sandra l. lefebvre, ; richard j. reid-smith, · 2019-10-11 · lefebvre’s present address is...

1404 ScientificReports JAVMA,Vol234,No.11,June1,2009

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Programs that involve animals visiting people in health-care facilities are common in North America, as such

programs have been shown to improve the functioning and general well-being of people with whom the animals interact.1–5 Dogs are the animals most commonly used in AAI programs,6 and in general, managers of AAI programs have acknowledged the need to ensure that participating dogs are unlikely to transmit infectious agents to the pa-tients with whom they interact. Consequently, most pro-tocols for selecting dogs to participate in AAI programs are designed to screen out animals that may be carrying zoonotic organisms (eg, roundworms) or potential disease vectors (eg, fleas and ticks).7–11 On the other hand, little attention has been paid to the pathogens that dogs might encounter via exposure to patients, staff, and the health-

Incidence of acquisition of methicillin-resistant Staphylococcus aureus, Clostridium difficile, and other health-care–associated pathogens

by dogs that participate in animal-assisted interventions

Sandra L. Lefebvre, dvm, phd; Richard J. Reid-Smith, dvm, dvsc; David Waltner-Toews, dvm, phd; J. Scott Weese, dvm, dvsc, dacvim

Objective—Todeterminewhetherdogsthatvisitedhumanhealth-carefacilitieswereatgreater risk of acquiring certain health-care–associatedpathogens, comparedwithdogsperforminganimal-assistedinterventionsinothersettings,andtoidentifyspecificbehav-iorsofdogsassociatedwithanincreasedriskofacquiringthesepathogens.Design—Prospectivecohortandnestedcase-controlstudies.Animals—96dogsthatvisitedhumanhealth-carefacilitiesand98dogsinvolvedinotheranimal-assistedinterventions.Procedures—Fecalsamplesandnasalswabspecimenswerecollectedfromdogsatthetimeofrecruitmentandevery2monthsfor1yearandweretestedformethicillin-resistantStaphylococcus aureus(MRSA),Clostridium difficile,andotherselectedbacteria.Informa-tionwasalsoobtainedonfacilitiesvisitedduringanimal-assistedinterventions,dogdiet,dogillnesses,andantimicrobialusewithinthehome.Attheendofthestudy,doghandlerswereaskedaboutthebehavioroftheirdogsduringvisitstohealth-carefacilities.Results—Ratesof acquisitionofMRSAand C difficilewere4.7 and2.4 times ashigh,respectively, among dogs that visited human health-care facilities, compared with ratesamongdogsinvolvedinotheranimal-assistedinterventions.Amongdogsthatvisitedhu-manhealth-carefacilities,thosethatlickedpatientsoracceptedtreatsduringvisitsweremorelikelytobepositiveforMRSAandC difficilethanweredogsthatdidnotlickpatientsoraccepttreats.Conclusions and Clinical Relevance—Results suggested that dogs that visited humanhealth-carefacilitieswereat riskofacquiringMRSAandC difficile,particularlywhentheylickedpatientsoracceptedtreatsduringvisits.(J Am Vet Med Assoc2009;234:1404–1417)

care environment. Dogs that become colonized by patho-gens encountered in health-care settings may be at risk for developing infections, may transmit pathogens to the

From the Departments of Population Medicine (Lefebvre, Reid-Smith, Waltner-Toews) and Pathobiology (Weese), Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada; and the Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, 110 Stone Rd W, Guelph, ON N1G 3W4, Canada (Reid-Smith). Dr. Lefebvre’s present address is AVMA, 1931 N Meacham Rd, Schaumburg, IL 60173.

Supported by grants from the Canadian Institutes of Health Research and the Pet Trust Fund of the Ontario Veterinary College.Presented in part at the 1st International Conference on MRSA in Animals, Liverpool, England, June 2006; the International Meeting on Emerg-

ing Diseases and Surveillance, Vienna, February 2007; the 107th General Meeting of the American Society for Microbiology, Toronto, May 2007; and the 2nd International Clostridium difficile Symposium, Maribor, Slovenia, June 2007.

The authors thank Nicol Janecko, Joyce Rousseau, and Dr. Patrick Boerlin for overseeing laboratory testing; Gabhan Chalmers, Lilla Yan, and Catherine Sabino for technical assistance; and William Sears for statistical assistance.

Address correspondence to Dr. Weese.

Abbreviations

AAI Animal-assistedinterventionCI ConfidenceintervalESBL Extended-spectrumβ-lactamaseGLMM GeneralizedlinearmixedmodelMRSA Methicillin-resistantStaphylococcus aureusMRSI Methicillin-resistantStaphylococcus intermediusOR OddsratioPFGE Pulsed-fieldgelelectrophoresisUTI UrinarytractinfectionVRE Vancomycin-resistantenterococci

JAVMA,Vol234,No.11,June1,2009 ScientificReports 1405

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patients they interact with, and may play a role in commu-nity dissemination of health-care–associated pathogens.

Health-care–associated pathogens to which dogs may be exposed during AAIs include MRSA, VRE, toxi-genic strains of Clostridium difficile, and ESBL-produc-ing and AmpC β-lactamase–producing strains of Esch-erichia coli. The first 3 of these are serious causes of morbidity and death among humans in North American health-care facilities12–14 and are frequently identified as environmental contaminants in health-care settings.15–17 Escherichia coli strains that produce ESBL or AmpC β-lactamase are growing concerns, particularly in inten-sive care units and long-term care facilities, because of challenges in treating infections caused by these highly drug-resistant organisms.18,19

Infections with all 5 of these pathogens have been identified in dogs.20–25 However, additional research is needed to establish whether the strains that dogs ac-quire are the same as those carried by humans with whom they have had contact. The strongest evidence supporting humans as sources of canine colonization or infection involves MRSA. Incidents of likely human-to-dog transmission of MRSA have been reported,26,27 and colonized dog owners were suspected to be the source of infection for their pets.28 Transmission from dogs back to humans has also been reported.29

Research into the potential for interspecies spread of C difficile has just begun. Available data suggest that dogs and humans that are not known to have contacted each other can harbor strains of C difficile that are indis-tinguishable by molecular analysis,30 although not all strains appear to have the ability to produce the toxins associated with disease. There is a report31 of a human epidemic strain of C difficile being detected in the feces of a dog participating in an AAI program that had visit-ed a hospital where human patients with C difficile–as-sociated disease were being treated. However, because the strain that caused the human cases of disease in the hospital was not identified, it is possible that the dog may have acquired the toxigenic strain from another source. Transmission of VRE or ESBL-producing or AmpC β-lactamase–producing strains of E coli between humans and dogs has not been reported to date but may be detected in the future because infections with these organisms are being more frequently identified in both species.32–34

Because pathogens that are believed to have origi-nated in health-care settings are being recognized more frequently as a cause of infections in otherwise healthy individuals in the community,35,36 the poten-tial role that animals involved in AAI programs may have in facilitating the spread of these pathogens must be considered. If dogs involved in AAI programs can indeed spread health-care–associated pathogens to people and other animals, then those at greatest risk would likely be those people and pets living in the same homes as dogs, especially given the close and frequent contact between most people and their pets.37 Evidence already exists to suggest that dogs owned by people who have had contact with health-care envi-ronments can serve as reservoirs of MRSA strains caus-ing human infection.29,38 If dogs shed pathogens they acquire from health-care environments in their feces, then humans and other animals exposed to the feces

may also be at risk. Because dogs participating in AAI programs may visit multiple facilities in conjunction with these programs, they may also expose vulnerable patients in other health-care settings to organisms ac-quired elsewhere.

