 1863-72.
Wright, JG, L A Tengelsen, K E Smith, J B Bender, R K Frank, J H Grendon, D H Rice,
A M Thiessen, C J Gilbertson, S Sivapalasingam, T J Barrett, T E Besser, D D Hancock,
F J Angulo. 2005. Multidrug-resistant Salmonella Typhimurium in four animal facilities.
Emerg Infect Dis, v. 11, p. 1235-41.
192 Table 5.1. Clinical signs of gastrointestinal (GI) or respiratory disease, or infectious
diseases associated with GI or respiratory tract reported by veterinarians (n=100)a in
southern Ontario that would require isolation or other infection control measures.
Gastrointestinal Veterinarians (%) Respiratory Veterinarians (%)
Canine parvovirus 56 Feline respiratory 77
disease
Canine diarrhea 49 Canine kennel cough 39
Any patient with diarrhea 37 Canine respiratory 31
disease
Feline diarrhea 36 Any sneezing patient 20
Any vomiting patient 30 Any coughing patient 13
Feline panleukopenia 7 Canine distemper 12
Giardiasis 5 Feline calicivirus 11
Disease associated with 4 Ocular discharge 10
Clostridial species
Salmonellosis 4 Nasal discharge 2
Dietary indiscretion 2 Clinical signs of upper 1 associated diarrhea respiratory tract disease
Coccidiosis 1
Any test positive patients 1 for GI zoonoses aOne veterinarian did not respond to this question.
193 Table 5.2. Veterinarian-reported reasons for having appropriate environmental cleaning and disinfection protocols with their veterinary clinic.
Reason Percentage of veterinarians
(n=100)a
Reduce the transmission of zoonotic diseases 66
Reduce the transmission of any infectious disease 63
Overall esthetics of the veterinary clinic 54
Reduce the transmission of disease between 45
patients
Reduce the occurrence of surgical site infections 10
Decrease the microbial burden within the practice 8
Reduce the cross contamination between 7
equipment or areas
Compliance with American Animal Hospital 7
Association regulations
Decrease the occurrence of antimicrobial 4 resistance
Reduce liability 4
*One veterinarian did not respond to this question.
194 Table 5.3. The reported frequency of specific products3 used for environmental
disinfection by veterinarians (n=100) and veterinary technicians (n=101) in veterinary
clinics in southern Ontario.
Disinfectant Individual Tables Kennels Runs Floors ffiFe
QAC" Veterinarian (%) 51 52 39 32 14
Technician (%) 63 65 56 42 24
c H202 Veterinarian (%) 25 22 14 10 5
Technician (%) 36 34 25 23 11
Bleach Veterinarian (%) 3 5 6 11 22
Technician (%) 1 6 15 15 32
Virkon®d Veterinarian (%) 0 1 0 0 2
Technician (%) 0 1 1 2 1
Phenolics Veterinarian (%) 0 0 1 0 0
Technician (%) 0 0 1 1 0
Generic Veterinarian (%) 0 0 0 15 1
Cleaners
Technician (%) 1 2 5 27 1
Do not Veterinarian (%) 20 24 41 40 60 know
Technician (%) 0 0 8 3 40
"Including exclusive use or in combination with other products. QAC- Quaternary ammonium
C compound products. H202- Peroxiguard®: 7% accelerated hydrogen peroxide based formulation. dPeroxygen compounds. "Product used for environmental disinfection of areas contaminated by infectious body fluids (IBF).
195 Table 5.4.The frequency of the methods of preparation of disinfectants for environmental
disinfection reported by veterinarians (n=100) and veterinary technicians (n=101) from
veterinary practices in southern Ontario.
Site Individual Per label Clinic specific Do not know how disinfectants are
instructions dilution prepared
Tables Veterinarian (%) 12 8 81
Technician (%) 21 56 24
Kennels Veterinarian (%) 10 6 85
Technician (%) 18 40 43
Runs Veterinarian (%) 6 6 89
Technician (%) 8 29 53
Floors Veterinarian (%) 3 11 87
Technician (%) 9 45 47
IBF8 Veterinarian (%) 3 3 95
Technician (%) 0 6 95 eThe method of preparation of a product used for environmental disinfection of areas contaminated by infectious body fluids (IBF).
196 Table 5.5. Antimicrobials used in clean non-elective surgical procedures reported by
veterinarians (n=100) from veterinary clinics in southern Ontario.
Antimicrobial Veterinarians (%)
Cephalosporins8 39
Ampicillin 17
Short-acting (procaine) penicillin 11
Amoxicillin-clavulanic acid 9
Amoxicillin 8
Long-acting (benzocaine) penicillin 8
Clindamycin 5
Enrofloxacin 4
Orbifloxacin 2
Unspecified fluoroquinolone 2
Ampicillin-sulbactam 1
Gentamicin 1
Cephalexin n=24, cefazolin n=8, unspecified cephalosporin n=5, cephalexin and cefazolin n=l, cephalexin and cefadroxil n=l.
197 Chapter 6 Summary Discussion and Conclusions
This thesis describes investigations into various components of the epidemiology of
antimicrobial use and resistance pertaining to companion animals, specifically those
associated with community veterinary practices. The specific objectives were: 1) to
compare the incidence of antimicrobial resistance in generic fecal E. coli isolates and the
isolation of C. difficile, Salmonella enterica, blacuY-2 positive £. coli, MRS A, MRSP and vancomycin resistant Enterococcus spp. (VRE) from dogs treated with antimicrobials to untreated, healthy dogs; 2) to determine the associations between antimicrobial treatment and the risk of antimicrobial resistance in fecal E. coli isolates and the isolation of fecal
C. difficile, Salmonella enterica, blacMY-2 positive E. coli, MRS A, MRSP and VRE; 3) to describe non-topical antimicrobial (oral and parenteral) use in dogs and cats; 4) to compare antimicrobial use in study animals against published formulary doses (mg/kg) and frequencies of administration; 5) to evaluate antimicrobial use in feline upper respiratory tract disease, feline lower urinary tract disease and canine infectious tracheobronchitis (diseases where antimicrobial use generally is not indicated); 6) to determine the environmental recovery of E. coli, C. difficile, Salmonella enterica,
MRSA, MRSP, feline calicivirus and canine parvovirus from veterinary hospitals and; 7 ) to evaluate specific infection control practices in veterinary practices including environmental disinfection, management of infectious patients and antimicrobial use in clean surgical procedures. The objectives for this thesis were fulfilled.
The most notable finding in this research was the association between treatment with cephalexin and selection of antimicrobial resistance in fecal E. coli isolates from dogs to
198 amoxicillin-clavulanic acid, cefoxitin, ceftriaxone, and ceftiofur (Chapter 3). As
cephalexin was the most commonly administered antimicrobial in dogs treated as
outpatients from community veterinary hospitals (Chapter 4), this may provide the
opportunity for significant selection pressure for the occurrence of antimicrobial resistance. This selective pressure may increase the risk of antimicrobial resistant infections in animals and have an adverse impact on public health through transmission to humans of antimicrobial resistant zoonotic pathogens (e.g., Salmonella enterica, potentially some E. coli clones) and commensal bacteria bearing transferable resistant determinants. Further research is required to characterize the potential impacts on animal and public health.
Antimicrobial treatment with amoxicillin-clavulanic acid was associated with an increased incidence of colonization with C. difficile in dogs, including ribotypes found in humans with C. difficile infection. The commonality of ribotypes between dogs and humans suggests that both species may be colonized through common sources, such as food (Rodriguez-Palacios et al., 2007, Rodriguez-Palacios et al., 2009). These findings also raise the possibility that companion animals may play a role as reservoirs for inter species transmission of C. difficile to humans (or vice-versa). In humans, C. difficile- infection (CDI) is an important clinical syndrome, especially in at-risk populations, but the degree to which C. difficile is zoonotic is unknown. Further research is required to determine how antimicrobial use in one group (human or animal) may contribute to colonization or CDI in the other group.
Antimicrobial therapy is sometimes critical to the treatment and prevention of infectious diseases, but unfortunately such therapy may increase the risk of antimicrobial
199 resistance, as was observed here. Dogs and cats get bacterial infections that warrant
antimicrobial therapy, therefore, it is not feasible to remove all risk associated with use of
cephalexin or other antimicrobials commonly used in companion animals. Nevertheless,
the results suggest that steps can be taken that may slow the progression of resistance
problems in companion animal medicine. One of these is to limit antimicrobial use in
disease conditions where it is not normally required. The study evaluating antimicrobial
drug use by companion animal veterinarians (Chapter 4) described a seemingly higher than necessary frequency of use of antimicrobials to treat feline upper respiratory tract
disease, feline lower urinary tract disease and canine infectious tracheobronchitis.
Additionally, 24% of the veterinarians surveyed reported using antimicrobials for clean elective sterilization surgeries where antimicrobial use as surgical prophylaxis is generally not recommended (Chapter 5). These data suggest that antimicrobials may be over-used in companion animal practice, at least for these disease conditions or clinical situations. For example, in cats, antimicrobial use could be reduced by up to 25% if most treatments with antimicrobials for feline respiratory disease or feline lower urinary tract disease were discontinued. This is an opportunity to substantially reduce unnecessary antimicrobial use in companion animal practice. It might be going too far to describe some of these uses as imprudent; definitive assessment of prudence of treatment may require detailed case reviews, including objective and subjective data, some of which may be difficult to interpret or classify. Additional studies, such as randomized controlled clinical trials designed to investigate antimicrobial use in surgical patients, may also be required. However, improving antimicrobial use behaviours of veterinarians may be challenging. Factors such as marketing and "education" through pharmaceutical
200 companies, perceived need to fulfill client expectations, fear of a treatment failure
particularly for disease conditions where a veterinarian has habitually used
antimicrobials, and possible litigation or disciplinary action in case of treatment failure
are potential barriers to improving antimicrobial use. The potential for treatment failure
and possible sequelae are likely to be perceived as more tangible risks than the risks of
increasing the frequency of antimicrobial resistance or other adverse events associated
with antimicrobial therapy (e.g., toxicity, allergic/anaphylactic reactions). Therefore
veterinarians may err on what they perceive as the side of caution, and incorporate
antimicrobials in the treatment plan, even though their use in a particular case may be not required.
