Methicillin-resistant staphylococci in companion animals.

We determined the molecular characteristics of methicillin-resistant staphylococci from animals and staff at a small animal and equine hospital, Methicillin-resistant Staphylococcus aureus (MRSA) identical to human EMRSA-15 was found in dogs and hospital staff. In contrast, 5 distinct MRSA strains were isolated from horses but not from hospital staff.

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Methicillin-resistant Staphylococcus aureus (MRSA) is among the most important causes of human healthcare associated infections. MRSA has also caused infections in dogs (1,2), and cases of human-to-dog transmission of MRSA in which dogs have acted as reservoirs for reinfection have been reported (3,4). MRSA and methicillin-resistant, coagulase-negative staphylococci (MRCNS) have also been reported in horses (5), including outbreaks in equine hospitals (6). In these cases, MRSA was thought to be of human origin (6); however, a Japanese study could not definitively relate equine to human MRSA with pulsed-field gel electrophoresis (PFGE) (7). At a Canadian equine hospital and thoroughbred farm, both horses and staff were positive for MRSA, and 96% and 93% of isolates, respectively, were subtypes of a rare Canadian MRSA-5 clone (8).

Horses, dogs, and cats in the community; animals treated at the University of Liverpool's Small Animal Hospital (SAH) and Philip Leverhulme Equine Hospital (PLEH); and staff at those hospitals were screened for MRSA. The molecular characteristics of MRSA in these populations were investigated to determine the source and routes of transmission. Animal samples were also screened for MRCNS.

The Study

Swabs were taken from the anterior nares of dogs, horses, and staff; nasal surface of cats; perineum of dogs, cats, and horses; and the neck skin surface of horses. All diagnostic submissions from both of these hospitals were screened for MRSA. Swab specimens were directly inoculated onto mannitol salt agar (LabM, Bury, UK) with aztreonam (2 mg/L) and oxacillin resistance-screening agar (Oxoid, Basingstoke, UK) and incubated at 37[degrees]C for [less than or equal to] 48 h. Staphylococci were identified by colony shape, Gram stain, staphylase test (Oxoid), and API staph kit (MR-CNS only) (bioMerieux, Basingstoke, UK). The disk-diffusion method (Mast, Liverpool, UK) was used to determine the susceptibility of all isolates to oxacillin, methicillin, gentamicin, vancomycin, rifampicin, ciprofloxacin, co-trimoxazole, fusidic acid, and tetracycline, according to the British Society for Antimicrobial Chemotherapy guidelines, by using S. aureus ATCC 26923, EMRSA-15, and EMRSA-16 as controls (9).

Cell lysates of all methicillin-resistant staphylococci were prepared as described previously (10). Cell lysates were also prepared from 3 control strains, EMRSA-15, EMRSA-16, and the Canadian epidemic strain, CMRSA-5, previously found in horses and humans (8). The presence of the mecA gene was determined with polymerase chain reaction (PCR) by using a modified method adapted from Vanuffel et al. (11), with a conventional thermocycler. PCR to detect the S. aureus femA gene was used to confirm isolates as MRSA (12). For all MRSA and equine MR-CNS isolates, the SCCmec cassette and the agr operon were analyzed as described previously (13). All MRSA isolates were screened for the gene encoding Panton-Valentine leukocidin by using the method of Lina et al. (14); a positive control for this reaction was provided by the Scottish MRSA Reference Laboratory. Macro-restriction of the genome and PFGE were conducted on all MRSA isolates according to the protocol described by Murchan et al. (15) and included on each gel with EMRSA-15 and -16 and CMRSA-5.

Swabs taken from cats (n = 50) and dogs (n = 55) treated at the SAH and cats within the community (February-March 2004) were negative for MRSA. One cat was positive for methicillin-resistant staphylococci, and 4 dogs were positive for MR-CNS, all of which were confirmed by PCR to be carrying the mecA gene. However, 3 dogs with clinical infections (a joint infection in January 2004, pleuropneumonia in March 2004, and a wound infection in June 2004) were positive for MRSA at the site of infection. The dog with the joint infection was also positive for nasal and fecal carriage of MRSA; a student who treated the dog had an MRSA-positive nasal swab in April 2004. Eleven staff provided nasal swabs, of which 2 were positive for MRSA in January 2004 (Table). All MRSA isolates were resistant to ciprofloxacin but sensitive to all other antimicrobial drugs tested. All MRSA isolates were positive for the mecA and femA genes, carried the SCCmec type IV cassette, and were agr operon group 1 strains but were negative for pvl genes. PFGE showed that the human and dog clinical MRSA isolates were identical to the human epidemic strain, EMRSA-15 (Figure).