The purpose of the study reported here was to de-termine whether dogs that visited human health-care facilities as part of an AAI program were at greater risk of acquiring certain health-care–associated pathogens, compared with dogs that performed AAIs in other set-tings. Bacteria of interest were MRSA, VRE, C difficile, and ESBL-producing and AmpC β-lactamase–produc-ing strains of E coli. In addition, if a difference in risk of acquiring these pathogens was identified between the 2 groups, we wanted to identify specific canine behav-iors associated with an increased risk that dogs visiting health-care facilities would acquire these pathogens. An additional objective was to determine whether people and pets living in the same household as dogs positive for MRSA were themselves colonized with MRSA. We also hypothesized that dogs that visited health-care fa-cilities would not differ from dogs performing AAIs in other settings with regard to their risk of acquisition of Salmonella spp or MRSI because these bacteria are not endemic to human health-care settings.

Materials and Methods

Animals—A prospective cohort study design was used, with 2 groups of dogs monitored over a 12-month period. Organizations in Ontario involved in AAI pro-grams incorporating dogs that had previously been iden-tified by our research group39 were sent a letter inviting them to participate in the study. Organization manag-ers were asked to advertise the study to their members, who were to advise their manager when they were inter-ested in participating. Managers subsequently informed our research group of their members’ interest. One co-hort (exposed dogs) consisted of dogs that had been recruited for AAIs in hospitals or long-term care facili-ties and that were scheduled to begin their visits within 1 week of enrollment in the study. The other cohort (unexposed dogs) consisted of dogs newly recruited to participate or actively participating in AAIs in non–health-care settings, such as schools and group homes. Dogs that were reported by their owners to have already visited human health-care facilities, as defined by the Ontario Hospital Association40 or the Ontario Ministry of Health and Long-Term Care,41 were excluded from both groups. The 2 groups were hypothesized to differ in their rates of acquisition of 5 pathogens endemic in some health-care settings: C difficile, MRSA, VRE, ESBL-producing E coli, and AmpC β-lactamase–producing E coli. To confirm that the groups were similar in every way except their exposure to health-care settings, ac-quisition of 2 other pathogens that were not endemic in human health-care facilities, MRSI and Salmonella spp, was also monitored.

Sample size was determined on the basis of prior estimates of the prevalence of the pathogens of interest in a similar population of dogs.42 It was assumed that re-peated sampling over 1 year would increase the chance of detecting rare pathogens such as MRSA or VRE and


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that the difference in acquisition rates between the 2 groups would be at least 15%. On the basis of these assumptions and an α value of 0.05 (2-tailed test) and β value of 0.20, it was calculated that 85 dogs would be required in each group without accounting for at-trition. Because of the restrictive inclusion criteria and difficulties in meeting sample size requirements, orga-nizations that sponsored AAI programs in Alberta that were identified through World Wide Web searches or through contact with Ontario organizations were also invited to participate. Alberta was chosen as opposed to other provinces because of the existence of several well-established AAI programs from which to select.

Informed consent was obtained from all participat-ing handlers. The study was approved by the Research Ethics Board and the Animal Care Committee of the University of Guelph.

Study design—Each dog owner was visited at the time of enrollment in the study for collection of base-line samples and to provide instructions on proper techniques for collection of subsequent samples; all vis-its were performed by a single individual (SLL). Own-ers were asked to provide a freshly voided fecal sample from their dog at this visit and were shown how to col-lect a nasal swab specimen. For collection of nasal swab specimens, owners were instructed to insert the tip of a sterile swab into the anterior nares of the dog and to rotate the swab against the inner wall; swabs were then placed in sterile transport medium.a Owners were also shown how to label all specimens and were instructed to store specimens in a cooler pending delivery to the laboratory.

Each owner enrolled in the study was provided with a kit of supplies and oral and written instructions for submitting to the research laboratory via expedited de-livery freshly collected nasal swab specimens and fecal samples every 2 months for 1 year. The 2-month time interval was chosen on the basis of available resources. Information on diet, breed, age, sex, and health status of the dogs was collected at the time of study enroll-ment. For dogs in the unexposed group, information on settings where AAIs were performed was also col-lected. To preserve anonymity, a code was assigned to each dog and coded labels were provided for owners to identify specimens they submitted. Each participating owner was also supplied with a logbook to accompany each set of specimens (ie, 6 logbooks total for the 1-year study) and was asked to record in the logbook informa-tion on antimicrobial use by animals and humans in the home, whether the dog consumed any raw meat (in-cluding poultry) or pig ears, any animal illnesses (ex-cluding preexisting conditions), and the locations and times where AAIs took place after study enrollment.

As each 2-month submission date approached, par-ticipating owners were reminded of the pending speci-men due date via electronic mail or telephone. Labora-tory records were monitored to ensure that specimens were submitted on time. Owners of dogs that did not complete the full year of surveillance were contacted to determine the reason.

During the cohort study, if a presumptive MRSA isolate was identified, a researcher visited the home to obtain a fresh fecal sample from the dog and nasal

swab specimens from all people and pets in the home, including any dogs in the home that were enrolled in the study. Specimens were stored in a cooler and trans-ported directly to the laboratory within 5 hours after collection. Owners were interviewed at that time to de-termine whether any characteristics of the dog or the people visited during AAIs may have provided an op-portunity for the dog to acquire the MRSA strain.

After the cohort study concluded, a nested case-control study was conducted to elucidate differences between dogs that visited health-care settings and ac-quired pathogens and those that visited health-care set-tings (sometimes the same facilities) and did not acquire pathogens. Owners of dogs that had visited a hospital or long-term care facility at any time during the previous year and had completed the study were mailed a pre-tested questionnaire to collect data on factors that had not been recorded in the logbooks but that might influ-ence the risk of dogs acquiring health-care–associated pathogens. Information requested included types of pa-tients visited (on respirators, receiving IV treatments, in isolation, with burns or open wounds, suspected of having infectious disease, or possibly incontinent); fre-quency with which the dog licked patients when visit-ing a health-care facility, received treats from patients, or went up on patients’ beds (scored on a scale from 0 to 4, where 0 = never, 1 = rarely, 2 = sometimes, 3 = of-ten, and 4 = usually); and whether the dog participated in activities that might have exposed it to the patho-gens of interest during the previous year (ie, drinking from toilets or coprophagia; ever or never). Owners of dogs positive for MRSA during the year were aware of the test status of their dogs before they completed the questionnaire; however, they were not aware of results of testing for other pathogens prior to completing the questionnaire.

Bacteriologic testing—As fecal samples were re-ceived by the laboratory, a portion of each was submit-ted for immediate culture for C difficile, MRSA, MRSI, and VRE by use of standard selective enrichment meth-ods and subsequent confirmation techniques, as de-scribed.43 Nasal swab specimens were tested for MRSA and MRSI only. Staphylococcus intermedius was discrim-inated from Staphylococcus aureus on the basis of nega-tive results for the S aureus latex agglutination testb and susceptibility to polymixin B and was discriminated from Staphylococcus schleiferi subsp coagulans on the basis of ability to ferment trehelose and mannitol.

