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Keywords:

  • community pathogens;
  • methicillin-resistance;
  • MRSA;
  • MRSI;
  • Staphylococcus spp

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To evaluate the prevalence of methicillin-resistant staphylococcal (MRS) colonization in clinically normal dogs and horses in the community.

Methods and Results:  Three hundred clinically normal horses and 200 clinically normal dogs were enrolled. One nasal swab was collected from each horse. Two swabs were taken from each dog: (i) from an anterior nare, and (ii) a combination of the perineal area and 0·5 cm into the anus. Enrichment cultures were performed. Methicillin-resistant Staphylococcus aureus (MRSA) was not identified. Methicillin-resistant Staphylococcus intermedius (MRSI) was isolated from the nasal swab from three dogs. Methicillin-resistant coagulase negative staphylococci (MRCoNS) were isolated from 126/300 (42%) horses and 26/200 (13%) dogs.

Conclusions:  At present MRSI is not considered to be a significant zoonotic concern; however, it may become an important pathogen in dogs. MRCoNS mostly cause disease in compromised human or animal hosts. However, these bacteria can serve as reservoirs of resistance determinants in the community, which could lead to the emergence of novel MRSA strains.

Significance and Impact of the Study:  This is the first report of the prevalence of MRS colonization in clinically normal dogs in a community setting. Continued surveillance is indicated to determine whether MRSA will emerge in the animal population and become a concern for animal disease and zoonotic infection.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Staphylococci are one of the major groups of bacterial commensals isolated from skin, skin glands, and mucous membranes of mammals (Kloos 1980). While a variety of staphylococcal species are present on or in clinically normal individuals, staphylococci are also opportunistic pathogens and leading causes of community-associated and hospital-associated disease in humans and animals worldwide (Kloos 1980; O'Mahony et al. 2005; Weese et al. 2005a, 2006a). Coagulase positive staphylococci (CoPS) (typically Staphylococcus aureus in humans, and Staph. aureus, Staphylococcus intermedius and Staphylococcus schleiferi ssp. coagulans in veterinary species) are most commonly implicated in disease (Kloos 1980; Cefai et al. 1994; Tomlin et al. 1999; May et al. 2005; O'Mahony et al. 2005; Weese et al. 2005a). Coagulase negative staphylococci (CoNS) (typically Staphylococcus epidermidis in humans) are considered less common causes of community-associated disease (Boyce 1997; Huebner and Goldmann 1999). While their role as hospital pathogens in human settings is becoming increasingly important, their zoonotic potential and importance in the veterinary medicine is still unclear (Boyce 1997; Huebner and Goldmann 1999; Yasuda et al. 2000, 2002; Busscher et al. 2006).

The emergence and dissemination of antimicrobial resistance amongst staphylococci is an important problem in human and veterinary medicine (Archer 1984; Parisi 1985; Kawano et al. 1996; Ayliffe 1997). In particular, methicillin-resistant Staph. aureus (MRSA) is a tremendous concern in human medicine worldwide (Ayliffe 1997; Melles et al. 2004), and is an emerging problem in veterinary species. Methicillin resistance in staphylococci is mediated by the mecA gene: a gene that encodes a novel penicillin binding protein (PBP2a). This penicillin binding protein mediates methicillin (oxacillin) resistance in staphylococci through a reduced affinity for beta-lactam antimicrobials (Chambers 1997). MRSA has been identified as a cause of disease in a variety of animal species including horses and dogs in Canada, the United States, the United Kingdom, the Netherlands and Ireland, and transmission of MRSA between humans and animals has been reported (Seguin et al. 1999; Guardabassi et al. 2004; van Duijkeren et al. 2005; O'Mahony et al. 2005; Weese et al. 2005a, 2006b,c). Other CoPS, such as Staph. intermedius and Staph. schleiferi have the potential to emerge as important multi-drug resistant pathogens in dogs (Gortel et al. 1999; Kania et al. 2004; May et al. 2005). Their potential for zoonotic transmission is currently unclear.