Another step that could be taken to improve antimicrobial use and reduce antimicrobial resistance selection pressure is increasing the frequency of bacterial culture and antimicrobial susceptibility testing. This is a reliable diagnostic tool that is available to assist veterinarians in determining the need for antimicrobial therapy and appropriate antimicrobial selection. In the study evaluating antimicrobial use by veterinarians
(Chapter 4), bacterial culture and antimicrobial susceptibility testing was performed in only 5% of the disease events in dogs and cats that were treated with antimicrobials. This frequency seems very low and suggests poor compliance with common recommendations regarding prudent use of antimicrobials. As one contributing factor, veterinarians may have simply considered it more expedient to immediately prescribe the antimicrobials that they considered most likely to be clinically effective. Other factors could also have contributed, including reluctance by owners to pay the extra costs of testing, length of time associated with testing, difficulty in obtaining a suitable diagnostic sample from
201 certain body sites (e.g., pulmonary tree, oral cavity), or inability to collect a diagnostic
sample at the time of presentation (e.g., urine). Investigations into these factors and perceptions of veterinarians and pet owners on the utility of bacterial culture and
antimicrobial therapy would be useful. As use of bacterial culture and antimicrobial
susceptibility testing is a universal recommendation in prudent use guidelines, understanding the factors associated with its use (or lack of use) as a diagnostic tool is important to developing interventions that may improve its uptake.
Also important in minimizing the occurrence of antimicrobial resistance is improving the representativeness of culture and susceptibility data reported by diagnostic laboratories. These data can be reported qualitatively (e.g., susceptible, intermediate or resistant (SIR)) or quantitatively with minimum inhibitory concentrations (MIC) (e.g., exact MIC or breakpoints), or measurements of the zone of inhibition (e.g., disc diffusion) that may or may not include a qualitative interpretation (e.g., SIR). Although many veterinarians may prefer the simplicity of SIR reporting, MICs are preferable as these data can be used by veterinarians to optimize therapy and possibly reduce the occurrence of antimicrobial resistance by combining the MIC data with the pharmacokinetic/pharmacodynaniic properties of an antimicrobial. Practically, veterinarians could use these data to optimize the dosing (mg/kg) of concentration- dependant antimicrobials (e.g., fluoroquinolones, aminoglycosides) or the frequency of administration of time-dependant antimicrobials (e.g., p-lactams, trimethoprim sulphonamide combinations). MIC data could also be useful for determining whether a concentration-dependant or time-dependant antimicrobial would be most appropriate, especially when considering factors like the mutant selection window or mutant
202 prevention concentration. However, understanding and using only quantitative
antimicrobial susceptibility data (without a quantitative interpretation) is perhaps beyond
the present-day capabilities of most veterinarians in community practice, and proper
interpretation probably requires input from microbiologists, pharmacologists, pharmacists
and clinical specialists. Unfortunately, this integration of is not yet widely available.
Although prudent use of antimicrobials is important to minimizing the selective
pressure of antimicrobials on resistance, it is also important to prevent or reduce the
frequency of infections in order to reduce the need for antimicrobial treatment in the first place, and this is an important purpose of hospital-based infection control practices. The findings of this thesis indicate that the veterinary hospital environment is a possible reservoir of potential pathogens, including zoonotic pathogens, in veterinary practices
(Chapter 2) and that factors related to environmental disinfection may need improvement
(Chapter 5). In human medicine, environmental sites, generally, are not considered important sources of hospital-associated infections (CDC 2003). However, the management, housing and behaviours of human patients are obviously very different from those of animal patients in veterinary hospitals. Normal exploratory behaviours of animals (e.g., sniffing) and, at times, poor hygiene (e.g., defecation on floors, in kennels, etc.) could place animals at risk of acquiring nosocomial infections. Further research is required to understand the contribution of environmental reservoirs in veterinary hospitals to hospital-associated infections in companion animals and people. Quantifying the role or determining the association between hospital-associated infections and environmental organisms (if any) is challenging since environmental contamination may
203 fit into many places within an epidemiological causal model of factors associated with
hospital-associated infections (HAIs).
At this time the contribution (if any) of environmental organisms to the
epidemiology of hospital-associated infection in companion animals is not well understood. Previously, outbreaks of hospital-acquired infections with a documented environmental reservoir have been reported (Boerlin et al., 2001, Sanchez et al., 2002,
Weese and Armstrong 2003); however, it is not understood whether the environmental reservoirs were associated with these outbreaks.
As antimicrobial use selects for antimicrobial resistance, so does hospitalization contribute to hospital-associated infections. Similarly, as imprudent use of antimicrobials contributes to excess occurrences of antimicrobial resistance, so do deficiencies in infection control practices contribute to excess hospital-acquired infections. In Chapter
5, study veterinarians described some of the components of an infection control program in their practices, however none had a defined, formal infection control program. This study also showed that improvements are needed in use of isolation facilities and in selection of environmental disinfectant products. It is not known whether these possible deficiencies contributed to the observed environmental reservoir of bacteria, or were associated with hospital-associated infections.
In human medicine, there is a perceived crisis concerning antimicrobial resistance among some hospital-associated infections, such as MRSA and VRE (Mulvey and Simor
2009). In contrast, in companion animal medicine there is not yet a perceived crisis.
However, with emerging pathogens, including methicillin-resistant Staphylococcus pseudintermedius (Weese and van Duijkeren, 2009), blacrx-u (Moreno et al., 2008,
204 Pomba et al., 2009) and blacuY-2 positive E. coli (Garcia-Femandez et al., 2008), and
zoonoses like methicillin-resistant Staphylococcus aureus (Weese and van Duijkeren,
2009), antimicrobial resistance is clearly an important animal and public health concern.
This research described a number of factors or component causes that may contribute to
the epidemiology of antimicrobial resistance in companion animal practice. One potentially important factor is the effect of antimicrobial therapy by companion animal
veterinarians on the selection of resistance in a variety of pathogens and commensals. A
second is the existence of an environmental reservoir of potential pathogens in veterinary hospitals, including antimicrobial resistant pathogens that may contribute to hospital- associated infections in animals and humans. In addition, there are clearly issues related to infection control in veterinary hospitals that need improvement in order to decrease hospital-associated infections and thereby decrease the need for antimicrobial drug use.
Further studies are required to better elucidate these factors, in particular to more thoroughly determine the effect of antimicrobial drug use in companion animals on antimicrobial drug resistant infections (companion animal and human), and to characterize and quantify imprudent use of antimicrobials by companion animal veterinarians and the contributions of imprudent use to antimicrobial resistance. More research is also required on methods to improve use by veterinarians of bacterial culture and antimicrobial susceptibility testing and to determine the type, frequency and risk factors for hospital-acquired infections, in particular the preventable fraction of these infections. It is also very important to provide the evidentiary base for infection control measures that reduce hospital-associated infections (companion animal and human) in veterinary hospitals.
205 References
Boerlin, P, S Eugster, F Gaschen, R Straub, P Schawalder. 2001. Transmission of
opportunistic pathogens in a veterinary teaching hospital Vet Microbiol, v. 82, p. 347-59.
Centers for Disease Control and Prevention (CDC). 2003. Recommendations of CDC and
the healthcare infection control practices advisory committee (HICPAC).
http://www.cdc.gov./ncidod/dhqp/gl_environinfection.html. Accessed February, 2009.
Christianson, S, G R Golding, J Campbell, M R Mulvey. 2007. Comparative genomics of
Canadian epidemic lineages of methicillin-resistant Staphylococcus aureus. J Clin
Microbiol, v. 45, p. 1904-11.
Garcia-Fernandez, A, G Chiaretto, A Bertini, L Villa, D Fortini, A Ricci, A Carattoli.
2008. Multilocus sequence typing of Incll plasmids carrying extended-spectrum beta-
lactamases in Escherichia coli and Salmonella of human and animal origin. J Antimicrob
Chemother, v. 61, p. 1229-33.
Government of Canada. 2008. Canadian Integrated Program for Antimicrobial Resistance
Surveillance (CIPARS) 2007- Preliminary Results. Public Health Agency of Canada.
Martin, H, B Willey, D E Low, H R Staempfli, A McGeer, P Boerlin, M Mulvey, J S
Weese. 2008. Characterization of Clostridium difficile strains isolated from patients in
Ontario, Canada, from 2004 to 2006. J Clin Microbiol, v. 46, p. 2999-3004.
206 Appendices Appendix A: Additional Documents Appendix A.2.1 Dear:
The Departments of Population Medicine, Clinical Studies, and Pathobiology of the Ontario Veterinary College are undertaking a study investigating the dispensing patterns of prescription medications by small animal veterinarians in Ontario. We would like to know if you have any interest in participating in two of them.
Last summer we contacted you looking for participation in a research study investigating the effect of antimicrobial therapy on the occurrence of antimicrobial resistance and carriage of specific pathogens like Clostridium difficile. We, again, are looking for your participation in this project. As you know, antibiotic/antimicrobial resistance (AMR) and Clostridium difficile are major problems in human medicine and of increasing concern in veterinary medicine. In 2002 we looked at AMR in healthy dogs and cats with no recent exposure to antimicrobials (your practice may have participated). Although the prevalence of AMR in fecal E. coli cultured fromdog s and cats was relatively uncommon compared to food animals, we did find multidrug-resistant strains, including strains resistant to eight antimicrobials, and strains confering resistance to extended- spectrum cephalosporins. These findings were concerning since these animals had not had recent exposure to antimicrobials. AMR was associated with various risk factors, including strong association with past treatment (greater than six weeks in the past) with antimicrobials including enrofloxacin, injectable penicillin given for surgical prophylaxis (e.g., spay, neuters), ampicillin, cephalexin and amoxicillin-clavulanic acid. In order to further clarify the epidemiology of AMR and C. difficile in companion animals, we are investigating the immediate and short-term effects of treatment with these antimicrobials on the development of AMR and colonization by specific pathogenic bacteria including C. difficile.
Your Role: We need serial fecal samples for culture & sensitivity from a sample of patients that you prescribe or treat with the antimicrobials being studied, and from controls not treated with antimicrobials. We will NOT influence your treatment of patients. Owners of eligible patients, identified and enrolled by your clinic, will be asked for written permission to include their dog or cat in the study, and for us to collect some information from the patient's medical records. They will also be given a brief questionnaire. All identifiable individual client/patient/clinic information will be kept confidential. Only aggregated or non-identified data will be published. Personal Information Privacy and Electronic Documents Act (PIPEDA) regulations will be observed. Once enrolled, clients will collect 5 fecal samples from their pet during and after the antimicrobial treatment period. We expect that each participating clinic will only have to enroll 5-10 patients and controls over the course of 12-18 months, so your time commitment for this project would be minimal.
207 2. The other study that may interest you, is an investigation of nosocomial agents and disinfection protocols in general practice. Recent studies have identified veterinary hospitals as a reservoir of bacterial and viral pathogens, including Salmonella, multiple-drug resistant E. coli and feline calicivirus (FCV). In one case, an outbreak of virulent, systemic FCV with a high mortality rate was associated with a nosocomial transmission n a veterinary hospital, with transfer from the hospital to client homes. There have also been several recent veterinary hospital associated outbreaks of salmonellosis affecting patients, staff, and, in a few cases, clients. Nosocomial infections are well understood in human medicine, but not in companion animal medicine. In human medicine, the risk of a nosocomial infection is associated with prolonged hospitalization and invasive procedures. Advances in veterinary medicine have lead to the treatment of companion animals who in the past would have been euthanized. Procedures such as intravenous and urinary catheterization, chemotherapy and the use of advanced broad-spectrum antimicrobials place our patients at risk of nosocomial infection. In order to better understand nosocomial infection in general practice we have undertaken a study of the occurrence of selected veterinary and zoonotic pathogens in private and referral companion animal practices, and evaluation of their disinfection procedures.