The existence of the vanA gene in resistant en-terococcal isolates was confirmed by use of a commer-cial, real-time PCR assay kitc used in accordance with the manufacturer’s instructions. Isolates identified as MRSA were further characterized by means of PFGE performed as described,44 with minor modifications. Modifications to the published procedure that were incorporated included adding 10 µL of lysostatin and 50 µL of lysozyme to the buffer that the casting plugs were in, rather than adding 2 µL of lysostatin directly to the plugs, and using switch times of 0.5 to 90 seconds (vs 5.3 to 34.9 seconds) when performing electropho-resis. Isolates were also tested for the Panton-Valentine leukocidin gene, a marker of the necrotoxic effects characteristic of community-associated MRSA infec-


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tions, with a real-time PCR assay, as described.45 Iso-lates identified as C difficile were further characterized with a PCR assay for detection of genes encoding toxins A,46 B,47 and CDT (binary toxin).48

For other bacterial cultures, a fecal slurry was pre-pared by aseptically homogenizing 10 g of the remaining fecal sample with 10 mL of sterile saline (0.85% NaCl) solution. Selective isolation of potential ESBL-produc-ing E coli strains and Salmonella spp was performed as described.43 For each fecal sample, 3 potential ESBL-producing E coli isolates were inoculated onto tryptic soya agar slants and submitted to the Laboratory for Foodborne Zoonoses of the Public Health Agency of Canada for antimicrobial susceptibility testing by use of a broth microdilution method.d Susceptibility to a panel of antimicrobials was assessed with breakpoints recommended by the Clinical and Laboratory Stan-dards Institute.49 Isolates were presumptively identified as ESBL-producing or AmpC β-lactamase–producing E coli when antimicrobial resistance patterns were con-sistent with laboratory standards for presumptive iden-tification.50 Specifically, isolates identified as ESBL-pro-ducing were required to have reduced susceptibility to ceftriaxone but not cefoxitin or amoxicillin-clavulanic acid. Isolates identified as AmpC β-lactamase–produc-ing were required to have reduced susceptibility to all 3 antimicrobials. Isolates presumptively identified as AmpC β-lactamase–producing E coli were confirmed to carry the AmpC β-lactamase (cephamycinase) bla

CMY-2 gene, which encodes for broad-spectrum ac-

tivity against β-lactam antimicrobials, cephalospo-rins, and β-lactamase inhibitors such as clavulanic acid,50 with a conventional PCR assay that had been validated by testing a collection of E coli isolates with known β-lactam susceptibility phenotypes and geno-types. Multiple positive and negative controls were included in the PCR assays, which were performed with an automated instrument.e

Statistical analysis—Summary statistics were cal-culated with commercially available software.f For each of the 5 pathogens of interest, the incidence rate was calculated by dividing the number of dogs that acquired the pathogen during the study by the total number of dog-years at risk. Total number of dog-years at risk was calculated as the sum of the time that each dog in the group participated in the study, with dogs censored at the time a positive test result was first obtained or at the end of the study period. Individual dogs were excluded from data used to calculate incidence rate of a particu-lar pathogen if that pathogen was isolated from baseline specimens. Incidence rate ratios and associated 95% CIs were calculated to compare rates between groups, with unexposed dogs serving as the referent group.

Additional statistical analyses were performed to identify risk factors significantly associated with acqui-sition of the pathogens of interest. Clustering of the data was assumed to occur at 2 levels. Dogs from a particular organization involved in AAI programs were expected to be more alike than dogs from different organizations because dogs in a specific organization typically shared the same geographic region and visitation practices and, in some situations, visited the same facilities. Similarly,

results of bacteriologic culture of specimens obtained from each dog were expected to be more alike than re-sults of bacteriologic culture of specimens from differ-ent dogs. Generalized linear mixed models were devel-oped with standard software51,g to account for these 2 levels of clustering; a manual approach to model build-ing was used. Initial models were developed to exam-ine the relationship between the primary predictor of interest (ie, exposure to human health-care settings [all health-care settings, acute care only, or long-term care only]) and each outcome (ie, whether dogs acquired each of the pathogens of interest). A random intercept was introduced to control for clustering by organiza-tion and was retained when the effect of including the intercept was significant. A first-order autoregressive covariance matrix was added to account for repeated measurements for each dog.

Subsequent models were developed to examine the relationship between each of the other variables of interest (ie, consumption of raw meat, coprophagia, drinking from toilets, exposure to antimicrobials, and exposure to groups of children) and each outcome. Variables for which the P value was ≤ 0.05 were consid-ered to be significantly associated with an outcome. All variables for which the P value was 0.2 were considered for inclusion in an expanded model that related health-care exposure to each outcome. Correlations between factors were examined to ensure that none were highly correlated (ie, | r | ≥ 0.80). In the event that 2 variables were correlated, the one with the stronger association with the outcome of interest was used in the expanded model and the other was rejected. Confounding was identified by adding variables to the expanded model one at a time and examining the effect addition of each variable had on the coefficients for other factors in the model, with a change of ≥ 20% in the other coefficients considered to be an indication that confounding was present. All confounding and significant factors were retained in the final model. Terms for biologically plau-sible interactions were then added to determine their effect on the final model. Significant interaction terms were retained, and their component factors were in-cluded even when those factors were not significant on their own. Unconditional associations between expo-sure to various factors (ie, health-care facilities, groups of children, antimicrobials, and raw meat) and concur-rent reports of dog illness were also examined in a simi-lar manner.

For the case-control study, a separate database was developed to record information relevant only to those dogs in the exposure group. Although it was recognized that certain risk factors for pathogen acquisition might have been shared for certain pathogens (eg, MRSA and C difficile) because common risk factors were unknown at the time of the study, a decision was made to analyze each pathogen separately, as opposed to defining a case dog as one that acquired ≥ 1 health-care–associated pathogen. Thus, separate analyses were performed for each pathogen of interest, with exposed dogs for which results of bacteriologic testing of baseline samples were negative for that pathogen but results for at least 1 sub-sequent sample were positive classified as cases, and exposed dogs for which results of bacteriologic testing


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of all samples were negative for that pathogen classified as controls. Scores for frequency of licking, accepting treats, and sitting on beds were dichotomized (ie, never or rarely vs sometimes, often, or usually) for inclusion in these analyses. Other variables such as coprophagia and drinking from toilets were treated as dichotomous (ever or never). Univariate logistic regression analyses performed with standard softwaref were used to exam-ine associations between questionnaire responses and case-control status. To determine whether shedding of one pathogen was associated with shedding of another, relationships between pairs of pathogens were also eval-uated. The Fisher exact test was used in circumstances when any cell in the 2 X 2 contingency table contained ≤ 10 observations. Multivariate logistic regression analy-ses were performed whenever adequate numbers of case dogs were identified for a particular pathogen.

Results

Dogs—A total of 200 dogs (100 in the exposed group and 100 in the unexposed group) were enrolled in the study between May and November 2005. Of these, 170 resided in Ontario and 30 resided in Alberta. Owners of 6 dogs (4 in the exposed group and 2 in the unexposed group) stopped participating after baseline specimens were collected, and these dogs were elimi-nated from the study. The remaining 194 dogs (96 in the exposed group and 98 in the unexposed group) were included in the study. Fourteen geographic regions (de-fined as the regions served by the various AAI organiza-tions) were represented (mean dogs/region, 14; median, 8; and range, 1 to 28). Dogs included in the study repre-sented 19 different organizations involved in AAI pro-grams (median number of dogs/organization, 8; range, 1 to 28). One hundred fifty-six dog owners participated in the study (median number of dogs/owner, 1; range, 1 to 4), and 1,324 sets of specimens (ie, a fecal sample and nasal swab specimen) were submitted for testing (median number of specimen sets/dog, 7; range, 2 to 7). None of the dog owners were employed in health-care–related fields at the time of the study; employment status of other individuals living in the same homes as study dogs was not known. Ten dogs dropped out of the study at various times during the 1-year study period for reasons unrelated to pathogen acquisition, includ-ing 2 dogs that died of noninfectious causes, 1 dog that was moved overseas, and 6 dogs that stopped partici-pating in AAI programs. These dogs were censored after the last set of specimens was submitted.

Once the study began, several dogs that had pre-viously been classified as unexposed started visiting health-care facilities, and several that had been clas-sified as exposed ceased visiting health-care facilities. Therefore, a decision was made to classify the exposure status of each dog at each sampling time on the basis of the reported visitation history for the 2 months pre-ceding specimen submission. In total, 116 of the 194 (59.8%) dogs were exposed to health-care facilities at some time during the study; however, the total time that these dogs were exposed to health-care facilities was only 79.7 years (958 months), whereas the total time these dogs participated in the study was 188.2 years (2,260 months). Median frequency with which

dogs engaged in AAIs during the study was 2 times/wk (range, 1 time/mo to 6 times/wk). Ninety-six of the 116 (82.8%) exposed dogs visited at least 2 health-care fa-cilities in any given month. Thirty of the 116 (25.9%) exposed dogs were also involved in AAI programs that involved children in the community.