Colonization by methicillin-resistant staphylococci (MRS) of any species may pose a risk for plasmid-encoded transfer of antimicrobial resistance determinants between staphylococci and other bacterial organisms (Archer 1984; Parisi 1985; Kawano et al. 1996; Yasuda et al. 2000). Also, the presence of MRS in animals in the community may pose a danger to veterinary care facilities because of the potential for adaptation of endemic MRS to animal species, and carries a substantial zoonotic potential. Therefore, this study was designed to investigate the prevalence of MRS in clinically normal dogs and horses in the community.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study population

A convenience sample of clinically normal horses (n = 300) and dogs (n = 200) was selected. horses selected were housed in public establishments used for tourist/show purposes (100), riding schools and recreational facilities (n = 100), or were competition horses (n = 100). Horses of various breeds from 14 farms were enrolled. Dogs of various breeds were selected from agility competitions (n = 70), rescue/working dog training camps (n = 70), and household pets (n = 60). Samples were collected from March 19 to June 12, 2005. Animals were swabbed only after owner's consent was given. All samples were collected by a veterinarian. The study was conducted in Slovenia.

Sample collection

A single nasal swab was collected from each horse. A cotton-tipped culture swab was inserted approximately 10 cm into one nasal passage and withdrawn with the swab in contact with the nasal mucosa.

Two swabs were taken from each dog. One cotton-tipped culture swab was inserted into one anterior nare, and a second swab was taken of the perineal region and also inserted up to 0·5 cm into the anus.

Swabs were promptly placed in liquid Stuart's medium (MEUS, Piove di Sacco, Italy) and kept at 4 °c until processing.

Culture techniques

Enrichment culture techniques were employed. Swabs were placed in 2 ml of enrichment broth consisting of 10 g l−1 Tryptone T, 75 g l−1 sodium chloride, 10 g l−1 mannitol and 2·5 g l−1 yeast extract, and incubated aerobically at 35 °C for 24 h. Approximately 100 μl of broth was then inoculated onto mannitol-salt agar with 2 μg ml−1 oxacillin and incubated aerobically at 35 °C for 48 h. Colonies consistent with staphylococci were subcultured onto blood agar for further identification via colony morphology, Gram stain appearance, catalase reaction, ability to ferment maltose, tube coagulase test and Staph. aureus latex agglutination test (Pastorex Staph Plus, Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). CoNS were not speciated.

Methicillin-resistance was confirmed by growth on Mueller–Hinton agar with 4% NaCl and 6 μg ml−1 oxacillin, as well as a positive PBP2a latex agglutination test (PBP2’ Test Kit, Oxoid, Hants, UK).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Horses

MRSA was not isolated from any sample. Methicillin-resistant CoNS (MRCoNS) were present in 126/300 (42%) horse samples.

Dogs

MRSA was not isolated from any sample. Methicillin-resistant Staph. intermedius (MRSI) was isolated from three dogs: from nasal and perineal/anal sites (n = 2), and nasal only (n = 1). There was no reported contact between the three dogs colonized with MRSI. Coagulase-negative MRS were isolated from 23 dogs: from both nasal and perianal/anal swab (n = 2), nasal swab only (n = 15), and perianal/anal swab only (n = 6).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Considering the importance of MRSA in human infections (Ayliffe 1997; Klavs et al. 2003; Melles et al. 2004), it was encouraging that MRSA was not isolated from any of the animals in this study. Many, if not most, dogs and horses have frequent and close contact with humans and other animals of same species, creating the potential for transmission of organisms such as MRS. A variety of reasons could explain the zero study prevalence, including a true zero population prevalence, prevalence below the detection threshold of this study or clustering of MRSA colonization among groups not sampled in this study. Isolation of MRSA or MRSI from veterinary clinical samples has not been reported in Slovenia. However, considering the situation in animals and humans in other countries, it is unlikely that MRSA truly does not exist in the animal population.

