Lack of transmission of methicillin-resistant (MRSA) between apparently healthy dogs in a rescue kennel

Lack of transmission of methicillin-resistant (MRSA) between apparently healthy dogs in a rescue kennel A. Loeffler, D.U. Pfeiffer, J.A. Lindsay, R. Soares-Magalhaes, D.H. Lloyd To cite this version: A. Loeffler, D.U. Pfeiffer, J.A. Lindsay, R. Soares-Magalhaes, D.H. Lloyd. Lack of transmission of methicillin-resistant (MRSA) between apparently healthy dogs in a rescue kennel. Veterinary Microbiology, Elsevier, 2010, 141 (1-2), pp.178. <10.1016/j.vetmic.2009.08.001>. <hal-00560846> HAL Id: hal-00560846 https://hal.archives-ouvertes.fr/hal-00560846 Submitted on 31 Jan 2011 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Title: Lack of transmission of methicillin-resistant Staphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel Authors: A. Loeffler, D.U. Pfeiffer, J.A. Lindsay, R. Soares-Magalhaes, D.H. Lloyd PII: S0378-1135(09)00362-9 DOI: doi:10.1016/j.vetmic.2009.08.001 Reference: VETMIC 4532 To appear in: VETMIC Received date: 14-4-2009 Revised date: 27-7-2009 Accepted date: 3-8-2009 Please cite this article as: Loeffler, A., Pfeiffer, D.U., Lindsay, J.A., Soares-Magalhaes, R., Lloyd, D.H., Lack of transmission of methicillin-resistant Staphylococcus aureus (MRSA) between apparently healthy dogs in a rescue kennel, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2009.08.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1 Lack of transmission of methicillin resistant Staphylococcus aureus 2 3 4 5 6 7 8 9 10 11 (MRSA) between apparently healthy dogs in a rescue kennel A. Loeffler a, *, D.U. Pfeiffer a, J.A. Lindsay b, R. Soares Magalhaes a, D.H. Lloyd a a Department of Veterinary Clinical Sciences, Royal Veterinary College, University of London, Hawkshead Lane, Hatfield, North Mymms, Hertfordshire AL9 7TA, UK b Centre for Infection, Department of Cellular and Molecular Medicine, St.George s, University of London * Corresponding author. Tel.:+44 1707 666234; fax: +44 1707 666298. E mail address: aloeffler@rvc.ac.uk (A. Loeffler). 1 Page 1 of 12

11 12 Abstract Although it is widely accepted that methicillin resistant Staphylococcus aureus (MRSA) can be 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 transmitted between humans and animals in both directions, little is known about the dynamics of animal to animal transfer. This study aimed to investigate aspects of dog to dog MRSA transfer in a rescue facility in the South East of England during an MRSA outbreak. One hundred and twenty nine apparently healthy dogs, mostly housed in pairs, were swabbed at nasal, oral, axillary and perianal sites. Swabs were enriched in selective broth and staphylococci identified using standard biological methods. MRSA isolates were confirmed by demonstration of the thermonuclease gene (nuc) and meca. After initial swabbing, a dog excluded from the study design but housed at the same facility was discovered to have a wound infection due to MRSA. MRSA carriage was identified in 10/129 dogs (7.8%) and all isolates were of the same lineage as the one isolated from the infected dog. All carrier dogs lived in shared kennels and their 16 kennel partners sampled negative on two occasions. Concurrently with successful antimicrobial treatment of the infected patient, MRSA carriage resolved spontaneously in all dogs within two weeks. In conclusion, MRSA did not transmit readily between apparently healthy dogs, MRSA carriage was not supported for long periods in a regularly cleaned environment and exposure alone may not lead to MRSA acquisition by dogs without the presence of additional risk factors. Keywords: MRSA; dogs; carriage; transmission 30 2 Page 2 of 12