[FIGURE OMITTED]

Of the 105 horses sampled, MRSA was isolated only from horses at PLEH. Of the 67 horses sampled at PLEH, 11 were positive (16%) for carriage and 3 had MRSA-associated clinical infections (pleuropneumonia, chronic septic arthritis, and chronic dermatitis). None of the isolates submitted from 12 staff members at the equine hospital were positive for MRSA. The horse MRSA isolates were resistant to gentamicin (100%), rifampicin (80%), ciprofloxacin (78%), fusidic acid (69%), co-trimoxazole (50%), and tetracycline (50%) but not to vancomycin. All MRSA isolates were positive for the mecA and femA genes and were agr group 1, except 2 that were agr group 2, but all were negative for the pvl genes. Like the human and dog isolates, all horse MRSA isolates except 3 (1 isolate had a variant of type II or III, and 2 isolates repeatedly failed to give PCR products for SCCmec cassettes), carried the SCCmec cassette type IV. MR-CNS was isolated from 19% of horses at the PLEH and 30% of horses in the community. All horse MR-CNS isolates (including those from PLEH) had different SCCmec cassettes than the MRSA isolates, and their banding patterns did not fully correspond to any of the known cassette types, giving a 209-bp band (types II and III) and a further band of 495 bp (type I). Twelve MRSA isolates from 7 horses were selected for PFGE based on differences in antibiogram and genes detected by PCR. This analysis showed 5 distinct strains. The same strain found in nasal samples, 1 skin sample from 3 horses, and 1 MRSA strain from a clinical case-patient were closely related to a nasal isolate from a different horse. None of the horse MRSA strains were related to EMRSA-15, EMRSA-16, or CMRSA-5 as demonstrated in the Figure.

Conclusions

This study documents MRSA transmission between humans and dogs; the same strain was found in 3 staff members and 3 dogs, all identical to the predominant human epidemic strain EMRSA-15. Two staff members and a student who treated 1 dog were positive for the same MRSA strain. Furthermore, MRSA was associated with clinical disease in 2 other dogs some months later; this finding could suggest a cycle of transmission between staff and animals. However, the origin of MRSA in the first dog is unknown and could have originated in either staff or the dog in question, with dog-to-human transmission or vice versa. This study suggests that dogs can act as reservoirs of MRSA, which can pose a public health risk to owners and veterinary staff, as well as limit the options for antimicrobial drug treatment of MRSA infections. Staff in veterinary hospitals could have an increased risk of carrying MRSA because of contact with infected animals and antimicrobial drugs in their work environment.

Contrary to SAH results of this study and previous work in Canada, no evidence was seen of MRSA transmission between staff and horses at PLEH, nor were any isolates related to the predominant UK human epidemic strains or CMRSA-5. However, 5 different horse MRSA strains were identified with unknown sources. The fact that different SCCmec cassettes were found in horse MR-CNS isolates than in MRSA isolates does not suggest that methicillin resistance had transferred from MR-CNS to MRSA. Furthermore, the prevalence of MR-CNS in horses in the community is almost double that which was found in horses at PLEH. This could suggest that MR-CNS may compete well with methicillin-sensitive CNS in an environment where antimicrobial drugs are not present. These results imply that MRSA is present in the general horse population and may represent a reservoir of new or rare MRSA strains that could be transmitted to humans.

Acknowledgments

We thank Caroline Janes, Katie Milner, Karl Faksvag, Emily Talbot, Jane Devaney, and Jackie Jones for their assistance throughout this project; Derek Knottenbelt, Malcolm Bennett, and John Cox for their advice and encouragement; and the Canadian MRSA reference laboratory for supplying the CMRSA-5 strain.

We gratefully acknowledge the Research Development Fund, University of Liverpool, for their financial support of this project.

References

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(3.) Manian FA. Asymptomatic nasal carriage of mupirocin, methicillin-resistant Staphylococcus aureus (MRSA) in a pet dog associated with MRSA infection in household contacts. Clin Infect Dis. 2003;36:e26-8. Epub 2003 Jan 6.

(4.) van Duijkeren E, Wolfhagen MJHM, Box ATA, Heck MEOC, Wannet WJB, Fluit AC. Human-to-dog transmission of methicillin-resistant Staphylococcus aureus. Emerg Infect Dis. 2004; 10:2235-7.

(5.) Hartmann FA, Trostle SS, Klohnen AA. Isolation of methicillin-resistant Staphylococcus aureus from a postoperative wound infection in a horse. J Am Vet Med Assoc. 1997;211:1558-61.