Of the 194 dogs included in the study, 22 (12.2%) were Golden Retrievers, 20 (10%) were of mixed breed-ing, and 19 (9.8%) were Labrador Retrievers. Two (1.0%) were sexually intact females, 99 (51.0%) were spayed females, and 93 (47.9%) were neutered males. Breed and sex distributions did not differ significantly between dogs recruited for the exposed group and dogs recruited for the unexposed groups. Similarly, mean age of dogs recruited for the exposed group (5.9 years; range, 0.9 to 16 years) was not significantly (P = 0.22) different from mean age of dogs recruited for the unex-posed group (5.5 years; range, 0.3 to 16 years).

Bacteriologic testing—Methicillin-resistant Staph-ylococcus intermedius was isolated from the baseline nasal swab specimen of 1 dog that was subsequently exposed to health-care facilities. Subsequent nasal swab specimens from this dog were negative for MRSI, and colonization with MRSI was not detected in any other dog during the study period. Salmonella organisms were recovered from baseline fecal samples from 5 dogs, none of which were subsequently exposed to health-care fa-cilities, and from an additional 42 fecal samples from 32 other dogs. Rate of detection of Salmonella spp in fecal samples did not differ significantly (P = 0.22) between exposure groups; however, it did differ significantly (P < 0.001) between dogs that had consumed raw meat in the 2 months before specimen submission and dogs that had not. Additional results regarding shedding of Salmonella spp are reported elsewhere.43

Methicillin-resistant S aureus was not detected in baseline specimens obtained from any of the dogs, but was subsequently recovered from nasal swab specimens from 3 dogs (2 dogs exposed to health-care facilities and 1 not exposed) and from fecal samples from 6 oth-er dogs (5 exposed to health-care facilities and 1 not exposed). Methicillin-resistant S aureus was not iso-lated from concurrent nasal swab specimens and fecal samples in any dogs, and MRSA was not isolated from any dog more than once. The Panton-Valentine leuko-cidin gene was detected in 2 fecal samples and 1 nasal swab specimen, all of which were obtained from dogs exposed to health-care facilities. The incidence rate of MRSA was 4.7 times as high among dogs that had visited health-care facilities in the 2 months prior to specimen submission as it was among dogs that had not visited health-care facilities (Table 1).

Three of the 9 MRSA isolates were classified as CMRSA-10 (USA300)48–50 by means of PFGE, and the remaining 6 were classified as CMRSA-2 (USA100).12,51 Four of the 7 dogs exposed to health-care facilities and from which MRSA was isolated had visited only long-term care facilities in the 2 months preceding speci-men submission, 2 had visited long-term care facilities and hospitals, and 1 had visited only hospitals. None of these dogs was reported to be ill in the 2 months preceding or the 2 months following submission of the specimens from which MRSA was isolated.


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Escherichia coli was isolated from 106 fecal sam-ples from 59 dogs; all isolates were identified as pro-ducing AmpC β-lactamase (ie, reduced susceptibility to cefoxitin, ceftriaxone, and amoxicillin-clavulanic acid) and possessed the blaCMY-2 (cephamycinase) gene. None of the isolates were identified as ESBL-produc-ing E coli strains. Isolates producing AmpC β-lactamase were recovered from baseline samples from 22 (11.3%) dogs, all of which had been recruited for the unexposed group. The remaining 84 isolates were recovered from subsequent fecal samples from 37 dogs. Sixteen of these dogs had not been exposed to health-care facilities in the 2 months prior to sample submission; the remain-ing 21 had.

Clostridium difficile was detected in baseline fecal samples from 9 (4.6%) dogs, all of which had been re-cruited for the unexposed group. During the following year, C difficile was recovered from 50 fecal samples from 39 other dogs. Sixteen of these dogs had not been exposed to health-care facilities during the 2 months prior to sample submission; the remaining 23 had. Only 1 of the 16 dogs that had not been exposed to health-care facilities had 2 consecutive positive test results, whereas 3 of the dogs that had been exposed to health-care facilities had 3 consecutive positive test results and 3 others had 2 consecutive positive test results. None of the dogs from which C difficile was recovered had diarrhea in the 2 months preceding or the 2 months following sample submission.

Results of the PCR assay for toxins A, B, and CDT indicated that 38 of the 59 (64.4%) C difficile isolates were toxigenic (ie, contained genes encoding at least 1 of those toxins). In total, 31 isolates, including 6 obtained from baseline samples, had genes for C difficile toxins A and B but not CDT (ie, A+B+CDT– profile), 3 isolates had genes for all 3 toxins (ie, A+B+CDT+ profile), 3 had the gene for toxin B but not toxin A or toxin CDT (ie, A–B+CDT– pro-file), and 1 had the gene for toxin CDT but not toxin A or toxin B (ie, A–B–CDT+ profile; Figure 1). Twenty-one of the 59 C difficile isolates, including 3 obtained from base-line samples, were nontoxigenic (ie, negative for the genes for toxins A, B, and CDT).

Vancomycin-resistant enterococci were recovered from 1 fecal sample during the study period. The dog had been exposed to a hospital approximately once a

week after baseline specimens were collected. Risk fac-tors for acquisition could not be evaluated because of the low incidence of VRE acquisition.

Detection rates of pathogens for dogs from Ontario were not significantly different from rates for dogs from Alberta. Scatterplots of the point prevalence of health-care–associated pathogens for each exposure group re-vealed high variability from one time point to the next, with no visually discernible pattern that would indicate an increase or decrease in prevalence over time.

Whether dogs had diarrhea in the 2 months pri-or to specimen submission was not significantly (P > 0.69) associated with whether they had positive test results for any of the health-care–associated pathogens evaluated. A review of the logbooks indicated that 16 of 21 (76%) owners of dogs with diarrhea and 8 of 9 (89%) owners of dogs with UTIs continued to partici-pate in AAIs with their dogs despite their dog being ill. Additional analysis of logbook data also indicated that dogs with diarrhea were 14.9 times as likely to have been treated with antimicrobials as were dogs without diarrhea (95% CI, 5.00 to 44.53; P < 0.001). Two of the dogs were treated with antimicrobials after the episode of diarrhea was recorded; 8 were treated in the week preceding the date when diarrhea was first identified.

Statistical modeling—Generalized linear mixed model analyses revealed that clustering by AAI orga-

Incidencerate (cases/dog-year)

No.(%)ofsamples No.ofdogs Exposed Unexposed IncidencePathogen withpositiveresults withpositiveresults dogs dogs rateratio 95%CI

Clostridium difficile 50 (4.4) 39 0.314 0.129 2.4* 1.22−5.07MRSA† 9 (0.8) 9 0.092 0.015 4.7‡ 0.91−47.10AmpC β-lactamase–producing 84 (7.4) 37 0.263 0.147 1.79 0.89−3.67 Escherichia coliVRE 1 (0.09) 1 0.013 0 NC NC

The incidence rate ratio represents the incidence of shedding of a particular pathogen in the exposed dogs, compared with the incidence in the unexposed dogs. Incidence rate was calculated by dividing the number of dogs that acquired the pathogen for the first time during the study by the total number of dog-years at risk. Exposed dogs were dogs that performed AAIs in human health-care facilities; unexposed dogs were dogs that performed AAIs in other settings.