Your Role: We will visit your clinic and conduct interviews on cleaning and disinfection practices with your and your staff. We will also test the practice facility for the presence of nosocomial agents. You will receive a confidential report on pathogens recovered, and an evaluation of cleaning and disinfection practices, with recommendations for change if any are required. The resulting study information will be used to generate continuing education materials (e.g., CD, manual) on cleaning and disinfection of veterinary practices.
If you would be willing to consider participation in ONE study or BOTH studies, please complete the attached brief questionnaire and return it by fax [(519) 763-3117 Attention Alyssa Calder] before May 27,2005. We will use this information to assemble the cross-section of practices we need for the study. If your practice fits the study profile, we will contact you by phone. At that time, we can give you more details about the project and vou can decide whether or not you wish to participate. Clinic visits and sample collection will take place between June 2005 and Dec. 2006.
Please do not hesitate to contact me by email at [email protected] or phone at (613) 961- 7466 if you have any questions. Thank you for your time and consideration of these studies. Sincerely,
Colleen Murphy DVM MSc For Drs. Scott McEwen, Richard Reid-Smith, Scott Weese, Patrick Boerlin and John Prescott
These studies are funded by OVCPet Trust and the Public Health Agency of Canada.
208 Please return by fax to Alyssa Calder 519-763-3117 by May 27 2005.
Name of Veterinary Practice:
Phone Number:
Contact Individual (Job Title):
Please check ONE:
Q YES, We are interested in participating in the antimicrobial resistance IC. difficle project. Please complete and return pages 3-5 as well
Q YES, We are interested in participating in the nosocomial pathogens/disinfection procedures project. Please complete and return Pages 3-5 as well
Q NO, We are not interested in participating in either research project
Remember, by selecting YES to any of the above, you are NOT committing to participation. We will contact you and discuss the details of each study further. You can then decide whether you wish to participate.
209 Clinic Questionnaire Individual clinic responses to this questionnaire are confidential; aggregated data may be used for analysis or presented.
1. Number of FULL-TIME Veterinarians:
2. Number of PART-TIME Veterinarians:
3. Number of FULL-TIME Veterinary Technicians (RVT, ACT, "in-house" trained etc.):
4. Number of PART-TIME Veterinary Technicians (RVT, ACT, "in-house" trained etc.):
5. How often does your practice prescribe to DOGS the following oral antimicrobials?
Amoxicillin-clavulanic acid (circle one) Daily Weekly Monthly Never
Fluoroquinolones (circle one) Daily Weekly Monthly Never (e.g., Baytril®™, Orbax®™, Zeniquin*™) 1st Generation cephalosporins Daily Weekly Monthly Never (e.g., cephalexin, cefadroxil)
6. How often does your practice prescribe to CATS the following oral antimicrobials?
Amoxicillin-clavulanic acid (circle one) Daily Weekly Monthly Never
Fluoroquinolones (circle one) Daily Weekly Monthly Never (e.g., Baytril®™, Orbax®™, Zeniquin®™)
1st Generation cephalosporins Daily Weekly Monthly Never (e.g., cephalexin, cefadroxil)
7. How often does your practice use ceftiofur(Excenel®)? Daily Weekly Monthly Never
8. a) Does your practice routinely use injectable penicillin for routine8 CANINE spays and castrations? YES NO
"A routine spay or castration is defined as a surgery performed on a young (less than 2 years old) dog or cat who has never had a litter (iffemale) and without concurrent disease at the time of surgery.
b) If YES, do ALL veterinarians in your practice use injectable penicillin for routine* CANINE spays and castrations? YES NO
c) If NO to a., does your practice routinely use another injectable antimicrobial routine8 CANINE spays and castrations? YES NO Please specify:
210 9. a) Does your practice routinely use injectable penicillin for routine* FELINE spays and castrations? YES NO
b) If YES, do ALL veterinarians in your practice use injectable penicillin for routine8 FELINE spays and castrations? YES NO
c) If NO to a., does your practice routinely use another injectable antimicrobial for routine" FELINE spays and castrations? YES NO Please specify:
10. At your practice, routine8 CANINE SPAYS are discharged (select one) a. the same day as the surgery is performed. b. the day after the surgery is performed. c. other (specify) d. depends on the veterinarian performing the surgery.
11. At your practice, routine8 CANINE CASTRATIONS are discharged (select one) a. the same day as the surgery is performed. b. the day after the surgery is performed. c. other (specify) d. depends on the veterinarian performing the surgery
12. At your practice, routine8 FELINE SPAYS are discharged (select one) a. the same day as the surgery is performed. b. the day after the surgery is performed. c. other (specify) d. depends on the veterinarian performing the surgery.
13. At your practice, routine8 FELINE CASTRATIONS are discharged (select one) a. the same day as the surgery is performed. b. the day after the surgery is performed. c. other (specify) d. depends on the veterinarian performing the surgery.
"A routine spay or castration is defined as a surgery performed on a young (less than 2 years old) dog or cat who has never had a litter (iffemale) and without concurrent disease at the time of surgery.
14. How many EXAM rooms does your clinic have?
15. How many KENNEL rooms does your clinic have (including canine, feline, patient, isolation, boarding, grooming, adoption etc- DO NOT include runs)?
16. Are your PATIENT kennel rooms separate from boarding, grooming, adoption? (circle one) YES NO
a) If YES, how many PATIENT KENNEL rooms does your clinic have?
211 17. Are your FELINE and CANINE PATIENT kennel rooms separate? (circle one) YES NO
a) If YES, how many CANINE PATIENT kennel rooms does your clinic have?
b) If YES, how many FELINE PATIENT kennel rooms does your clinic have?
18. Does your clinic have a designated ISOLATION ward? (circle one) YES NO 19. How many CANINE runs does your clinic have (including patient, isolation, boarding, grooming, adoption etc)?
20. Are your PATIENT runs separate from isolation, boarding, grooming, adoption? (circle one) YES NO 21. How often does your clinic see pocket pets (rodents, rabbits, hedgehogs)? (circle one) Daily Weekly Monthly Never 22. How often does your clinic see reptiles? (circle one) Daily Weekly Monthly Never 23. Are you aware of any of your clients feeding raw food diets (e.g., BARF), either commercial or home made, to dogs or cats? YES NO
If you have any questions or comments, please feel free to list them below or contact me at: [email protected] (613)961-7466
Questions/Comments:
Thank you for your input! Colleen Murphy DVM MSc (PhD Candidate)
212 Appendix A.2.2
QUESTIONNAIRE Environmental recovery and antimicrobial susceptibility of selected veterinary and zoonotic pathogens, and evaluation of disinfection procedures in private and referral companion animal veterinary practices in Ontario.
VETERINARIAN COPY
213 All questions refer to environmental cleaning and disinfection unless otherwise stated.
1) Name of Hospital
2) Number of Veterinarians
Full-time
Part-time
3) Number of Veterinary Technicians (RVT, ACT, in-house trained etc.)
Full-time
Part-time
4) Number of additional staff (reception, office, kennel etc.)
Full-time
Part-time
5) Is your practice:
0. Companion Animal only 1. Mixed Animal 6) How many hospitalized CANINE patients (not including boarding, grooming etc.) does your practice have on normal day?
7) How many hospitalized FELINE patients (not including boarding, grooming etc.) does your practice have on normal day?
8) How many CANINE appointments does your practice have on a normal day?
9) How many FELINE appointments does your practice have on a normal day?
214 10) How recently have you had the following species hospitalized in your practice?
-0- -1- -2- -3- -4- -5- -6- -7- Species Never Greater 6-12 Within Within the Within the Within the Daily than 12 months the last last 3 last month last week months ago 6 months ago months
Food animals (please detail) Pet birds Pet reptiles Pet rodents Pet rabbits Other pet exotics (please detail)
Wild birds Other wild animals (please detail)
215 11) Does your practice provide grooming services?
0. NO (proceed to QUESTION 13) 1. YES (proceed to QUESTION 12)
12) If YES, are the healthy grooming animals kennelled separately from the hospitalized patients (please select one)?
0. YES- healthy grooming clients have a designated kennel area, separate from hospital patients and the boarding and adoption kennels
1. YES- healthy grooming clients have a designated kennel area separate from hospital patients. However the kennels are shared with the boarding and/or adoption kennels.
2. NO- the grooming kennels are shared with the hospital patients.
3. Other (please describe)
13) Does your practice provide boarding services?
0. NO (proceed to QUESTION 15) 1. YES (proceed to QUESTION 14)
14) If YES, are the healthy boarding animals kennelled separately from the hospitalized patients (please select one)?
0. YES- healthy boarding animals have a designated kennel area, separate from hospital patients and the grooming and adoption kennels
1. YES- healthy boarding clients have a designated kennel area separate from hospital patients. However the kennels are shared with the grooming and/or adoption kennels.
2. NO- the boarding kennels are shared with the hospital patients.
3. Other (please describe)
216 15) Does your practice provide adoption services?
0. NO (proceed to QUESTION 17) 1. YES (proceed to QUESTION 16)
16) If YES, are the healthy adoption animals kennelled separately from the hospitalized patients (please select one)?
0. YES- healthy adoption animals have a designated kennel area, separate from hospital patients and the grooming and boarding kennels
1. YES- healthy adoption animals have a designated kennel area separate from hospital patients. However the kennels are shared with the grooming and/or boarding kennels.
4. NO- the adoption kennels are shared with the hospital patients.
5. Other (please describe)
17) Please rate the importance of using appropriate cleaning and disinfection protocols in your practice (please circle one).
12 3 4 not important low moderate high importance
18) List up to three reasons why it is important to use appropriate cleaning and disinfection protocols in your practice.
1) 2) 3)
217 19) Please list up to five pathogens (bacterial, viral, parasitic), or diseases that you consider significant to control to reduce the risk of nosocomial transmission within your practice.
1) 2) 3) 4) 5)
20) Please rate your knowledge of proper hand washing technique (not surgical scrub technique) for cleaning and sanitation (please circle one).
12 3 4 no knowledge low moderate excellent knowledge
21) Please list the hand cleansers or sanitizers used by your practice (not including those used exclusively for surgical scrub). 1. 2. 3. 4. 5.
22) Please describe the hand washing technique used at your practice for routine cleaning and sanitation (not including surgical scrub). Please be as specific as possible including products, contact times, water temperature, type of towel for drying etc.
