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Methicillin-resistant Staphylococcus aureus (MRSA) on the skin of long-term hospitalised horses
The Veterinary Journal 193 (2012) 408–411
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The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Methicillin-resistant Staphylococcus aureus (MRSA) on the skin of long-term hospitalised horses A. Van den Eede a,⇑, K. Hermans b, A. Van den Abeele c, K. Floré d, J. Dewulf e, W. Vanderhaeghen f, F. Crombé f, P. Butaye b,f, F. Gasthuys a, F. Haesebrouck b, A. Martens a a
Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Department of Medical Microbiology, AZ St.-Lucas, Groenebriel 1, 9000 Ghent, Belgium d Department of Medical Microbiology, AZ St.-Lucas, St.-Lucaslaan 29, 8310 Bruges, Belgium e Veterinary Epidemiology Unit, Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium f Veterinary and Agrochemical Research Centre, CODA-CERVA-VAR, Groeselenberg 99, 1180 Ukkel, Belgium b c
a r t i c l e
i n f o
Article history: Accepted 14 December 2011
Keywords: MRSA Bacterial screening Skin locations Horse Hospitalisation
a b s t r a c t Given the significance of methicillin-resistant Staphylococcus aureus (MRSA) infections for both horses and staff in equine veterinary hospitals, protocols are required to minimise the risk of nosocomial transmission, including the screening of the skin and nasal chambers of equine patients for evidence of infection. The objective of this study was to clarify the potential existence and extent of MRSA on the skin of horses requiring long-term hospitalisation (P6 months). Thirty such horses were sampled at eight different locations on their skin and from their nasal chambers. MRSA was isolated from 12 animals (40%), with all sample sites testing positive on at least one occasion. Organisms were most frequently detected in the nasal chambers (relative sensitivity, 83.3%; 34.5% positive horses; isolation rate 33.3%). Skin presence was found in 30% of animals with the highest isolation rates found at the carpus (16.7%), neck, withers and croup (13.3% each). To achieve a relative screening sensitivity of >90%, at least one skin site was required in addition to nasal sampling. This evidence of skin as well as nasal reservoirs of MRSA in long-term hospitalised horses should facilitate the design of effective screening and containment protocols. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in equine hospitals has highlighted the need for the implementation of containment strategies that minimise the risk of nosocomial transmission (Tschudin-Sutter et al., 2010; van Duijkeren et al., 2010; Weese, 2010b), which can result in the infection of both hospital personnel and patients (van Duijkeren et al., 2010; Weese, 2010a; Weese and van Duijkeren, 2010). To facilitate the introduction of evidence-based, effective MRSA containment protocols (Kampf and Kramer, 2004; Lukas et al., 2010; Tschudin-Sutter et al., 2010), the factors that contribute to nosocomial transmission require examination and analysis. Sources of MRSA contamination in equine hospitals include the environment, humans and horses (Weese et al., 2004; Van den Eede, 2008; Van den Eede et al., 2009). Up to 14% of hospital personnel can be carriers of MRSA (Weese and van Duijkeren, 2010)
⇑ Corresponding author. Tel.: +32 9 264 76 18. E-mail address: [email protected] (A. Van den Eede). 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.12.004
and equine clinic environments can be extensively contaminated (Weese et al., 2004; Cimolai, 2008; van Duijkeren et al., 2010). Hospitalised horses can also form a substantial infection reservoir with nasal detection rates of up to 10.9% and 42% on admission and during hospitalisation, respectively (Weese et al., 2006; Van den Eede et al., 2009; van Duijkeren et al., 2010). It is likely that S. aureus colonises the nasal mucous membrane and skin of animals in a manner analogous to humans (Williams, 1963; Quinn et al., 2007; Leonard and Markey, 2008; Bitterman et al., 2010; Weese and van Duijkeren, 2010). Although the presence of MRSA on the skin of horses in the absence of clinical signs has not been demonstrated (Busscher et al., 2006; van Duijkeren et al., 2010; Weese and van Duijkeren, 2010), such an occurrence would be important in nosocomial transmission, especially when hospitalised horses are frequently handled (Kampf and Kramer, 2004; Anderson et al., 2008; Tschudin-Sutter et al., 2010). The objective of the present study was to determine if MRSA is resident on the healthy skin as well as on the nasal mucosa of hospitalised horses. Animals were sampled at several skin sites on the body and two different types of swab were used in order to optimise detection.