30 31 Introduction Methicillin resistant Staphylococcus aureus (MRSA) is now widely accepted as a human and animal 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 pathogen. For dogs in particular, indirect evidence from case reports and epidemiological strain typing suggests that MRSA transmission between human and animal hosts can occur in both directions (Baptiste et al., 2005; Hanselman et al., 2008; Loeffler et al., 2005; van Duijkeren et al., 2004; Weese et al., 2006; Cefai et al., 1994; Manian, 2003). Thus, dogs are considered as potential vectors and reservoirs for MRSA with implications for public health (Tisinger, 2008). In contrast, little is known about the epidemiology of MRSA within the dog population and whether dogs can support MRSA carriage or contamination over time. While the term reservoir may imply the spread of an organism within the reservoir host to maintain the pathogen indefinitely (Ashford, 2003), this has not been established for MRSA in animals. Unfortunately, temporal studies of the duration of MRSA carriage and studies on transmission of MRSA between animals in privately owned dogs is associated with practical and ethical difficulties as leaving any detected carrier animals or infected pets untreated may allow the spread of a human pathogen. In one case, MRSA carriage was maintained in a healthy dog over several weeks but the dog lived with immunocompromised owners previously infected with MRSA and continuing to suffer from open wounds. Therefore, contamination of the environment was highly likely and could have accounted for positive samples recovered from the dog (Manian, 2003). In horses, it has been proposed that MRSA carriage is short lived, but again specific longitudinal studies on animal carriage have not been published (Weese and Rousseau, 2005). Such information is urgently needed though to evaluate whether decolonisation of carrier animals is possible and required. Decolonisation is used in humans as part of comprehensive infection control strategies to prevent MRSA infection. However, its value remains controversial 52 even in people due to inconsistent long term efficacy and increasing drug resistance amongst MRSA. 53 54 The aim of this study was to examine the potential for animal to animal transmission of MRSA in apparently healthy carrier dogs housed in shared kennels during an MRSA outbreak. 3 Page 3 of 12

55 56 Materials and methods Study design 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Apparently healthy dogs at a large rescue centre in the Greater London area were sampled for MRSA carriage as part of a cross sectional prevalence study in companion animals. Dogs were considered healthy if they were (1) free of non neoplastic skin disease, fresh wounds and abnormal discharge from body openings on inspection,(2) had not received within the previous three weeks either antimicrobial or immunosuppressive therapy, (3) had not required within the previous three weeks veterinary attention other than for routine health checks, vaccination, neutering and antiparasitic treatment administered by the facility s own veterinary surgeon on site and (4) were considered fit for rehoming. Dogs were sampled irrespective of their length of stay at the facility and dogs in contact with diseased animals were excluded from the screening. The precision of the prevalence estimate was expressed using the 95% confidence interval (WinEpiscope 2.0) and groups were compared using the chi squared test with a P value of less than 0.05 considered as significant. A dog (index case) with a surgical wound infection had been housed at the premises for five weeks before study begin. Four days after the first prevalence screening, MRSA isolation from the wound was reported. An epidemiological link between the MRSA carriage rate in apparently healthy dogs and the infected dog was proposed based on the temporal and geographical overlap and both were investigated as an outbreak. The infected dog and its kennel partner were sampled for carriage as part of this outbreak investigation. Both dogs had been kennelled in a wing separate from the healthy screened dogs but communal areas and staff movement along shared corridors provided communication between the wings. 76 77 78 79 Most dogs were housed in pairs without the opportunity for contact with neighbouring kennels. Equipment such as food bowls and toys were either dog or dog pair specific or they were washed between use. Kennels were cleaned and disinfected (TriGene, Medichem International, Sevenoaks, UK) twice daily. Infection control policies at the facility remained unchanged during the study period. 4 Page 4 of 12

80 81 To minimise contamination during sampling, hands were washed when macroscopically dirty or after handling a group of animals; alcohol hand gel was used before and after handling of each dog. All 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 investigators handling swabs or broth vials confirmed fortnightly that their own nasal swabs were MRSA negative following the methods described for animal samples. The study was approved by the Royal Veterinary College Ethics and Welfare Committee. Animals positive for MRSA were re sampled seven to ten days after the first swab and if follow up swabs were also positive, topical antimicrobial therapy with products licensed for use in dogs would be initiated to pursue elimination of MRSA from carrier sites before rehoming. Swab collection and identification of MRSA Saline moistened swabs from both nostrils, the buccal mucosa, the axillary skin and the anal mucosa were pooled immediately tryptone soya broth (Oxoid, Basingstoke, UK) supplemented with sodium chloride (Sigma Aldrich, Gillingham, UK) to a total salt concentration of 10% and incubated at 37 C for 48 h. Samples were streaked out and incubated at 37 C on blood agar (Oxoid) containing 5% ovine blood (TCS Biosciences, Buckingham, UK) for isolation of total staphylococci and on mannitol salt agar (Oxoid) with 6 mg/l oxacillin (MSA)(Sigma Aldrich) for selective isolation of MRSA and examined after 24 and 48 h respectively. Colonies from MSA with oxacillin were subcultured on blood agar for further identification. Presumptive staphylococci were identified based on colony morphology, haemolysis, Gram staining and salt resistance. S. aureus was identified phenotypically by positive results for DNase, clumping factor with dog plasma or, if negative, tube coagulase using rabbit plasma, Voges Proskauer reaction (VP A and VP B, biomérieux, France) and fermentation tests with trehalose and lactose (Barrow, 1993). Resistance to methicillin was verified by disc 101 102 diffusion tests according to CLSI guidelines on Mueller Hinton agar using 5μg methicillin discs (Oxoid) and overnight incubation at 30 C (CLSI, 2004). 5 Page 5 of 12