218 23) Does your practice have a written documents) outlining the cleaning and disinfection of areas (i.e. exam rooms, kennels, tables, reception) of your practice? (circle one)
0. YES (if YES, obtain a copy of the document(s) if possible) 1. NO
24) Does your practice have a written documents) outlining the cleaning and disinfection of equipment and tools (not including sterilization) (i.e. thermometers, otoscopes, stethoscopes, endoscopes, ultrasound probes) of your practice? (circle one)
0. YES (if YES, obtain a copy of the document(s) if possible) l.NO
25) Does your practice have a written documents) outlining the sterilization of equipment and tools (i.e. surgical instruments, dental tools) of your practice? (circle one)
0. YES (if YES, obtain a copy of the document(s) if possible) l.NO
26) Does your practice have a written documents) outlining hand washing technique (not including surgical scrub) and protocol? (circle one)
0. YES (if YES, obtain a copy of the document(s) if possible) 1. NO
219 27) Who is mainly responsible for the routine cleaning of specific areas of the ractice? (please check all that apply)
-0- -1- -2- -3- -4- -5- Veterinary Cleaning Veterinarian Fonnally trained Informally The most Technician Service (using specific trained, non convenient (including RVT, protocols), non medical individual or ACT, in-house medical personnel personnel no trained) (i.e. students, (i.e. students, specifically groomer) groomer, designated receptionist) personnel Exam Rooms (including tables, equipment, floor etc.)
Treatment Area (including tables, equipment, kennels etc.)
Surgery Room
Kennel Room (including kennels, counter, floor etc.)
Runs
Boarding Kennels or Room
Isolation Kennels or Room
Lab Area
Reception Area
Office Area
Kitchen Area
220 28) What products do you use and how are these used for routine cleaning/disinfection (when no gross feces, blood etc. is present) of specific hospital areas? (If more than product is used or more than one method in an individual area, please describe on the back). Be as specific as possible.
Method of Application Method of Removal (i.e. Other Preparation for Use (i.e. spray bottle, Contact Product single use paper towel, air (please detail) (i.e. specific dilution) pressure washer at Time dry) specific psi, mop)
Exam Room Tables
Exam Room Counters
Exam Room Floor
Treatment Room Tables
Treatment Room Counters
Treatment Room Floor
Surgery Table
221 Method of Application Method of Removal (i.e. Other Preparation for Use (i.e. spray bottle, Contact Product single use paper towel, air (please detail) (i.e. specific dilution) pressure washer at Time dry) specific psi, mop)
Hospitalization Kennels
Kennel room counters
Kennel room floors
INDOOR Runs (if present)
OUTDOOR Runs (if present)
Boarding and grooming kennels
Laboratory Workspace
222 29) Do you have different protocols for cleaning and disinfection when blood, feces, respiratory secretions or other body fluids are present?
0. YES (if YES, please complete Question 34) 1. NO (move to Question 35)
30) Please describe in detail the changes in cleaning and disinfection procedures for each area when blood, feces or other body fluids are present. If the protocol changes for a specific contaminant (i.e. feces versus blood), please detail as well. If more room is required, please use back or attach a separate sheet. Be as specific as possible.
No Modifications from above change Exam Room Tables
Exam Room Counters
Exam Room Floor
Treatment Room Tables
Treatment Room Counters
Treatment Room Floor
Surgery Table
Hospitalization Kennels
Kennel room counters
Kennel room floors
Runs (if present)
Boarding and grooming kennels
Laboratory Workspace
223 31) Does your practice have a separate isolation area (separate from hospital, boarding and grooming kennels) used for animals that have a suspected or confirmed infectious agent (not including non-vaccinated animals)?
0. YES ( please complete QUESTION 36) 1. NO (proceed to QUESTION 37)
32) Is your isolation area ONLY used for animals with a suspected or confirmed infectious agent? (proceed to QUESTION 38)
0. YES 1. NO
33) If your practice has no separate isolation area, please describe what measures are taken when an animal with suspected or confirmed infectious agent is hospitalized (not including non-vaccinated animals).
224 34) Please list infectious agents or specific clinical signs that would result in an animals being placed in isolation or have special measures taken when no isolation area is available, (please check all that apply and do not prompt the interviewee)
a Bordetella bronchiseptica a Feline calicivirus a Clostridium associated diarrhea a Coccidia a Canine distempter virus a Cats with feline immunodeficiency virus • Cats with feline leukemia virus • Fleas • Giardia a Leptospirosis a Nosocomial acquired infections a Canine parvovirus • Feline parvovirus (panleukopenia/feline distemper) a Rabies a Ringworm a Salmonella associated diarrhea a Uninvestigated dermatological lesions • Dogs with uninvestigated diarrhea a Cats with uninvestigated diarrhea a Dogs with uninvestigated respiratory infections • Cats with uninvestigated respiratory infections a Cats with upper respiratory infection a Unvaccinated animals a Other (please describe)
225 39. Does your practice routinely use antimicrobials for elective sterilization surgery (spays, neuters)?
0-No (Proceed to Question 41) 1-Yes (Proceed to Question 40)
40. If YES, please list the antimicrobials that are routinely used.
41. Does your practice routinely use antimicrobials for non-elective clean surgeries (lumps, orthopedic procedures)?
0-No (Proceed to Question 43) 1-Yes (Proceed to Question 42)
42. If YES, please list the antimicrobials that are routinely used.
43. Does your practice routinely use antimicrobials for clean/contaminated or contaminated surgeries?
0-No (Proceed to Question 45) 1-Yes (Proceed to Question 44)
44. If YES, please list the antimicrobials that are routinely used.
45. What injectable antimicrobials do you use for in-hospital use (please list)?
46. What oral antimicrobials do you use for in-hospital use (please list)?
226 Appendix A.2.3
QUESTIONNAIRE Environmental recovery and antimicrobial susceptibility of selected veterinary and zoonotic pathogens, and evaluation of disinfection procedures in private and referral companion-animal veterinary practices in Southern Ontario.
TECHNICIAN COPY
227 35) Who is mainly responsible for the routine cleaning of specific areas of the practice? (please check all that apply)
-0- -1- -2- -3- -4- -5- Veterinary C leaning Veterinarian Formally trained Informally trained, The most Technician Service (using specific non medical convenient (including protocols), non personnel individual or RVT, ACT, in- medical personnel (i.e. students, no house trained) (i.e. students, groomer) specifically groomer) designated personnel Exam Rooms (including tables, equipment, floor etc.)
Treatment Area (including tables, equipment, kennels etc.)
Surgery Room
Kennel Room (including kennels, counter, floor etc.)
Runs
Boarding Kennels or Room
Isolation Kennels or Room
Lab Area
Reception Area
Office Area
Kitchen Area |
228 36) What products do you use and how are these used for routine cleaning/disinfection (when no gross feces, blood etc. is present) of specific hospital areas? (If more than product is used or more than one method in an individual area, please describe on the back). Be as specific as possible.
Method of Application Method of Removal (i.e. Other Preparation for Use (i.e. spray bottle, Contact Product single use paper towel, air (please detail) (i.e. specific dilution) pressure washer at Time dry) specific psi, mop)
Exam Room Tables
Exam Room Counters
Exam Room Floor
Treatment Room Tables
Treatment Room Counters
Treatment Room Floor
Surgery Table
229 Method of Application Method of Removal (i.e. Other Preparation for Use (i.e. spray bottle, Contact Product single use paper towel, air (please detail) (i.e. specific dilution) pressure washer at Time dry) specific psi, mop)
Hospitalization Kennels
Kennel room counters
Kennel room floors
INDOOR Runs (if present)
OUTDOOR Runs (if present)
Boarding and grooming kennels
Laboratory Workspace
230 37) Do you have different protocols for cleaning and disinfection when blood, feces, respiratory secretions or other body fluids are present?
0. YES (if YES, please complete Question 28) 1. NO (move to Question 29)
38) Please describe in detail the changes in cleaning and disinfection procedures for each area when blood, feces or other body fluids are present. If the protocol changes for a specific contaminant (i.e. feces versus blood), please detail as well. If more room is required, please use back or attach a separate sheet. Be as specific as possible. No Modifications from above change Exam Room Tables
Exam Room Counters
Exam Room Floor
Treatment Room Tables
Treatment Room Counters
Treatment Room Floor
Surgery Table
Hospitalization Kennels
Kennel room counters
Kennel room floors
Runs (if present)
Boarding and grooming kennels
Laboratory Workspace
231 5) Please list the hand cleansers or sanitizers used by your practice (not including those used for surgical scrub). 1. 2. 3. 4. 5.
Please describe the hand washing technique used at your practice for routine cleaning and sanitation (not including surgical scrub). Please be as specific as possible including products, contact times, water temperature, type of towel for drying etc.
232 ID label Appendix A.3.1
Antibiotic Resistance Study: Companion Animals Client Survey
233 Owner Questionnaire
1) Name Only enter pet's name not surname Pet's Name Your Surname
2) Pet's Age Do not enter years months weeks (please circle one)
3) Pet's Breed
2) Sex of your Pet:(please choose one)
Male Male Neutered Female Female Spayed
5) Do you breed dogs at your home? YES NO
6) Are there other pets in the home? YES NO If Yes, please check all that apply and indicate the number of additional pets. Other Dogs (number) Cats (number) Other (number) (number)
234 Diet 7) What is the main daily diet of this pet (excluding treats) (please choose all that apply)? Commercial (kibble, moist, semi-moist) dog food Homemade diet as directed by my veterinarian Homemade "BARF" diet Commercial "BARF" diet Other (please describe)
8) Do you feed this pet treats in addition to the main diet? YES NO If Yes, please answer the following. If No, please proceed to Question 10 (pg 4)
9) Do you feed the following treats?(please circle all that apply)
Commercial "dog" biscuits NO YES
Homemade, cooked treats NO YES
Homemade, raw meat treats NO YES
Table scraps NO YES
Pig ear treats NO YES
Rawhide treats NO YES
Pet store-purchased beef bones NO YES
Pet store purchased pork bones NO YES
Other treats (please describe)
235 Hunting
10) Does this pet hunt and catch or eat prey (e.g., mice, rodents, rabbits, squirrels etc)? YES NO 11) Does your pet eat its own feces? YES NO 12) Does your pet eat the feces of other animals (e.g, livestock, wildlife, cats)? YES NO
Water 13) What is the primary source of water for this pet Municipal tap water Well tap water Filtered municipal tap water (e.g., Brita, in-line filter) Filtered well tap water (e.g., Brita, in-line filter) U-V filtered well tap water Bottled water Other- please describe 14) Does this pet drink regularly out of the toilet? YES NO
15) In the previous 6 weeks, did your pet have access to these water sources either for drinking or swimming?(please circle all that apply)
Lakes, rivers or creeks YES NO Water in ditches, puddles YES NO
236 Raw Meat 16) Has this pet ever consmed Raw Meat within the last year (excluding wild caught animals)? YES NO If Yes, please answer the following. If No, please proceed to Question 18 (pg 5)
17) Which type of raw meat has your pet consumed? (Please check all that apply) Raw Chicken YES NO Raw Beef YES NO Raw Pork YES NO Other "non-game" raw meat (please describe)
Bones
18) Has this pet ate raw butcher or meat bones (not purchased from a pet store) within the last year? YES NO If Yes, please answer the following. If No, please proceed to Question 20 (pg6)
19) Which kind of raw bone has your pet ate? (Please check all that apply)
Raw Chicken YES NO Raw Beef YES NO Raw Pork YES NO Other type of raw bone
237 20) Has this pet had contact with livestock within the last year? YES NO If Yes, please answer the following. If No, please proceed to Question 22 (pg6)
21) Which type of livestock has your pet had contact with?