(6.) Seguin JC, Walker RD, Caron JP, Kloos WE, George CG, Hollis RJ, et al. Methicillin-resistant Staphylococcus aureus outbreak in a veterinary teaching hospital: potential human-to-animal-transmission. J Clin Microbiol. 1999;37:1459-63.

(7.) Shimizu A, Kawano J, Yamamoto C, Kakutani O, Anzai T, Kamada M. Genetic analysis of equine methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis. J Vet Med Sci. 1997;59:935-7.

(8.) Weese JS, Archambault M, Willey BM, Dick H, Hearn P, Kreiswirth BN, et al. Methicillin-resistant Staphylococcus aureus in horses and horse personnel, 2000-2002. Emerg Infect Dis. 2005;11:430-5.

(9.) Andrews JM, BSAC Working Party on Susceptibility Testing. BSAC standardized disc susceptibility testing method. J Antimicrob Chemother. 2001;48(Suppl 1):43-57.

(10.) Senna JPM, Pinto CA, Carvalho LPS, Santos DS. Comparison of pulsed-field gel electrophoresis and PCR analysis of polymorphisms on the mec hypervariable region for typing methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2002;40:2254-6.

(11.) Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, et al. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol. 1995;33:2864-7.

(12.) Francois P, Pittet D, Bento M, Pepey B, Vaudaux P, Lew D, et al. Rapid detection of methicillin-resistant Staphylococcus aureus directly from sterile or non-sterile clinical samples by a new molecular assay. J Clin Microbiol. 2003;41:254-60.

(13.) Corkill JE, Anson JJ, Griffiths P, Hart CA. Detection of elements of the staphylococcal cassette chromosome (SCC) in a methicillin-susceptible (mecA gene negative) homologue of a fucidin-resistant MRSA. J Antimicrob Chemother. 2004;54:229-31.

(14.) Lina G; Piemont Y, Godail-Gamot F, Bes M, Peter MO, Gauduchon V, et al. Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clin Infect Dis. 1999;29:1128-32.

(15.) Murchan S, Kaufmann ME, Deplano A, Ryck R, Struelens M, Zinn CE, et al. Harmonisation of pulsed field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J Clin Microbiol. 2003;41:1574-85.

Keith E. Baptiste, * Kerry Williams, ([dagger]) Nicola J. Willams, * Andrew Wattret, * Peter D. Clegg, * Susan Dawson, * John E. Corkill, ([dagger]) Turlough O'Neill, * and C. Anthony Hart ([dagger])

* University of Liverpool, Leahurst, United Kingdom; and Royal Liverpool University Hospital, Liverpool, United Kingdom

Address for correspondence: Nicola Williams, Department of Veterinary Pathology, Faculty of Veterinary Science, Chester High Rd, Leahurst, United Kingdom CH64 7TE; fax: [phone]; email: [e-mail]

Dr Baptiste is a lecturer of veterinary public health and epidemiology in the Department of Veterinary Pathology, Faculty of Veterinary Sciences, University of Liverpool. His current research interests include associations and origins of antimicrobial drug-resistant bacteria in animals.

Table. Isolate test results for methicillin-resistant Staphylococcus
aureus (MRSA) *

                                   No. samples positive for
                                      MRSA ([dagger]) (%)

                          No.
                        sampled   Nasal    Perineum    Skin

Dogs
  Clinical cases           3        1         1         1
  SAH                     32        0         0         0
  Community               22        0         0         0
Cats
  SAH                     26        0         0         0
  Community               24        0         0         0
SAH veterinary staff      11      3 (27)      NT        NT
Horses
  Clinical cases           3        1         NT        1
  PLEH                    67      8 (12)      0       2 (3)
  Community               40        0         0         0
PLEH Veterinary staff     12        0         NT        NT

                                             No. samples positive for
                        Other (clinical)       MR-CNS ([dagger]) (%)

Dogs
  Clinical cases        Joint and pleural     NT       NT      NT
  SAH                     fluid, feces       2 (6)    1 (0)     0
  Community                                  1 (5)      0       0
Cats
  SAH                                          0        0       0
  Community                                  1 (5)    1 (5)     0
SAH veterinary staff                          NT       NT      NT
Horses
  Clinical cases           Pleural and        NT       NT      NT
  PLEH                     joint fluid       6 (9)    3 (5)   5 (8)
  Community                                 12 (30)     0     1 (3)
PLEH Veterinary staff                         NT       NT      NT

* SAH, small animal hospital: PLEH, Philip Leverhulme Equine Hospital;
NT, not tested, MR-CNS, methicillin-resistant, coagulase-negative
staphylococci. ([dagger]) Some animals were positive for >1 body site.