103 104 MRSA were confirmed genotypically by the demonstration of the S. aureus specific thermonuclease gene (nuc) and meca after gene replication by polymerase chain reaction (PCR) (Becker et al., 2005; 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 Brakstad et al., 1993) and typed by determination of the S. aureus lineage specific restriction modification enzyme system (Cockfield et al., 2007). Reference strains for bacteriological and genetic assays were ATCC29663, PM62 (Moore and Lindsay, 2002), ATCC49545, LMG22219. Results Carrier animals A total of 129 dogs was sampled on two days during December 2006 and January 2007. These accounted for 71.7% of the 180 dogs present at the facility during the study period while 51 dogs, including the index case and its kennel partner, were excluded from the screening due to concurrent or preceding disease, behavioural problems or imminent rehoming. The first sampling involved 84 dogs of which six (7.1%) were positive for MRSA; on the second study day, three weeks later, 4/45 dogs (8.9%) yielded MRSA. Proportions of positive dogs were similar on the two days (P=0.72) and a prevalence estimate of 7.8% MRSA carriage was calculated for the total population of 129 dogs (standard error 2.5%, confidence interval 95%). The 129 dogs sampled included 82 crossbred dogs and 47 presumed pure breeds (27 terriers, 5 spaniels, 18 large breeds such as Rottweiler, German shepherd dog, Doberman, Setter, Border collie and 3 toy dog breeds). Sixty seven dogs were male (34 neutered) and 62 female (33 neutered). Their approximate ages ranged from three months to 16 years (mean 2.1 years) and at the time of sampling their mean length of stay at the premises was 3.6 weeks (range between six hours to three months). 124 125 126 Of the ten carrier dogs, none was housed alone, five shared a kennel with one other dog and three shared with two other dogs. The remaining two MRSA carriers were two of seven three month old puppies from the same litter, all kept in the same kennel. They were swabbed on the day of arrival at 6 Page 6 of 12

127 128 the centre after they had been rescued and transported to the facility in the same container over a period of 48 hours. Thus, sixteen dogs in total had close direct contact with at least one of the ten 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 MRSA carrier dogs; all sixteen in contacts sampled negative for MRSA on the same occasion. Follow up swabs taken from the ten original carrier dogs and from the sixteen in contact kennel mates which had remained in the same kennels were all negative for MRSA; samples from these 26 dogs all yielded non MRSA staphylococci. Index case The infected dog and its kennel partner were sampled for carriage nine days after the first screening of healthy dogs. Systemic antimicrobial treatment of the infected wound with trimethoprimsulfamethoxazole had been initiated five days earlier. While the infected dog also carried MRSA in the nostrils and on the perineum, swabs from its axilla and the mouth as well as all four swabs from the in contact dog were negative. Due to the need for prompt rehoming and the zoonotic risk of MRSA, the dog was isolated and decolonisation using fusidic acid and chlorhexidine topically was started. MRSA was no longer isolated from samples taken one week later, nor from a swab of the closed wound on the dorsum. Topical antimicrobial therapy was discontinued five days and systemic treatment fifteen days after the last sampling. A final follow up sample from carrier sites was submitted prior to rehoming one month later and was again negative. Genotyping The infection isolate was obtained from the veterinary laboratory, confirmed as MRSA and typed as described above. All ten carriage isolates from apparently healthy dogs, the MRSA from the wound and the two MRSA isolates from the infected dog s nostrils and perineum were of clonal cluster 22, 148 represented in the UK by EMRSA 15 (Johnson et al., 2001). 7 Page 7 of 12