Cattle YES NO
Pigs YES NO
Chickens YES NO
Turkeys YES NO
Horses YES NO
Sheep YES NO
Goats YES NO
Other (please describe)
22) Within the last year, had your dog been a "therapy" dog (e.g., visits to nursing homes, hospitals, schools)? YES NO
238 Appendix A.4.1.
Dear Veterinarian:
The Departments of Population Medicine, Clinical Studies, and Pathobiology of the Ontario Veterinary College are undertaking a study investigating the use of selected prescription medications by small animal veterinarians in Ontario. We would like to know if you have any interest in participating. The use of prescription pharmaceuticals is critical to the maintenance of health and management of illness in our patients. Medications such as insulin offer longevity and quality of life and antibiotics manage or treat infectious diseases that otherwise could kill. However, in veterinary medicine there is little published data that provides insight into how veterinarians use these medications in practice, and the conditions that are treated or managed by these drugs. Your Role: We need veterinarians to complete a "prescription journal" approximately once a month for one year. The prescription journal will gather simple data on the new prescriptions that you dispense or prescribe on your journal days. No identifying patient or client information will be collected, and all veterinarian and veterinary clinic information will be strictly confidential. It is anticipated that it will take approximately 30-60 minutes to complete the journal, and the study will take place over 12 months. Therefore, every veterinarian in the study will complete approximately 12 journals (once monthly) for the duration of the study. We are mainly collecting data on prescription medications dispensed to out-patients, such as antimicrobials, antiparasitics, medications for treatment of metabolic conditions (i.e. hypo/hyperthyroidism), and analgesics. We will not be collecting data on vaccinations, medications for anesthesia or fluid therapy. Please complete the attached brief questionnaire and return it by fax [(519) 763-3117 Attention Nicol Janecko before Friday June 29, 2007. All information gathered is confidential and only aggregated data will be analyzed or presented. We will use this information to assemble the cross- section of veterinarians we need for the study. If your information fits the study profile, we will contact you by phone. At that time, we can give you more details about the project and vou can decide whether you wish to participate.
Please do not hesitate to contact me by email at [email protected] or phone at (613) 961- 7466 if you have any questions. Thank you for your time and consideration of this study.
Sincerely,
Colleen Murphy DVM MSc (PhD candidate) For Drs. Scott McEwen, Richard Reid-Smith, Scott Weese, Patrick Boerlin and John Prescott This study has partial financial support from the Public Health Agency of Canada.
239 Please return by fax to Nicol Janecko 519-763-3117 by Friday June 29, 2007. 1. Name (please print) :
2. Contact telephone number:
3. Email address: 4. Gender (please circle) a. Male b. Female 5. Age: 6. From which University did you receive your degree in veterinary medicine?
7. Which year did you graduate?
8. Are you a board certified or board eligible specialist (i.e. ACVP, ACVIM) (please circle one) a. No b. Yes- please describe specialty or board certification
9. Are you a (please circle one) a. Practice Owner /partner b. Associate c. Locum d. Other (please describe)
10. The type of practice that you primarily practice in is: a. Companion animal general veterinary practice b. Mixed animal general practice c. Veterinary referral practice (please describe referral service(s))
d. Veterinary emergency practice e. Other (please describe)
11. What percentage of your time do you practice on: Dogs % Cats %
Please check ONE:
Q YES, I am interested in participating in the study on dispensing patterns of selected prescription medications (By selecting YES, you are NOT committing to participation. We will contact you and discuss the details of this study further. You can decide whether you with to participate at that time)
Q NO, I am not interested in participating in this research project, but I may be interested in participating in other companion animal research studies.
Q NO, I am not interested in participating in this research project or other companion animal research studies.
240 ANIMAL ILLNESS AND PRESCRIPTION DRUG USE STUDY
Appendix A.4.2 FEEDBACK questionnaire
1) Did you understand which patients were eligible or ineligible from the Patient and Prescription Eligibility Guide? a. Yes b. tio-please describe
2) Are there any other patient situations that need to be addressed in the Patient and Prescription Eligibility Guide? a. No b. Yes-please describe
3) Did you understand which prescription medications were eligible or ineligible from the Patient and Prescription Eligibility Guide? a. Yes b. No-please describe
4) Are there any additional prescription medications or situations should be addressed in the Patient and Prescription Eligibility Guide? a. No b. Yes-please describe
S) Did you have any difficulty understanding the Journal Entry form? a. No b. Yes- please describe
6) Did you have any difficulty in completing the patient or prescription data on the Journal Entry form? a. No b. Yes-please describe
7) Approximately how long did it take to complete the Journal Entry form for one patient?
8) If you collected data on all eligible patients seen during your journal day, approximately how many Journal Entry forms would you expect complete?
9) General Comments or Concerns about the study procedures:
241 Appendix A.4.3 ANIMAL ILLNESS AND PRESCRIPTION DRUG USE STUDY JOURNAL ENTRY FORM
_Patient (select one) ID 2Uf 3D 4D 5D DO NOT USE ABBREVIATIONS
Species Canine D Feline • Breed: Sex MD MN 0 FD FS D
Age (select unit) Weeks Months Years Weight (select unit) kgs lbs
Detail all NEW Disease Events) (complete as many as required)
NEW Disease event 1 (limit one): NEW Disease event 2(limit one): NEW Disease event 3(limit one):
Condition (select all that apply) Condition (select all that apply) Condition (select all that apply)
Acute • Chronic D Recurrent D Acute D Chronic D Recurrent D Acute • Chronic 0 Recurrent •
Severity (select one) Severity (select one) Severity (select one)
Mild D Moderate Severe • Mild D Moderate D Severe O Mild • Moderate • Severe D o This patient is (select one) Stable D Unstable D
List pre-existing disease conditions (if any) e.g., diabetes, hypothyroid, allergies, dental disease:
Tests to diagnose the NEW Disease Event(s) (check all that apply; indicate corresponding Disease Event 1,2,3)
History Physical Exam Hematology Biochemistry Urine sediment Urine dipstick Fecal flotation ID 2D 30 ID 2D 3D ID 2D 3D ID 2D 3D ID 2D 3D ID 2D 3D ID 2D 3D
Radiographs Ultrasound 10-:- 2D. 3D ID- u 2D 30-
Culture and Sensitivity Endocrinology ID:::.: 20 30 ID,:-.-..,•• 20 30-
Antibody Titre Antigen Testing ID-.V,-;,\ 2D - 3D 10s-.-,.'.,\ 20, 3D
Histology or Cytology (specify test and site) Other (describe in detail) 10.v>i;l:-,U.-V 2CK-M.,-V -;i.' 3D: 10 20 3D
Type of patient contact (check one) Scheduled office appointment 0 Walk-in appointment 0 Emergency: In-hours D Emergency: Out of hours 0
House call D Consultation with no patient contact 0 Other (please describe)
Treatment for NEW Disease Eventsfcheck all that apply) Treated with prescription medications O No prescription medications required D (complete pg 2 if medication fulfills eligibility criteria) Treated with non-prescription medication 0 Treatment pending results of Owner declined Euthanized 0 (do not complete page 2) diagnostic tests D treatment D
Please return by mail in envelope provided OR fax to (519) 763-3117 OR Email: [email protected]
242 Appendix A.4.3 ANIMAL ILLNESS AND PRESCRIPTION DRUG USE STUDY JOURNAL ENTRY FORM
Veterinarian ID Date Patient (select one) ID 2D 3D 4D 5D
PLILS, TABLETS, CAPSULES AND CAPLET S Include all units ag., ms, nib Treatment for which disease event (select all that apply) Event 1 • Event 2 • Event 3 D Name (trade or generic): Concentration: # pills per dose: Duration ofTreatment (select one): Days O Weeks D
Frequency (select one): 1 x daily D 2 x daily • 3 x daily • 4 x daily • Other (describe)
Route of Administration: Oral D Other (describe) Medication is (select one): Manufactured D Compounded D Indicate where compounded In-house D Human pharmacy Q Compounding Other (describe) (select one): pharmacy (human or veterinary) • Prescription filled (select one): In-house 0 Human pharmacy D Internet pharmacy Other (describe) D Treatment for which disease event (select all that Event 1 D Event 2 • Event 3 • apply) Name (trade or generic): Concentration: # pills per dose: Duration ofTreatment (select one): Days D Weeks O
Frequency (select one): 1 x daily • 2 x daily D 3 x daily • 4 x daily D Other (describe)
Route of Administration: Oral D Other (describe) Medication is (select one): Manufactured D Compounded D Indicate where compounded In-house D Human pharmacy • Compounding Other (describe) (select one): pharmacy (human or veterinary) D Prescription filled (select one): In-house D Human pharmacy D Internet pharmacy Other (describe) D INJECTIONS, SUSPENSIONS, OINTMENTS, CREAMS OR PATCHES * Treatment for which disease event (select all that Event 1 D Event 2 • Event 3 D apply) Name (trade or generic): I Concentration: Volume per dose: Duration of Treatment (select one): Days D Weeks a Frequency (select one): 1 x daily • 2 x daily • 3 x daily D 4xDdaily • Other (describe)
Route of Administration: OralD SQD IMD IV D OticD Ophthalmic D SkinD Other (describe)
Medication is (select one): Manufactured D Compounded 0 Indicate where In-house D Human pharmacy • Compounding pharmacy Other (describe) compounded (select one): (human or veterinary) D Prescription filled In-house D Human pharmacy D Internet pharmacy D Other (describe) (select one): Treatment for which disease event (select all the t Event 1 D Event 2 • Event 3 D apply) Name (trade or generic): Concentration: Volume per dose: Duration ofTreatment (select one): Days a Weeks U
Frequency (select one): 1 x daily D 2 x daily • 3 x daily D 4 x daily • Other (describe)
Route of Administration: OralD SQD IMD IV D OticD Ophthalmic D SkinD Other (describe)
Medication is (select one): Manufactured D Compounded D Indicate where In-house D Human pharmacy D Compounding pharmacy Other (describe) compounded (select one): (human or veterinary) D Prescription filled In-house • Human pharmacy D Internet pharmacy D Other (describe) (select one): NOTE: If you require additional entries for medication data, please complete and attach another journal entry form.If the prescription format for an eligible medication is not compatible with this form (e.g. tapering dose of steroids), please describe missing data on a separate sheet (paper or prescription label) and attach to the journal entry form.