*Incidence rate ratio is significantly (P 0.001) different from 1 by use of a Fisher exact test. †For detection of MRSA only, fecal samples and nasal swab specimens were collected. ‡Incidence rate ratio is significantly (P 0.05) different from 1 by use of a Fisher exact test.

NC = Not calculated.

Table1—Incidenceofisolationofselectedhealth-care–associatedpathogensfromfecalsamples(n=1,130)collectedevery2monthsfor1yearfrom194dogsthatparticipatedinAAIsinOntarioandAlbertaduring2005and2006.

Figure1—Toxinprofilesof50Clostridium difficileisolatesrecov-eredovera1-yearperiodfrom1,130fecalsamplesofdogsthatparticipatedinAAIsandthatwere(greybars)orwerenot(blackbars)exposedtohumanhealth-carefacilitiesduringthe2monthspriortosamplesubmission.ErrorbarsrepresentSE.*Percent-agewassignificantly(P=0.01)differentbetweengroups.


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nization was significant for all associations between acquisition of AmpC β-lactamase–producing E coli or C difficile and all variables examined. Clustering by AAI organization was not significant for acquisition of MRSA because no 1 organization had more than 1 dog positive for MRSA. Models testing the relationship between acquisition of MRSA and antimicrobial use did not converge because none of the dogs positive for MRSA had a history of antimicrobial exposure, either through direct administration or through administra-tion to other animals or people in the same household. None of the created interaction terms were significant in any of the analyses.

Univariate GLMM analysis revealed that exposure to health-care facilities, specifically long-term care fa-cilities, was significantly associated with MRSA ac-quisition (Table 2). Exposure to groups of children in settings where MRSA was not believed to be endemic, such as schools or libraries, was also a significant risk factor for MRSA acquisition. When exposure to groups of children and exposure to health-care facilities were included in a multivariate model, both factors were still significant, although the strength of association with exposure to health-care facilities was somewhat less (OR, 5.2; 95% CI, 1.06 to 25.49; P = 0.04). On the other hand, controlling for exposure to children elimi-nated the significant association between acquisition of MRSA and exposure to long-term care facilities (OR, 4.0; 95% CI, 0.98 to 16.46; P = 0.05).

Exposure to health-care facilities was not a signifi-cant risk factor for acquisition of AmpC β-lactamase–producing E coli; however, consumption of raw meat was (Table 2). Of the 59 dogs that acquired AmpC β-lactamase–producing E coli, 11 (18.6%) had been treated with antimicrobials within the 2 months prior to submission of the fecal sample from which the or-ganism was isolated. Three (5.1%) dogs reportedly had an eye infection in the 2 months prior to sample sub-mission, and 2 dogs reportedly had a UTI.

Univariate GLMM analysis revealed that exposure to health-care facilities, whether hospitals or long-term care facilities, and exposure to groups of children were significant risk factors for acquiring C difficile (Table 2). Direct and indirect exposure to antimicrobials was also significantly associated with acquisition of C difficile. Multivariate analysis revealed that the association be-tween exposure to health-care facilities and acquisition of C difficile was maintained when exposure to children and direct and indirect exposure to antimicrobials were controlled for (Table 3). Similar results were obtained when the type of health-care facility was specified.

Univariate GLMM analysis revealed a significant association between acquisition of A+B+CDT– strains of C difficile and exposure to health-care facilities (OR, 2.5; 95% CI, 1.23 to 5.20; P = 0.01). When the model was modified to specify the type of health-care facil-ity, exposure to hospitals was not significant (OR, 1.4; 95% CI, 0.54 to 3.75; P = 0.48); however, exposure to

Factor OR 95%CI P value

Dogs positive for C difficile Exposure to human health-care facilities 3.3 2.16−3.39 0.001 Exposure to hospitals only 2.2 1.18−3.95 0.01 Exposure to long-term care facilities only 2.3 1.48−3.55 0.001 Exposure to groups of children 3.5 2.06−5.85 0.001 Antimicrobial use 2.2 1.34−3.55 0.002 Others in home treated with antimicrobials 3.2 1.97−5.24 0.001 Fed raw meat 0.8 0.28−2.14 0.62

Dogs positive for MRSA Exposure to human health-care facilities 6.3 1.29−30.26 0.02 Exposure to hospitals only 3.7 0.91−14.81 0.07 Exposure to long-term care facilities only 4.9 1.21−19.59 0.02 Exposure to groups of children 7.1 1.87−26.61 0.004 Antimicrobial use NC NC NC Others in home treated with antimicrobials NC NC NC Fed raw meat 1.4 0.28−6.58 0.70

Dogs positive for AmpC β-lactamase–producing E coli Exposure to human health-care facilities 1.3 0.89−2.00 0.17 Exposure to hospitals only 1.2 0.63−2.25 0.60 Exposure to long-term care facilities only 1.5 0.93−2.27 0.10 Exposure to groups of children 1.2 0.70−2.07 0.50 Antimicrobial use 2.6 1.55−4.48 0.001 Others in home treated with antimicrobials 1.6 0.92−2.67 0.10 Fed raw meat 17.2 9.35−32.26 0.001

Data represent results of bacterial culture of 1,324 sets of fecal samples and nasal swab specimens from 194 dogs. Analyses were adjusted for clustering within dogs (ie, collection of multiple samples from individual dogs) and within organiza-tions sponsoring the AAI program. The OR represents the odds that dogs exposed to the factor of interest would acquire the pathogen, compared with the odds for dogs not exposed to the factor of interest. Dogs were classified as exposed if they had been exposed to the factor at any time during the 2 months prior to specimen submission.

NC = Not calculated.

Table2—Resultsofunivariateanalysesoffactorsassociatedwithisolationofvarioushealth-care–associatedpathogensfromdogsthatparticipatedinAAIs.


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long-term care facilities was significant (OR, 2.6; 95% CI, 1.29 to 5.38; P = 0.008). No other associations be-tween toxin profiles and exposure to health-care facili-ties were detected.

When predictor variables were screened for inclu-sion in multivariate analyses, dogs that were exposed to health-care facilities were identified as 2.0 times as like-ly to also visit with groups of children than were unex-posed dogs (95% CI, 1.37 to 2.78; P < 0.001), although the correlation between the 2 predictors was weak (r = 0.10). In addition, dogs that were treated with antimi-crobials were 2.6 times as likely to have been in a home where others were treated with antimicrobials as were dogs that were not treated (95% CI, 1.50 to 4.54; P = 0.001). Again, the correlation between the factors was low (r = 0.10).

Dogs that visited health-care facilities during any given 2-month period were 3.9 times as likely to have had diarrhea during that same period as were dogs that were not exposed to health-care facilities (95% CI, 1.44 to 10.42; P = 0.007). Dogs that had diarrhea during the 2 months before specimen submission were more likely to be shedding Salmonella organisms than were dogs without diarrhea (OR, 3.0; 95% CI, 1.29 to 7.06; P = 0.01), but they were not more likely to be shedding other pathogens. Exposed dogs were also more likely to have had a UTI than were unexposed dogs (OR, 6.5; 95% CI, 1.77 to 23.63; P = 0.005). However, having a UTI in the 2 months prior to specimen submission was not significantly associated with identification of any pathogen, although the presence of a UTI in the 2 months following specimen submission was strongly associated with detection of AmpC β-lactamase–pro-ducing E coli in feces (OR, 8.2; 95% CI, 3.13 to 17.64; P < 0.001). Logbook records were not detailed enough to indicate the agents that caused UTIs. Exposure to health-care facilities was not associated with any other reported illness.