149 150 Discussion The study findings indicate that MRSA is not always acquired by healthy dogs despite exposure and 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 that it is not transmitted readily between in contact dogs. In particular, the dog sharing a kennel with the infected animal for four weeks prior to isolation and therefore the animal thought to be most at risk from exposure sampled negative at all four carrier sites. Although the sensitivity of surface swabbing may be similar to the 77% quoted for human nasal sampling (Sewell et al., 1993), false negative results cannot be ruled out especially since the use of a high salt concentration in enrichment broth (10%) and oxacillin rather than cefoxitin discs may have underestimated the occurrence of MRSA (Pottumarthy et al, 2005). However, the lack of MRSA isolation from this incontact dog strongly suggests that exposure alone does not necessarily result in carriage in healthy dogs and that other factors may be required to facilitate MRSA acquisition. Similarly, another sixteen in contact kennel partner dogs, each sharing a kennel with at least one MRSA carrier, did not yield MRSA when sampled on two occasions. While the differences between exposure, contamination, carriage and colonisation may be ill defined, the lack of MRSA isolation from those 16 in contact dogs suggests that dog to dog transmission of MRSA is infrequent. Dog todog transmission cannot be excluded in the two cohabitating puppies though, but as they were sampled within hours of arrival at the facility, separate MRSA acquisition prior to or during transport is also possible. In people, contact with other carriers or infected patients, antimicrobial treatment, advanced age and association with hospitals or healthcare facilities are well documented as the main risk factors for MRSA carriage (Harbarth et al., 2008). None of these was identified in the in contact animals but unfortunately only limited histories were available for most rescue dogs. 170 171 172 173 MRSA exposure may have occurred from a contaminated environment, from human vectors, from direct contact with the infected dog or with carrier dogs or via inanimate objects, and all routes would be compatible with the studied outbreak scenario. In people, MRSA is most frequently transferred by hand contact but acquisition from contaminated environments and inanimate objects 8 Page 8 of 12

174 175 has also been documented (Sexton et al., 2006). Although environmental swabs were not taken, the regular use of detergents and disinfectants is likely to have reduced environmental contamination. 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 As dog to dog transmission could not be demonstrated, human vectors or inanimate objects may have been the main routes of transfer that led to the high MRSA prevalence detected. Although MRSA exposure may have been reduced in the dogs sampled on the second study day as antimicrobial treatment had by then been initiated for the index case, carriage rates were similar for both groups (7.1% and 8.9% respectively; P=0.72). The combined MRSA carriage prevalence of 7.8% amongst apparently healthy dogs in a rescue facility in the South East of England was alarmingly high. A previous study from the North West of England did not detect any MRSA carriers in 22 dogs sampled in the community (Baptiste et al., 2005). Similarly, MRSA carriage was rare in healthy dogs in Hong Kong (6/815, 0.72%)(Boost et al., 2007) and not detected in Slovenia (n=200)(vengust et al., 2006). In contrast, MRSA was isolated from 14% of dogs at a university referral hospital in the South East of England but all were diseased and had received veterinary treatment prior to referral (Loeffler et al., 2005). These data suggest that the prevalence of MRSA carriage amongst healthy dogs is low unless risk factors predispose or if particular routes of transmission, as in the presented study, exist. In line with other MRSA reports from UK dogs, all MRSA isolated in this study were typed as EMRSA 15 (Baptiste et al., 2005, Loeffler et al., 2005). This lineage is considered highly clonal in the UK so that further genotypic distinction of isolates by additional typing methods would not have been helpful. More discriminatory genetic information on isolates within this lineage could, for example, be gained from comparative genome microarray analysis and this might have helped to support a 195 196 common origin of the two isolates from puppies. However, it would not have contributed to the study objective which was to investigate dog to dog transmission of MRSA. 9 Page 9 of 12

197 198 Conclusions MRSA carriage in all healthy carrier dogs was transient and resolved without antimicrobial 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 decolonisation therapy in the regularly cleaned and disinfected environment. In addition, MRSA was not transferred readily between healthy dogs despite close contact. This is the first study to describe the dynamics of MRSA transmission in a dog kennel environment; similar research is now required within a home environment. Acknowledgements This work was funded by PetPlan Charitable Trust, UK, as part of a larger study into the prevalence of MRSA carriage in companion animals. The authors are very grateful to Chris Laurence, Dogs Trust, for his supportive collaboration and to Sian Buckingham for her skilled help with the sampling. References Ashford, R.W., 2003. When is a reservoir not a reservoir? Emerg. Infect. Dis. 9, 1495 1496. Baptiste, K.E., Williams, K., Willams, N.J., Wattret, A., Clegg, P.D., Dawson, S., Corkill, J.E., O'Neill, T., Hart, C.A., 2005. Methicillin resistant staphylococci in companion animals. Emerg. Infect. Dis. 11, 1942 1944. Barrow, G.J., Feltham, R. K., A., 1993, Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd edition Edition. Cambridge University Press. Becker, K., von Eiff, C., Keller, B., Bruck, M., Etienne, J., Peters, G., 2005. Thermonuclease gene as a target for specific identification of Staphylococcus intermedius isolates: Use of a PCR DNA enzyme immunoassay. Diagn. Microbiol. Infect. Dis. 51, 237. Boost, M.V., O'Donoghue, M.M., Siu, K.H., 2007. Characterisation of methicillin resistant 218 219 Staphylococcus aureus isolates from dogs and their owners. Clin Microbiol Infect 13, 731 733. 10 Page 10 of 12

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