243 Appendix B
Figure A.3.1. Kaplan-Meier estimator of the survival probability of antimicrobial susceptibility of E. coli isolates from dogs treated with antimicrobials and untreated dogs8, where none of the estimated survival probabilities were significantly different.
Susceptibility to cefoxitin Susceptibility to ciprofloxacin A.3i:la Mib i L S 0.75 "i_.
g 0.50 3
0,25 | 0,5
1—I—I r - -'•• "•' •••- T" • 0 7 14 28 90 0 7 14 28 90 Days after enrolment in the study Days after enrolment in the study
A.3.1c Susceptibility to gentamicin A.3.1d Susceptibility to kanamycin 1.00-1-
fo.75
0 7 14 28 90 0 7 14 26 90 Days after enrolment in the study Days after enrolment in the study
' Susceptibility to nalidixic-acid A.3. If Susceptibility to streptomycin loo 1.00
s' 75 1 J 1 °' " 1 1 1 1 1 0.50 1 1 I 0.25-
0.00- 0 7 14 28 90 0 7 14 28 90 Days after enrolment in the study Days after enrolment in the study
Legend
— Amoxicillin-clavulanicacid — Cephalexin — Fluoroquinolones — Penicillin — Control
244 A.3.1gg Susceptibility to sutfisoxazole A.3.1 n Susceptibility to trimethoprim-sulfamethoxazole 1.00 ^=i «• 0.75
5 0.50 ? 0.50 3 I 03 -I 0.25-
0 7 14 28 90 0 7 14 28 90 Days after enrolment in the study Days after enrolment in the study
A.3. li Susceptibility to tetracycline 1.00 l-.U
I 0.50 1 1 0.25
0 7 14 28 90 Days after enrolment In the study
"For dogs treated with antimicrobials, the day 0 sample was collected prior to antimicrobial therapy and antimicrobial treatment started within 12 hours of collection of the day 0 sample. For the untreated control dogs, the day 0 sample was collected on the day of enrolment.
Legend — Amoxicillin-clavulanic acid — Cephalexin — Fluoroquinolones •— Penicillin — Control
245 Figure A.3.2. Kaplan-Meier estimator of the survival probability of not isolating E. coli
isolates with an blacuy-2 phenotype from dogs treated with antimicrobials and untreated
dogsa'b
1.00- n^ L_ £< 1 0.75- 1 I .. , __ prob a
•—•? 5 •3g 0.50- 1 1 0.25- LU
0.00- 1—1—1 1 1 1 0 7 14 28 90 180 Days after enrolment in the study
Legend — Amoxicillin-clavulanic acid — Cephalexin — Fluoroquinolones • Penicillin — Control aFor dogs treated with antimicrobials, the day 0 sample was collected prior to antimicrobial therapy and antimicrobial treatment started within 12 hours of collection of the day 0 sample. For the untreated control dogs, the day 0 sample was collected on the day of enrolment. bCephalexin treatment compared to controls: Log rank test p=0.Q7, Wilcoxin test^=0.07,Peto-Prentice test/>=0.07; Amoxicillin-clavulanic acid treatment compared to controls: Log rank test/7=0.50, Wilcoxin test/j=0.50, Peto- Prentice test^?=0.50; Fluoroquinolone treatment compared to controls: Log rank test p=0.34, Wilcoxin testp=033, Peto-Prentice test^7=0.45; Penicillin treatment compared to controls: Log rank test/?=0.09, Wilcoxin test/?=0.12, Peto-Prentice test/7=0.08.
246 Figure A.3.3. Kaplan-Meier estimator of the survival probability of not isolating
Salmonella enterica isolates from dogs treated with antimicrobials and untreated dogsa'b.
1.00H
I 0.75 flj np
"g 0.50 3 (0 1 (0 I 0.25 HI
o.ooH 0 7 14 28 90 180 Days after enrolment in the study
Legend — Amoxicillin-clavulanic acid — Cephalexin — Fluoroquinolones Penicillin — Control
aFor dogs treated with antimicrobials, the day 0 sample was collected prior to antimicrobial therapy and antimicrobial treatment started within 12 hours of collection of the day 0 sample. For the untreated control dogs, the day 0 sample was collected on the day of enrolment. bCephalexin treatment compared to controls: Log rank test/y=0.51, Wilcoxin test/?=0.25, Peto-Prentice testp=0.27; Amoxicillin-clavulanic acid treatment compared to controls: Log rank test/?=0.16, Wilcoxin test/?=0.10, Peto-Prentice test p=0.14; Fluoroquinolone treatment compared to controls: Log rank test/?=0.73, Wilcoxin testp=0.36, Peto-Prentice test/?=0.52; Penicillin treatment compared to controls: Log rank test/?=0.73, Wilcoxin testp=0.63, Peto-Prentice testp=0.63.
247 Appendix C
Table A.3.1. Signalment of dogs in antimicrobial treatment and untreated cohorts.
Cohort N Age" Weight9 Mai Castrated Female Spayed
e male (%) female
(%) (%) (%)
Amoxicillin- 12 7 17 0C 17c 8C 67c clavulanic acidb (0.3-16)c (7-32)
Cephalexin 33 6C 30 6C 33c 18c 42c
(0.33- (2,57)c
12)
Fluoroquinolone 12 5 26.5 8C 25° 0C 67c s (l,14)c (12,46)c
Penicillin 9 1 22 22c 0C 67c llc
(0.42-5)c (3-32)
Untreated 8 4 16 0 25 0 75
(0.5-6) (3-38) aMedian (range) age in years, median (range) weight in kilograms. "Sex was not provided for one dog in the amoxicillin-clavulanic acid treated cohort. cMedian values are significantly different (p<0.05) than the values reported in the untreated dogs using the
Mann Whitney U test.
248 Table A.3.2. The dose, frequency and duration of treatment for dogs treated with oral amoxicillin- clavulanic acid, oral cephalexin, oral fluoroquinolones and injectable penicillin.
Antimicrobial3 Dose8 Frequency" (%) Duration' (%)
(Range) Once Twice Three 5 7 10 14 21 42
Amoxicillin- 13 8 92 50 8 33 clavulanic acid (8-19)
(n=12)
Cephalexin (n=33) 21 82 18 6 18 15 61
(11-42)
Fluoroquinolones
(n=12)
Enrofloxacin 5.4 67 33 11 22 22 33 11
(n=9) (3.1-9.0)
Orbifloxacin 3.2 100 50 50
(n=2) (2.9-3.5)
Marbofloxacin 2.53 100 100
(n=l)
Penicillin (n=9) 28,412
(9, 677-
46, 875)
"Median value; mg/kg for oral amoxicillin-clavulanic acid, oral cephalexin, and oral fluoroquinolones and IU/kg for injectable penicillin. bFrequency of administration per day. cDuration of treatment in days.
249 Table A.3.3. The frequency of responses to a questionnaire completed by owners of dogs treated
with antimicrobials and untreated dogs.
Exposure Amoxicillin- Cephalexin Fluoroquinolone Penicillin Untreated
clavulanic acid Cohort Cohort (n==11 ) Cohort Cohort
cohort (n=10) (n=26) (n=7) (n=7)
Main Diet8
Commercial 88% 100% 100% 85% 100%
Homemadeb 8% 0 0 0 0
Homemade 4% 0 0 0 0
BARFC
Commercial 0 0 0 15% 0
BARF0
Treats 0 0 0 0 0
Pigearsd 34% 44% 40% 28% 28%
Rawhided 12% 22% 10% 0 14%
Raw meat 8% 30% 0 0 14%
Raw bones 12% 10% 0 0 14%
Drinking Water
Municipal 42% 60% 82% 57% 100%
tap water
Well water 50% 20% 9% 14% 0
Brita filtered 4% 20%
water
250 Exposure Amoxicillin- Cephalexin Fluoroquinolone Penicillin Untreated
clavulanic acid Cohort Cohort (n=ll) Cohort Cohort
cohort (n=10) (n=26) (n=7) (n=7)
UV filtered 4% 0 0 0 0
well water
Bottled 0 9% 29%
water
Participates in 0 11% 9% 14 coprophagia
Drinks water 0 19% 18% 14% from a toilet
Has contact 22% 11% 29% 29% with livestock6
Participates as 0 4% 29% a therapy dogd
aIn the amoxicillin-clavulanic acid cohort, 9 of the submitted questionnaires (n=10) completed
this question. In the penicillin cohort, 6 of the submitted questionnaires (n=7) completed this
question. bHomemade diet as directed by a veterinarian "BARF: Bones And Raw Food Diet. dIn
the amoxicillin-clavulanic acid cohort, 9 of the submitted questionnaires (n=10) completed this
question. In the fluoroquinolone cohort, 10 of the submitted questionnaires (n=l 1) completed this
question. In the amoxicillin-clavulanic acid cohort, 9 of the submitted questionnaires (n=10)
completed this question.
251 Table A.3.4. Distribution (%) of antimicrobial minimum inhibitory concentrations of fecal Escherichia coli isolates (n=1002) from dogs untreated and treated with antimicrobials from community veterinary practices in southern Ontario.
Antimicrobial3 Distribution of minimum inhibitory concentrations b,c (jig/ml) Total Resistant
0.015 0.03 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256
AMC 15 49 12 n 19 19(17,22) AMK 37 54 7 0.8 0 (0,0.4)e
AMP 44 21 1 0.2 28 28 (25, 31)
CHL 53 41 3 (2,4)
CIP 95 0.3 0.3 0.6 0.3 0.6 0.6 (0.2,1)
CRO 83 0.2 0.1 0.3 2 3(2,5)
FOX 1 23 50 8 16 16(14,19)
GEN 20 71 7 0.6 0.1 2 (0.9,3)
KAN 97 0.5 0.1 | 2 2(1,3)
NAL8 16f 76 5 0.4 2 2(1,3) g sox 76 16 1 ;f o (o, o.4)e
252 Antimicrobial3 Distribution of minimum inhibitory concentrations b'c (ug/ml) Total Resistant
0.015 0.03 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256
STRg 95 5 (4,7)
SXT8 83 11 0.4 5 (4,7)
no 48 33 0.2 13 13 (12,16)
TCY 90 0.1 10 10(8,12)
"Antimicrobial Abbreviations: AMC- amoxicillin-clavulanic acid, AMK- amikacin, AMP-ampicillin, CHL-chloramphenicol, CIP- ciprofloxacin, CRO-ceftriaxone, FOX-cefoxitin, GEN-gentamicin, KAN-kanamycin, NAL-nalidixic acid, SOX-sulfizoxazole, STR- streptomycin, SXT-trimethoprim-sulphamethoxazole, TlO-ceftiofur, TCY-tetracycline. bMinimum Inhibitory Concentration distribution: The unshaded fields indicate the MIC range tested for each antimicrobial in the plate configuration. The MICs at the upper or lower bound of the distribution are censored. The values at the upper bound are > to the value presented and the values at the lower bound are < the value presented. Trouble bar represents the resistant breakpoint. Single bar represents the susceptible breakpoint. 'Values in brackets are 95% confidence intervals. Total resistant may not equal values presented in table due to rounding error. eOne sided 97.5% confidence interval. fExact MIC. 6Susceptible breakpoint-NAL: <16 ug/ml, Susceptible breakpoint-SOX:
<256 ug/ml, Resistant breakpoint-SOX: >512 ug/ml, Susceptible breakpoint-STR: <32 ug/ml, Susceptible breakpoint-SXT: <2 ug/ml.