Case-control analyses—Questionnaires were mailed to owners of all 114 dogs that completed the study and had visited health-care facilities at some time during the study period. All questionnaires were returned with all questions answered. Six of the 114 (5.3%) owners reported being encouraged to take their dogs into isolation rooms, where gowning and gloving of health-care workers was required. None of these dogs acquired any health-care–associated pathogens during the study, although it was not known whether the patients in isolation rooms that were visited were infected with any of the pathogens evaluated in the study. Owners were asked whether they had changed the

way that their dogs interacted with patients at any time during the previous year. One replied that she had stopped her dog from licking patients; another replied that she had stopped her dog from accepting treats. Neither of the dogs tested positive for any of the pathogens evaluated during the study period. For statistical purposes, both dogs were treated as if these behaviors had not stopped.

Eight of the owner-dog teams involved in the case-control portion of the present study had participated in a 2004 study39 in which data about dogs involved in AAI programs had been collected but had not quali-fied for inclusion in analyses performed in that study because the dogs were visiting retirement residences, as opposed to health-care facilities, at the time. To evaluate the stability of behaviors from year to year and, hence, the reliability of the questionnaire, questionnaire re-sponses from the previous study were compared with responses provided for the questionnaire in the pres-ent study. For these 8 dogs, answers regarding whether dogs went up on patients’ beds or licked patients when visiting a health-care facility and on coprophagia were identical for the 2 questionnaires.

Univariate logistic regression analyses revealed that licking patients and receiving treats from patients were significant risk factors for acquisition of MRSA during the study period and that licking patients and going up on patients’ beds were significant risk factors for acquisition of C difficile (Table 4). Consuming feces from other animals was identified as protective against acquisition of C difficile. Fecal samples that contained one type of pathogen were not more likely to contain another type of pathogen. The number of dogs positive for MRSA was too small to support the development of a stable multivariate model. When licking patients was controlled for in a multivariate model, the association between going up on patients’ beds and acquisition of C difficile remained significant (OR, 1.4; 95% CI, 1.01 to 2.00; P = 0.049); however, when going up on patients’ beds was controlled for in the multivariate model, lick-ing patients was no longer significantly associated with acquisition of C difficile (OR, 2.1; 95% CI, 0.79 to 5.68; P = 0.14).

MRSA community transmission—Methicillin-re-sistant S aureus was not recovered from specimens col-lected from people and other pets in homes of dogs from which MRSA was isolated. Interviews with owners re-vealed that 6 of the 7 dogs positive for MRSA frequently licked people, including the hands and faces of health-care workers and patients or residents in health-care facilities. Three of the 7 dogs that visited health-care facilities were reported to go up on patients’ beds when interacting with patients. These reports were consistent with owners’ responses on questionnaires submitted at the conclusion of the study. One of the dogs that were positive for MRSA was known to have had contact with 2 patients positive for MRSA. Consequently, swab spec-imens were obtained from the 2 patients and submitted for bacterial culture. Molecular characterization of the isolates by means of PFGE revealed that the strain of MRSA recovered from the dog was not the same as the strain recovered from these 2 patients. The owner of one of the 2 unexposed dogs from which MRSA was isolated reported that the dog regularly consumed raw

Factor OR 95%CI Pvalue

Exposure to human health-care 2.2 1.43−3.50 0.001 facilitiesExposure to groups of children 2.4 1.40−4.16 0.002Antimicrobial use 1.8 1.09−2.92 0.02Others in home treated 2.2 1.30−3.61 0.003 with antimicrobials

See Table 2 for key.

Table3—ResultsofmultivariategeneralizedlinearmixedmodelanalysisoffactorsindependentlyassociatedwithacquisitionofC difficilebydogsthatparticipatedinAAIs.


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poultry, raw fish, and horse feces. No potential source of MRSA was identified for the second unexposed dog.

Discussion

Results of the present study suggested that dogs that visit health-care facilities when participating in AAIs were more likely to acquire MRSA and C difficile than were dogs that performed AAIs in other settings. In addition, for dogs that visited health-care facilities, licking patients and receiving treats from patients were significant risk factors for acquisition of MRSA and licking patients and going up on patients’ beds were significant risk factors for acquisition of C difficile.

Given the previous evidence29,38 that pets of health-care workers colonized or infected with MRSA could also acquire MRSA, we expected that dogs that visited pa-tients and health-care workers in conjunction with AAI programs would also be vulnerable to acquiring health-care–associated pathogens. Therefore, the significant as-sociation between exposure to health-care facilities and acquisition of MRSA was not surprising. What we did not expect was the high incidence rate for acquisition of MRSA. The total time that dogs participating in the present study were exposed to health-care facilities was 79.7 dog-years, and during this time, 7 dogs acquired MRSA, representing 9% of dogs/y. Estimates of the point prevalence MRSA carriage in a variety of populations of dogs in Ontario have ranged from 0% to 0.5%.42,52,h Al-though the point prevalence of MRSA carriage did not exceed 2% at any time during the present study, the high

incidence rate is cause for concern, given the zoonotic potential of this pathogen. More frequent sampling may have revealed other carriers but was not possible because of resource constraints. Data regarding antimicrobial use by people in the home did not support a hypothesis that dogs were infected by individuals living in the same household that were known to be infected with MRSA. Despite the lack of evidence that any of the patients visited by dogs in the present study were infected with MRSA, our results suggest a need to include MRSA in the list of those zoonotic agents targeted in infection preven-tion and control policies for AAI programs. The available evidence also supports the inclusion of animal contact as relevant information in the investigation of nosocomial MRSA infection in humans. Because MRSA coloniza-tion has been associated with an increased likelihood of MRSA infection when humans and horses are admitted to hospitals,53,54 dogs that visit health-care facilities may be more vulnerable than other dogs to MRSA infection. However, additional research is required to evaluate this hypothesis.

The small number of dogs positive for MRSA pre-cluded further analysis of whether the risk of acquisition was associated with molecular type of the MRSA strain. However, the usefulness of such an exercise would have been questionable anyway because molecular typing alone can provide only limited information regarding the origin of any MRSA recovered. Establishing the ori-gin of a bacterial strain is important from epidemiologic and clinical perspectives, but doing so can be challeng-ing without strong data (eg, data connecting dogs to

No.(%)ofdogs No.(%)ofdogsFactor exposedtofactor notexposedtofactor OR 95%CI P value

Dogs positive for C difficile Coprophagia 0 (0) 24 (20.3) 0.1* 0.00–0.63 0.02 Drank from toilets 1 (4.2) 8 (12.5) 0.3 0.01–2.52 0.25 Visited incontinent patients 15 (62.5) 33 (51.6) 1.6 0.54–4.67 0.36 Licked patients 13 (54.2) 16 (2.89) 3.5 1.18–10.62 0.01 Received treats from patients 13 (54.2) 31 (48.4) 1.3 0.44–3.61 0.63 Went up on patients’ beds† 10 (54.2) 18 (28.1) 1.5 1.02–8.92 0.02

Dogs positive for MRSA Coprophagia 2 (28.6) 14 (21.2) 1.5 0.13–10.27 0.66 Drank from toilets 1 (14.3) 8 (12.1) 1.2 0.02–12.24 0.87 Visited incontinent patients 6 (85.7) 34 (51.5) 5.6 0.62–267.67 0.08 Licked patients 6 (85.7) 16 (24.2) 18.8 1.95–878.88 0.001 Received treats from patients 6 (85.7) 23 (34.9) 11.2 1.21–527.55 0.009 Went up on patients’ beds† 4 (57.1) 18 (27.3) 3.6 0.53–26.18 0.10

Dogs positive for AmpC β-lactamase–producing E coli Coprophagia 9 (34.6) 13 (20.3) 2.1 0.65–6.35 0.15 Drank from toilets 3 (11.5) 8 (12.5) 0.9 0.14–4.26 0.89 Visited incontinent patients 19 (73.1) 33 (51.6) 2.5 0.87–8.13 0.06 Licked patients 8 (30.8) 16 (25.0) 1.3 0.42–4.02 0.58 Received treats from patients 9 (34.6) 23 (35.9) 0.9 0.32–2.68 0.91 Went up on patients’ beds† 8 (30.8) 18 (28.1) 1.1 0.36–3.37 0.80

Samples were collected once every 2 months for 1 year. Coprophagia and drinking from toilets refer to activities away from the health-care facility; the other factors refer to activities while at the health-care facility.