To the authors’ knowledge this is the first report of the prevalence of MRS colonization in clinically normal dogs in a community setting. No MRSA was isolated from dogs in the present study. To date most MRSA isolates in dogs have been associated with clinical samples from surgical site infections, wound infections, catheter site infections, urinary tract infections, pneumonia, and skin infections (Tomlin et al. 1999; van Duijkeren et al. 2004; O'Mahony et al. 2005). In a recent survey in a small animal referral hospital in the UK, 9% of dogs had MRSA-positive nasal swabs. Colonized hospital staff and environmental sites were also identified (Loeffler et al. 2005). However, hospital based studies must be interpreted with caution because of potential hospital-associated colonization and selection bias, and likely do not represent the true population prevalence.

The lack of identification of MRSA in horses in the community in Slovenia is in contrast to a report from Ontario, Canada and New York, USA, which found a MRSA prevalence of 4·7% in horses on local horse farms (Weese et al. 2005a). The intriguing part of the same report was the predilection of a particular MRSA clone, Canadian epidemic MRSA-5 (ST8;MRSA:SCCmecIV), to colonize horses. This clone was considered uncommon in humans in the same geographical area (Weese et al. 2005a). Similarly, a predilection for a particular MRSA clone to colonize horses was reported in Ireland; however, that study only isolated MRSA from clinical samples (O'Mahony et al. 2005). In the Netherlands, no MRSA was isolated from healthy horses, although the sample population was relatively small (Busscher et al. 2006). In both Ireland and the Netherlands, the proportion of human Staph. aureus isolates that are methicillin resistant differs from that in Slovenia. In Slovenia, the proportion of human MRSA isolates dropped from 22% in 2000 to 15% in 2002, while in Ireland and the Netherlands this proportion ranged from 39% in 2000 to 45% in 2002, and 0·4% in 2000 to 1% in 2002, respectively (Tiemersma et al. 2004). All three countries participate in the European Antimicrobial Resistance Surveillance System (Tiemersma et al. 2004). Data from other participating countries may provide further insight into the relationship between human and animal MRSA colonization.

MRSI was identified in dogs in this study, which is not surprising as Staph. intermedius is the most commonly isolated coagulase-positive species of Staphylococcus in dogs and cats (Phillips and Williams 1984; Cox et al. 1988). MRSI has been previously isolated from dogs with clinical infections (Gortel et al. 1999; Kania et al. 2004), but colonization of dogs in the general population has not been evaluated. The transfer of antimicrobial-resistant Staph. intermedius strains from dogs with deep pyoderma to their owners had been demonstrated (Guardabassi et al. 2004), although MRSI is not currently considered to be a significant zoonotic concern.

Isolation of MRCoNS from dogs and especially from horses was expected (Yasuda et al. 2000, 2002; Busscher et al. 2006). The prevalence of MRCoNS colonization in horses (42%) was higher in this study compared with 29·5% reported from Japan (Yasuda et al. 2000), and lower than that reported from the Netherlands (56·5%) (Busscher et al. 2006). In humans CoNS are becoming increasingly important hospital pathogens [Hamory et al. 1987; Boyce 1997; National Nosocomial Infections Surveillance (NNIS) 1999]. No analysis of the importance of CoNS hospital infection in veterinary medicine or their potential for zoonotic infection is available to date.

It has become apparent that MRSA can be readily transmitted between humans and animals in the community (Cefai et al. 1994; Seguin et al. 1999; van Duijkeren et al. 2004, 2005; Weese et al. 2005a,b, 2006b). Possible mechanisms of future emergence of MRSA in animals in Slovenia, and perhaps other countries, include importation of animal-specific clones, and development of new MRSA clones in animals by horizontal transmission of mecA from MRCoNS to commensal Staph. aureus. Because colonization with staphylococci is more common than infection, both active and passive surveillance will continue to be necessary to monitor the potential emergence of this important pathogen.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors gratefully acknowledge the assistance of the Slovenian Kennel club and their sections. We would also like to thank Dr Janez Ves, Dr Joze Drobnic and all the horse and dog owners for assisting us with this study. This study was supported by the Slovenian Research Agency grant P4-0053.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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