253 Table A.4.1. Categorization of body sites or disease conditions in study dogs that were created by aggregating the incident disease events description reported by veterinarians in the submitted journals.
Category Disease events included within category
(Body Site or Disease condition)
Anal gland Anal gland abscess, anal gland infection, anal gland
sacculitis
Digits Infected digit, infected nail bed, ingrown nail, nail
fracture, swollen toe, torn nail
Dental/Oral Dental abscess, dental disease, gingivitis, oral
infection, oral mass, oral pain, periodontal disease,
salivary mucocele, stomatitis, tooth abscess, tooth
infection
Ears Bacterial otitis, ear infection, otitis, otitis externa,
sore ear
Eyes Blepharitis, conjunctivitis, corneal ulcer
Gastrointestinal bloody diarrhea, colitis, coccidiosis, gastroenteritis,
hematochezia, large bowel diarrhea, pancreatitis,
small bowel diarrhea, vomiting, vomiting and
diarrhea
Genitalia Balanoposthitis, dystocia, pyometra, swollen
prepuce, vaginal discharge, vulvar discharge,
254 Category Disease events included within category
(Body Site or Disease condition)
Respiratory Cough, infectious tracheobronchitis, infectious
tracheitis, epistaxis, kennel cough, nasal discharge,
rhinitis, pneumonia, sinusitis, tracheobronchitis
Skin Acute bacterial dermatitis, acute moist dermatitis,
atopy, bacterial dermatitis, bite (raccoon, dog),
cellulitis, chronic Staphylococcus dermatitis, cysts
(infected, ruptured, sebaceous), demodicosis,
folliculitis, hot spots, interdigital dermatitis,
interdigital pododermatitis, interdigital
pyodermatitis, lacerations (digital, foot), lick
granulomas, moist dermatitis, pododermatitis,
pyoderma, pyodermatitis, sarcoptic mange,
seborrhea, skin abscess, skin infection,
Staphylococcus dermatitis, superficial pyoderma,
wounds,
Urinary Bacterial cystitis, cystitis, polyuria,
polyuria/polydypsia, urinary tract infection, urinary
tract infection with hematuria
255 Table A.4.2. Categorization of body sites or disease conditions in study cats that were
created by aggregating the incident disease events description reported by veterinarians in the submitted journals.
Category Disease events included within category
(Body Site or Disease condition)
Abscess Abscess repair, facial abscess, lip abscess, tail
abscess, ruptured abscess
Anal Gland Anal sac abscess, anal sacculitis
Bite Bite abscess, bite wound, cat bite abscess, fight
wound
Dental Broken tooth, dental abscess, dental disease,
gingivitis, gingivostomatitis, periodontal disease
Digit Infected toe, ingrown toe, swollen toe, torn nail
Ear Ear infection, otitis externa
Gastrointestinal Colitis, diarrhea, gastritis, melena, soft stool,
vomiting
Respiratory Asthma, bronchitis, coughing, nasal discharge,
pneumonia, rhinitis, sinusitis, purulent nasal
discharge, tracheitis, upper respiratory tract
congestion, upper respiratory tract disease, upper
respiratory tract infection, upper respiratory tract
inflammation,
256 Category Disease events included within category
(Body Site or Disease condition)
Skin Acne, cellulitis, dermatitis, eczema, flea allergy
dermatitis, folliculitis, moist dermatitis, perianal
dermatitis, skin infection
Urinary Bacterial cystitis, crystalluria, cystitis, hematuria,
urinary tract infection, pollakiuria
Wound Laceration, puncture wound, unhealed skin wound,
wound
257 Table A.4.3. The incident disease event descriptions reported by veterinarians that were
aggregated for the evaluation of antimicrobial use in feline lower urinary tract disease,
feline upper respiratory tract disease and canine infectious tracheobronchitis.
Disease Disease event descriptions
Feline lower urinary tract Bacterial cystitis, crystalluria, cystitis, hematuria,
disease poUakiuria, urinary tract infection.
Feline upper respiratory Nasal discharge, rhinitis, sinusitis, tracheitis, upper
tract disease respiratory congestion, upper respiratory disease, upper
respiratory tract infection, upper respiratory tract
inflammation
Canine infectious Cough, infectious tracheobronchitis, infectious tracheitis, tracheobronchitis kennel cough, tracheobronchitis
258 Table A.4.4. The age, sex and weight distributions of the dogs and cats with incident
disease events treated with non-topical antimicrobials reported in the journals submitted
by veterinarians.
Dog Cat
Age8 5 years (2 weeks-18 years) 6 years (1 week-22 years)
Sex
Neutered female 42% 35%
Neutered male 38% 51%
Intact female 8% 6%
Intact male 12% 8%
Weight3 19 kg (1kg-72 kg) 5kg(0.3kg-15kg) aMedian value (Range).
259 Table A.4.5. Distribution of the description of the incident disease events in dogs and cats treated with non-topical antimicrobials reported by veterinarians in the submitted journals.
Disease Description Dogs Cats
Condition9
Acute 75% 80%
Chronic 15% 12%
Recurrent 8% 7%
Severityb
Mild 31% 25%
Moderate 58% 57%
Severe 11% 18%
"Ndog^M, Ncat=179. Other disease conditions descriptions were reported (e.g., acute and
b recurrent, chronic and recurrent) at a frequency of less than 2%. NCanine=405, Nfcune=175.
260 Table A.4.6. Other incident disease events treated with non-topical antimicrobials in dogs.
Number of Antimicrobials
reports
Atypical behaviour 1 Enrofloxacin
Anorexia 2 Enrofloxacin
Body mass 1 Amoxicillin-clavulanic acid
Cholangiohepatitis 2 Amoxicillin (n=l), Metronidazole
(n=l)
Circulatory compromise of foot 1 Amoxicillin-clavulanic acid associated with an elastic band
Congestive heart failure 1 Orbifloxacin
Cutaneous mass 1 Cephalexin
Cuterebra 2 Amoxicillin-clavulanic acid (n=l),
Cephalexin (n=l)
Difficulty eating 1 Marbofloxacin
Discoid lupus 1 Tetracycline
Foreign body ingestion 1 Amoxicillin-clavulanic acid
Hepatic disease 1 Enrofloxacin
Mammary mass 1 Cephalexin
Pain 1 Cefovecin
Pulmonary neoplasia 1 Amoxicillin
Seizures 1 Ampicillin
261 Number of Antimicrobials
reports
Septic arthritis 1 Cephalexin
Stick in the mouth 2 Amoxicillin (n=l), Amoxicillin-
clavulanic acid (n=l)
Swollen face 1 Amoxicillin-clavulanic acid
Swollen lymph node 1 Amoxicillin
Swollen mandible 2 Amoxicillin
Swollen nose 1 Amoxicillin-clavulanic acid
Swollen paw 1 Amoxicillin
Torn tendons 2 Cefovecin (n=l), cephalexin (n=l)
Weight loss 1 Clindamycin
262 Table A.4.7. Other incident disease events treated by non-topical antimicrobials in cats.
Number Antimicrobials
of reports
Anemia Tetracycline hydrochloride, novobiocin sodium
and prednisolone combination (Delta albaplex®)
Blood on nose 1 Procaine and benzathine penicillin
Chelitis 1 Amoxicillin-clavulanic acid
Cholangiohepatitis 1 Metronidazole
Diabetes mellitus 1 Cefovecin
Enlarged chin 1 Cefovecin
Fever 3 Amoxicillin-clavulanic acid (n=3), cefovecin (n=l)
Granuloma complex 1 Clindamycin
Inappropriate urination 2 Amoxicillin (n=l), amoxicillin-clavulanic acid
(n=l)
Liver tumour 1 Procaine and benzathine penicillin
Paw injury 1 Cefovecin
Pyometra 1 Marbofloxacin
Swollen face 1 Amoxicillin
Vaginal discharge 1 Amoxicillin-clavulanic acid
263 Appendix D
The prevalence of bacterial contamination of surgical cold sterile
solutions from community companion animal veterinary practices in
southern Ontario.
As accepted for publication in the Canadian Veterinary Journal
Abstract
Surgical cold sterile solutions are commonly used in veterinary practice, yet sterility
cannot be verified under practical clinical conditions. Surgical cold sterile solutions were
sampled and bacteria were recovered from 13% of the sampled solutions including
opportunistic pathogens. Attempts to sterilize surgical instruments with cold sterile
solutions should be avoided.
1.0. Introduction
Sterilization is a standard practice for surgical instruments and is typically
achieved by steam under pressure (autoclave). For instruments that cannot withstand
steam sterilization, such as endoscopes, there are other sterilization methods including
peroxide vapour, ethylene oxide gas and chemical sterilant solutions (commonly called
"cold sterilization"). Cold sterilization involves immersion of items in a sterilant solution,
such as glutaraldehyde or alcohol, for a predetermined period of time. Some cold sterile solutions and protocols are able to achieve sterilization or high level disinfection; however not all disinfectants and practices can effectively eliminate all microbial contaminants.
264 Although there are no published data describing the use of surgical cold sterile
solutions in veterinary medicine, in the authors' experience, surgical cold sterile solutions
are used in community veterinary practices to sterilize dental instruments, suture needles
and suture material and surgical instruments for minor surgical procedures including
clean surgical procedures (e.g., lump removal), clean contaminated surgical procedures
(e.g., feline castration, feline onychectomy and wound debridement) and dirty surgeries
(e.g., lancing an abscess). In addition, instruments may be obtained from a surgical cold
sterile solution to replace surgical instruments from a sterile surgical pack that may have become contaminated during sterile surgical procedures.
There are numerous concerns regarding the use of cold sterile solutions, particularly for surgical instruments that are used in sterile body sites. Sterilization using cold sterile solutions is a lengthy procedure; under normal clinical conditions, it takes approximately 10 hours for an instrument to become sterile (Rutala and Weber, 1999).
Also, cold sterile solutions can be easily contaminated through the introduction of particulate or organic matter. This can occur when cold sterile solutions are open and exposed to air, through the addition of improperly cleaned instruments and the retrieval of instruments using contaminated objects, including fingers. Furthermore, cold sterile solutions need to be managed properly so that alterations in dilution, pH, temperature, contact time, organic load and frequency of use do not reduce the effectiveness of these solutions.