*Odds ratio was derived from a Cornfield estimate of the value of a 50% CI. †Without a barrier between the dog and the sheets on the bed.

See Table 2 for remainder of key.

Table 4—Results of univariate analyses of factors associated with isolation of various health-care–associated pathogens from fecal specimens (C difficile, MRSA, and AmpC β-lactamase–producingE coli)andnasalswabspecimens(MRSAonly)atleastonceduringthepreviousyearamong114dogsthatparticipatedinAAIsathumanhealth-carefacilitiesduringthesameyear.


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patients with the same MRSA strains). Three dogs in the present study had the CMRSA-10 (USA300) strain, which has been associated with infections of communi-ty origin.55–57 However, this does not necessarily mean that the dogs could not have acquired the organism from hospitalized patients because this strain has become an important cause of health-care–associated infections.58 In addition, the finding that 6 of the MRSA isolates pos-sessed the PFGE profile of a clone that originated in hospitals (CMRSA-2 [USA100]) does not rule out the possibility that the dogs acquired the organism outside health-care facilities because this clone is also a com-mon community-associated pathogen in Canada59 and has been identified in dogs that have had no contact with human health-care settings.27 The present study failed to connect any of the dogs positive for MRSA with a specific patient source, despite indications that 1 dog had visited with patients known to be colonized with MRSA. Nevertheless, it seems likely that unidenti-fied colonized or infected patients or health-care work-ers or the health-care facility environment itself was the source of the organism in the dogs.

The inability to detect MRSA in people and pets liv-ing in the same homes as dogs positive for MRSA sup-ports the supposition that the home environment was not the source of the organism, although this possibility could not be ruled out because of the small sample size and the time lag between submission of specimens from which MRSA was isolated and subsequent collection of specimens from people and pets in the same home. Reports29,38 of dogs serving as reservoirs of MRSA for colonized or infected humans suggested that there may be a risk that dogs involved in AAI programs may trans-mit the organism to their owners or other individuals in the family as a result of close and sustained contact between dogs and their owners. Because of the afore-mentioned limitations, the potential for dogs involved in AAI programs to spread MRSA into the community also cannot be dismissed. Follow-up testing of fam-ily members for VRE, AmpC β-lactamase–producing E coli, toxigenic strains of C difficile, and Salmonella spp was not performed because of several months’ delay be-tween specimen submission and laboratory confirma-tion of isolate identification. Such follow-up testing would have helped to support or refute the hypothesis that dogs that acquire health-care–associated pathogens are at risk of spreading them in the community.

The finding that MRSA was isolated more often from fecal samples than from nasal swab specimens in the present study was interesting given that the ante-rior nares appears to be the most frequently reported site for recovering MRSA from healthy dogs.52,60,61 The difference between findings of the present and previ-ous studies may be associated with route of acquisi-tion. Perhaps dogs that ingest MRSA (eg, via licking) become colonized intestinally, while those that acquire MRSA through contact become colonized nasally. Al-ternatively, in previous studies,52,60,61 perineal or rectal swab specimens, but not fecal samples, were tested for MRSA, and perhaps bacterial culture of feces is a more sensitive technique for detecting MRSA.

Several factors were strongly associated with ac-quisition of C difficile in multivariate analyses in the

present study. Antimicrobial treatment, particularly with agents that disrupt the normal anaerobic gut flora, is an established risk factor for colonization and infec-tion with C difficile in humans and other animals,62–65 and it follows that dogs involved in AAI programs that are treated with antimicrobials and exposed to health-care facilities may be vulnerable to C difficile–associated disease. For this reason and because of the finding in the present study that dogs treated with antimicrobials are more likely to shed resistant strains of E coli, dogs participating in AAIs should be temporarily suspended from visiting health-care facilities while being treated with antimicrobials.

The most common toxin profile for C difficile iso-lates in the present study, A+B+CDT–, was also the most common profile among isolates from hospitalized peo-ple in Ontario during the same period.i Therefore, it makes sense that dogs engaged in AAIs at health-care facilities were at greater risk of acquiring this particular type of C difficile, relative to unexposed dogs. On the other hand, this toxin profile also appears to be com-mon in humans and animals outside health-care set-tings.30,66 Strains with the ability to produce all 3 tox-ins are the second most common in Ontario,i and had more dogs acquired strains with the A+B+CDT+ profile, an association with health-care facilities may have been detected. The other toxigenic strains are also of clinical relevance, although they seem to be less common.67,i

An interesting result of the case-control portion of the present study was the observation that copropha-gia had a significant protective effect on dogs acquiring C difficile. This association is unlikely to be spurious because it was also detected in another study42 involv-ing a similar population of dogs. Perhaps the feces of other animals has a probiotic effect, providing a source of intestinal flora that effectively prevent C difficile from colonizing.

The significant relationship between exposure to groups of children and acquisition of C difficile and MRSA in the present study was unexpected but not unexplainable. For C difficile, high prevalences of col-onization among healthy children ≤ 5 years old have been reported,68,69 with prevalence diminishing as age increased. Reports of MRSA infection in children are becoming more common, although they were not com-mon when the present study was designed, and out-breaks of MRSA infection and colonization have been reported in children’s day care settings.70,71 Some re-searchers have also reported high rates of colonization in other populations of children. For example, a study72 of MRSA nasal colonization in 500 healthy children be-tween 2 weeks and 21 years of age undergoing routine health evaluations in Tennessee revealed an increase in prevalence over time, with prevalences of 0.8% in 2001 and 9.2% in 2004, and a US study73 that was part of the 2001–2002 National Health and Nutrition Examina-tion Survey revealed a prevalence of 0.8% during 2001. Even if the prevalence of colonization in children is low, poor hygiene habits may increase the probability that dogs exposed to children will be colonized with the or-ganism. On the other hand, the associations between exposure to children and pathogen acquisition by dogs in the present study may have been, at least in part,


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an artifact of the recruitment process because dogs that visited health-care facilities were twice as likely to visit with children, compared with unexposed dogs. How-ever, the fact that results of multivariate analyses indi-cated that visiting health-care facilities and exposure to children were independent risk factors for acquisition of pathogens suggests that detected associations were unlikely to be artifactual. Additional research involving dogs engaged in AAIs at health-care facilities that do not interact with groups of children may better clarify the impact that visiting health-care facilities has on the likelihood of pathogen acquisition.

For dogs in the present study, visiting long-term care facilities was associated with a stronger probability of acquiring health-care–associated pathogens than was visiting hospitals. This was not particularly surprising. Although these pathogens can be endemic in both types of facility, infection control practices likely vary greatly and the prevalence of colonization with certain patho-gens is likely higher for residents of long-term care fa-cilities. Furthermore, long-term care facilities are more likely to house elderly individuals, who have higher risks of nasal colonization with MRSA73 and C difficile–associated diarrhea74,75 than do younger individuals, who may also have deficient hygiene practices.

Acquisition of AmpC β-lactamase–producing E coli by dogs in the present study appeared to be unrelated to exposure to health-care facilities. In fact, the only risk factors for acquisition of AmpC β-lactamase–produc-ing E coli that were identified in the present study (ie, antimicrobial treatment and consumption of raw meat) were related to exposures outside health-care facilities. No ESBL-producing E coli were identified, which was unexpected given the major role ESBL-producing E coli plays in nosocomial infections in many North Ameri-can health-care settings.18,19 However, the prevalence of ESBL-producing E coli in the health-care facilities vis-ited by dogs in the present study was unknown. On the other hand, the fact that ESBL-producing E coli strains were not isolated from dogs that were not exposed to health-care settings was predictable, especially given that a previous studyh of 188 healthy dogs in Ontario also failed to detect any ESBL-producing E coli strains in their feces and a study42 of carriage by Ontario dogs actively involved in hospital visitation detected ESBL-producing E coli in only 1 of 102 (1%) dogs. Isolates fitting the AmpC β-lactamase–producing resistance profile were recovered in all 3 studies, so it is unlikely that the lack of ESBL-producing strains was due to in-sensitive culture techniques.