This study aimed to determine the prevalence of surgical cold sterile solutions in community companion animal veterinary practices and the prevalence of bacterial
265 contamination of these solutions. In addition, this study examined associations between
bacterial recovery from cold sterile solutions and specific practice demographics.
2.0. Materials and Methods
Clinic selection and sampling was done during the summer of 2005. Veterinary
hospitals in Southern Ontario licensed as companion animal hospitals or offices,
including those with additional licenses for food animal or equine hospital or mobile
(mixed-animal practices), by the College of Veterinarians of Ontario in 2005 were
eligible for recruitment. A recruitment letter was mailed to these practices (n=766)
describing the study objectives. Practices willing to participate were asked to respond by
mail, fax, or telephone with a completed practice-demographic survey. Based on
previous experience in community-based research in companion veterinary practices in
southern Ontario, we sought to enroll 100 veterinary practices. This also provided
sufficient statistical power to detect bacterial contamination at a prevalence of 10%.
Cold sterile solutions in the surgery room of the study practices were sampled.
Samples were not collected from cold sterile solutions in other areas of the practice (e.g., treatment room, exam rooms) and samples were not collected from cold sterile solutions used exclusively for instruments used in dental procedures. Data was not collected on the type of chemical sterilants used in the cold sterile solutions. The solutions were sampled
(approximately l-3ml) aseptically using a syringe. Approximately 16 to 24 h after sampling, 100 ul aliquots of cold sterile solution were streaked onto two blood agar plates. One plate was incubated aerobically at 35°C and the other anaerobically at 37°C.
Isolates were identified using Gram stain and appropriate biochemical tests followed by use of commercial identification kits for Staphylococcus spp. (API Staph, BioMerieux
266 Canada Inc., St Laurent, Quebec ), Streptococcus spp (API 20 Strep, BioMerieux Canada
Inc.) or Gram negatives (BBL Enterotube II, Becton, Dickinson and Co., Franklin Lakes,
New Jersey, USA).
Descriptive statistics were used to determine the prevalence of cold sterile
solutions in veterinary practices, the prevalence of specific bacteria recovered and overall
prevalence of bacterial recovery. All variables were categorical and Fisher's exact test
was used to determine associations between overall bacterial recovery and these practice
demographics: practice type (companion animal or mixed animal), number of
hospitalized patients per day, number of appointments per day, number of staff and
presence of a "Standard Operating Procedure" for sterilization practices. Statistical
significance was determined using a/7-value <0.05 and a 95% confidence interval for the
odds ratio that did not include the null. Statistical analysis was performed using Microsoft
Excel 2003 (Microsoft Corporation, Redmond Washington USA) and Intercooled Stata
10.0 software (StataCorp, Collage Station Texas USA).
3.0. Results
One hundred and twenty-one clinics responded with interest to the recruitment
letter (response rate 16%). Twelve of these practices could not be sampled because of time limitations and 8 practices were out of the geographic sampling region. Therefore
from the initial 121 clinics that responded with interest, the study population of 101 veterinary practices was enrolled. Of the 101 practices, 90 practices were companion animal, ten were mixed animal, and one practice treated primarily exotic animals.
The prevalence (and one sided 97.5% confidence interval) of use of surgical cold sterile solutions in community veterinary practices was 100% (96%, 100%) and all
267 practices had only one surgical cold sterile solution each. The prevalence (and 95%
confidence interval) of aerobes and anaerobes recovered from surgical cold sterile
solutions was 11% (6%, 19%) and 5% (2%, 11%), respectively. When combined, 13%
(7%, 21%) of the cold sterile solutions yielded bacterial growth (aerobic or anaerobic),
including veterinary and human opportunistic pathogens (Table 1). There were no
significant associations (/7-values >0.5) between bacterial recovery and the tested practice demographics.
4.0. Discussion
In 1968, Earle H. Spaulding (Spaulding, 1968) devised a classification system to guide hospital disinfection and sterilization based on the risk of infection to the human patient. This system classified any object that entered sterile tissue or the vascular system as "critical". Autoclaving was the recommended method of sterilization for
"critical items". The recommended use of chemical sterilants or cold sterile solutions was limited to instruments where sterilization by other means was unsuitable. The same principles are still recommended (Rutala, 1999) and use of cold sterile solutions for instruments used in sterile surgical procedures is not recommended (Fossum, 2007) because sterility cannot, for practical purposes, be verified when using cold sterile solutions. In other sterilization methods, objective evaluations of sterility can be easily performed, such as the use of indicator strips and biological indicators for autoclaves
(Rutala and Weber, 1999).
The observed high prevalence of bacterial recovery, including some opportunistic pathogens, from surgical cold sterile solutions was striking. This is especially remarkable since methods were not employed to neutralize the chemical sterilants within the cold
268 sterile solutions prior to bacterial isolation. Furthermore, the time from sample collection
to culture was greater than 10 h, and this time lag should have been sufficient for the
solution to become sterile if it had been contaminated, even by normal procedures like
the addition of clean instruments, at the time of sampling. This suggests that factors, such
as inappropriate dilution or pH or contamination with particulate or organic matter, may
have reduced the efficacy of the sampled surgical cold sterile solutions. However, this
time-lag may have resulted in the elimination of some bacteria that are typically
susceptible to disinfection like Gram-positive bacteria. Consequently, we may have
underestimated the prevalence of bacterial contamination and the diversity of viable pathogens in surgical cold sterile solutions.
A diverse group of bacteria were recovered; isolation of sporeformers {Bacillus and Clostridium spp.) was not surprising since bacterial spores are highly resistant to disinfectants (Rutala and Weber, 1999). Similarly, Serratia spp. can be relatively tolerant to disinfectants and have a propensity for colonization of solutions (van der Vorm and
Woldring-Zwaan, 2002). Yet, the recovery of important opportunistic pathogens, such as
Staphylococcus and Acinetobacter spp., is concerning because these organisms are generally susceptible to disinfectants and chemical sterilants. The origin (human or animal) of the bacterial contaminates is unknown. Since some of these organisms are components of human skin (Cogen et al., 2008) and fecal (Cummings et al., 2004) flora, contamination may have occurred through mishandling or inadequate hand hygiene.
However, some of the bacterial isolates could be of animal origin (e.g., Burkholderia cepacia) (Authier et al., 2006) and may have been introduced into the solution through improperly cleaned instruments.
269 Some the organisms recovered in this study have been associated with severe
infections in dogs such as septicemia (Galarneau et al., 2003, Armstrong, 1984) and
surgical site infections (Boerlin et al., 2001) including an infection of a surgical implant
(Peremans et al., 2002). Therefore, the bacteria recovered from the surgical cold sterile
solutions pose a potential health risk to surgical patients. In addition, such infections
could contribute to the overall epidemiology of opportunistic bacteria in the community
through zoonotic transmission of an animal infection to a person. The magnitude of these
potential risks are unknown as there is a general lack of information on the epidemiology
of hospital associated infections, including SSI in companion animal veterinary medicine
and the risk they pose to animal and human health. Reducing these risks is achieved by
adherence to aseptic principles. The results of this study indicate that the use of surgical instruments from a cold sterile solution contravenes aseptic principles. In addition to the possible negative animal and human health consequences associated with this contravention, there could also be legal and professional consequences for veterinarians.
Minimizing adverse surgical events in companion animal veterinary medicine is one goal of a effective infection control program. Strict adherence to aseptic surgical principles is imperative to achieving this goal. The use of cold sterile solutions should be limited in veterinary medicine and sterilization of instruments or equipment for sterile procedures by these solutions should be avoided.
Acknowledgements
We would like to acknowledge the following for their contributions to this study: Alyssa
Calder, Karlee Thomas and Adriana Sage for clinic sampling; Joyce Rousseau for bacterial isolation and identification and the veterinary practices for participating in the
270 project. This study was funded by the Ontario Veterinary College Pet Trust Fund and the
Public Health Agency of Canada. Colleen Murphy was a recipient of the Ontario
Veterinary College Graduate Student Fellowship.
271 References
Armstrong, PJ. 1984. Systemic Serratia marcescens infections in a dog and a cat. J Am
Vet Med Assoc, v. 184, p. 1154-8.
Boerlin, P, S Eugster, F Gaschen, R Straub, P Schawalder. 2001. Transmission of
opportunistic pathogens in a veterinary teaching hospital. Vet Microbiol, v. 82, p. 347-59.
Cogen, AL, V Nizet, R L Gallo. 2008. Skin microbiota: a source of disease or defence?
Br J Dermatol, v. 158, p. 442-55.
Cummings, JH, J M Antoine, F Azpiroz, R Bourdet-Sicard, P Brandtzaeg, P C Calder, G
R Gibson, F Guarner, E Isolauri, D Pannemans, C Shortt, S Tuijtelaars, B Watzl. 2004.
PASSCLAIM~gut health and immunity. Eur J Nutr. v. 43 Suppl 2, p. Ill 18-11173.
Fossum T. 2007. Small Animal Surgery 3rd Edition. Mosby, St Louis Missouri,.
Galarneau, JR, M Fortin, J M Lapointe, C Girard. 2003. Citrobacter freundii septicemia in two dogs. J Vet Diagn Invest, v. 15, p. 297-9.
Peremans, K, F De Winter, L Janssens, F Dumont, H Van Bree, R Dierckx. 2002. An infected hip prosthesis in a dog diagnosed with a 99mTc-ciprofloxacin (infecton) scan.
Vet Radiol Ultrasound, v. 43, p. 178-82.
Rutala, WA, D J Weber. 1999. Infection control: the role of disinfection and sterilization.
272 J Hosp Infect, v. 43 Suppl, p. S43-55.
Spaulding EH. 1968. Disinfection, Sterilization and Preservation, Lea & Febiger,
Philadelphia, Pennsylvania. van der Vorm, ER, C Woldring-Zwaan. 2002. Source, carriers, and management of a
Serratia marcescens outbreak on a pulmonary unit. J Hosp Infect, v. 52, p. 263-7.
273 Table A.D.I. The prevalence of bacterial growth from surgical cold sterile solutions
(n=101) from community companion animal veterinary practices (n=101) in Southern
Ontario.
Organism Prevalence (%)
(95% Confidence Interval)
Aerobes Mannheimia hemolytica 3 (0.6, 8)
Burkholderia cepacia 3 (0.6, 8)
Shigella species 1 (0.02, 5)
Coagulase negative 1 (0.02, 5)
Staphylococcus species
Serratia marcescens 1 (0.02, 5)
Citrobacterfreundii 1 (0.02, 5)
Acinetobacter Iwcffii 1 (0.02, 5)
Anaerobes Bacillus species 3 (0.6, 8)
Clostridium fallax 1 (0.02, 5)
274