No conclusions could be made regarding isolation of VRE from dogs exposed to health-care facilities in the present study. However, if dogs were truly acquiring health-care–associated pathogens when visiting health-care facilities, we would have expected them to be vul-nerable to acquiring VRE, which is known for its har-diness and pervasiveness on skin and in environments where VRE is endemic.63 One possible explanation for the fact that VRE was isolated from only 1 dog in the present study could be that dogs visited facilities in which VRE was not endemic, and the same explanation could apply to our failure to identify dogs that acquired ESBL-producing E coli during the study. One way to in-

vestigate this hypothesis would have been to contact infection control personnel in the relevant health-care facilities and ask what their experiences had been with these pathogens. However, this was not done because of the reluctance of health-care facilities to share these data.

The lack of information regarding endemic preva-lences of infection or colonization of patients and of en-vironmental contamination with pathogens in health-care facilities visited by dogs in the study limits the extent to which our results can be generalized to other geographic regions, other facilities, or even other areas within the same facilities visited. Presumably, the risk that dogs will acquire pathogens is contingent on the risk of exposure. Thus, the risk of exposure should be considered when making decisions regarding suitabil-ity of facilities for AAIs.

It was reassuring to find that dogs in the present study that acquired pathogens were not any more likely to be sick in the 2 months prior to specimen submis-sion than were dogs that did not acquire pathogens. This finding may imply that visiting health-care facili-ties did not pose a health risk to the dogs, regardless of their degree of contact with patients and the environ-ment. However, when pathogen status was ignored and only exposure to health-care facilities was examined for an association with illness, dogs exposed to health-care facilities were more likely to develop diarrhea and UTIs than were dogs that performed AAIs in other set-tings. In fact, by searching logbooks for diseases that followed, rather than preceded a positive test result, we found that having a positive test result for AmpC β-lactamase–producing E coli in the preceding 2 months was a significant risk factor for development of a UTI. The question remains whether these dogs actually had an increased likelihood of developing illness or other factors better explain the association. Dogs may have acquired agents other than those evaluated in the pres-ent study or the stress of visiting health-care facilities may have made some dogs more vulnerable to infec-tion. More research is needed to clarify the mechanism underlying this association between animal illness and exposure to health-care facilities.

Dog diet was monitored in the present study be-cause of the potential confounding effect of consump-tion of raw foods of animal origin on pathogen acquisi-tion. Studies from North America76,77 and elsewhere78–83 have revealed that raw meat and natural pet treats and chews are a potential source of all of the health-care– associated pathogens examined in the present study, and results of our study indicated that consuming raw foods of animal origin increased the likelihood that dogs would acquire AmpC β-lactamase–producing E coli but not MRSA or C difficile. This finding is sup-ported by other researchh that revealed that dogs that con-sume pig ear treats or raw bones are at increased likeli-hood of shedding antimicrobial-resistant strains of E coli, relative to other dogs in Ontario. Because of the higher probability that dogs fed raw meat will shed AmpC β-lactamase–producing E coli and salmonellae re-ported here and elsewhere,43 managers of AAI pro-grams should reevaluate their policies for permitting volunteers to feed these items to dogs that visit any


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population of patients with increased vulnerability to infection.

The questionnaire used in the present study re-vealed some useful information for the development of infection prevention and control protocols to minimize the probability that dogs involved in AAI programs would acquire health-care–associated pathogens dur-ing visits. Dogs exposed to health-care facilities were more likely to acquire MRSA and C difficile if they were allowed to lick patients and were more likely to acquire C difficile if they were allowed to sit on patients’ beds without a barrier between them and the bedding. The skin of patients15,84 and workers85 in health-care facili-ties is vulnerable to contamination with health-care–associated pathogens, and various surfaces that dogs may come into contact with when visiting health-care facilities, such as bed linens, bed rails, and floors, can be contaminated.15,17,86 Thus, the wisdom of allowing intimate contact between dogs and patients is question-able, both from the perspective of canine health and from the perspective of patient health. Licking has been implicated as a cause of serious infections in vulnerable individuals.87–89 Furthermore, allowing dogs onto beds may provide opportunities for dogs to deposit whatever pathogens have collected on their hair or feet onto the bedding.j

The present study had several important limita-tions. Biases introduced by the retrospective nature of the questionnaire and the reclassification of dogs as exposed or unexposed would generally have tended to make it more likely that we would have accepted, rather than rejected, the null hypothesis. It would have been interesting to investigate the influence that amount of exposure to health-care facilities would have had on the probability of dogs acquiring health-care–associ-ated pathogens; however, the variability in the amount of detail recorded in the logbooks precluded us from doing so.

A few general findings of the present study war-rant discussion. The first is that many dog owners con-tinued to visit health-care facilities even when their dogs had diarrhea. Although having diarrhea was not associated with shedding of any of the health-care– associated pathogens of interest, it was associated with an increased risk of shedding Salmonella spp. Salmo-nella spp may not be considered important health-care– associated pathogens in humans, but exposure should be of concern because of the potential for increased sus-ceptibility and more severe disease in immunocompro-mised individuals. It seems likely that diarrhea was not of infectious origin in many dogs in the present study, but infectious diarrhea caused by zoonotic agents such as Salmonella spp, Campylobacter spp, Toxocara canis, and Ancylostoma spp has been identified in dogs. Con-sequently, dogs involved in AAI programs that develop diarrhea of unknown origin should be temporarily sus-pended from visiting health-care facilities until clinical signs have resolved for at least 1 week.

We also found in the present study that many dogs visited ≥ 1 health-care facility and interacted with people in health-care facilities and in community set-tings when performing AAIs. Because dogs that visited health-care facilities in our study had an increased risk

of acquiring some health-care–associated pathogens, the scope of infection prevention and control protocols for health-care facilities should be expanded to pre-vent the spread of agents between facilities and into the community.

In the present study, dogs that participated in AAIs were more likely to acquire MRSA when they visited health-care facilities than when they interacted with people in other settings. Consequently, veterinarians treating such dogs should consider submitting appro-priate samples for bacteriologic culture and susceptibil-ity testing whenever these dogs develop opportunistic infections that may be caused by MRSA or other hos-pital-associated pathogens. If the laboratory does not routinely include methicillin or oxacillin in its antimi-crobial susceptibility testing profile, the veterinarian should include specific instructions to test for resis-tance to methicillin when S aureus is isolated. Infection with multidrug-resistant organisms may cause UTI, pneumonia, bacteremia, infection of surgical sites and other wounds, and general skin and soft tissue infec-tions. Given the possibility that animals participating in AAI programs may carry MRSA or C difficile, veterinary personnel caring for these animals should take steps to protect themselves and their other patients by practic-ing hand hygiene and following other routine infec-tion prevention and control measures. Dogs that visit health-care facilities should be prevented from licking patients, accepting treats from patients, and sitting di-rectly on patients’ beds without a barrier. Furthermore, dogs need to be considered as potential sources of health-care–associated pathogens when investigating causes of nosocomial infections in humans.

a. BBL culture swab, Becton Dixon, Sparks, Md.b. Pastorex Staph-Plus kit, Bio-Rad, Hercules, Calif.c. LightCycler VRE detection kit, Roche Diagnostics, Penzberg,

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