A. Van den Eede et al. / The Veterinary Journal 193 (2012) 408–411 Materials and methods Sample collection During February 2009, 30 clinically normal horses resident at the large animal hospital of Ghent University (Belgium) for P6 months were screened for MRSA. These animals, used for both educational and research purposes, were being accommodated at three separate locations within the hospital (surgery, internal medicine and obstetrics), and had been permanently indoors for >1 month prior to sampling. All were swabbed at nine body locations (Fig. 1) and nasal samples were taken from 10 cm within the left ventral meatus as previously described (Weese et al., 2005, 2006; Van den Eede et al., 2009). The skin sites chosen for sampling were those frequently touched by human hands and by the noses of horse during individual and mutual grooming. Since horses are conventionally handled from the left, skin locations on this side were favoured (Fig. 1). Swabs were stroked, whilst being rotated, at least 10 times over a 5 10 cm area of skin, which had been neither cleaned nor disinfected prior to sampling. Two different types of swab were used consecutively at each location: a ‘classic’ cotton-tipped swab embedded in solid Stuart’s medium (swab A) (UniTer Amies CLR, MEUS); and a nylon-flocked elution swab (ESwab, Copan) immersed in liquid Amies medium (swab B). The sampling sequence (swab A–swab B, swab B– swab A) was alternated randomly between horses and all specimens were held at 4 °C overnight prior to further processing. Isolation, identification and typing of MRSA isolates Either the classic swab or a volume of 50 lL of the ESwab transport medium was transferred to a 0.001% colistin and nalidixic acid containing brain heart infusion (BHI) enrichment broth. Further isolation, phenotypic identification and confirmation of oxacillin resistance were performed as previously described (Van den Eede et al., 2009), with the modification of inoculating 50 lL (instead of 1 lL) of the enrichment broth onto the ChromID MRSA plates (Biomérieux). This modification was designed to improve the bacterial detection limit. A triplex PCR targeting the 16S rRNA, mecA and nuc genes (Maes et al., 2002), was used to confirm the presence of MRSA. When available, a nasal and randomly chosen skin isolate were selected from each positive horse for spa (Harmsen et al., 2003) and SCCmec (Vanderhaeghen et al., 2010a) typing. Non-typable SCCmec cassettes were further determined according to Kondo et al. (2007). Statistical analysis The relative sensitivity (RS) and 95% confidence interval (CI) for MRSA detection was calculated for each skin site using the ‘gold standard’ of isolation at any location from at least one swab type (Lautenbach et al., 2009). The same procedure was followed for all combinations of two or three locations. The isolation rate/location (IR) was calculated as the proportion of animals testing positive at a particular location when the results of both swab types were pooled. Differences in IRs, swab types, sequences of sampling and hospital location were examined using logistic regression including an odds ratio calculation with a 95% CI. The SPSS Statistics 17.0 package (IBM Corporation) was used and a significance level (a) of 0.05 was applied. To quantify any agreement between the different swab types and sequence of sampling, kappa values (j) were also calculated and interpreted according to the scale proposed by Viera and Garrett (2005).
Results Culture-positive horses and sampling sites A total of 538 samples (269 classic and 269 ESwabs) were taken as nasal samples could not be obtained from one of the 30 horses.
Fig. 1. Diagram highlighting the selected sampling locations on the left side of the body: within the nasal chambers and at eight positions on the skin surface.
409
Twelve animals (40%) tested positive for at least one anatomical location. MRSA was detected in the nasal chambers and on the skin of 34.5% (10/29) and 30% (9/30) of horses respectively and two of the skin-positive animals had negative nasal samples (Fig. 2). All skin locations sampled had at least one positive result although isolation rates (IR) varied between these sites. Bacteria were most commonly recovered from nasal samples (IR, 33.3%), whereas the highest IRs for the sampled skin regions were found at the carpus (16.7%), and neck, withers and croup (13.3% in each case). Other skin locations such as the perineum and pastern (10%, P = 0.04), and flank and thigh (6.7%, P = 0.02) tested positive significantly less frequently than the nasal chambers. The likelihood of detecting a positive animal by screening a single location was highest for the nasal chambers (RS = 83.3%, 95% CI 69.8–96.9). Combining nasal sampling with samples from either the carpus, neck, withers, pastern or perineum increased the likelihood of detection (RS = 91.7%, 95% CI 81.6–100), whereas additional sampling of the croup, flank or thigh did not. The proportion of positive animals was significantly higher in those hospitalised in the surgery areas (60% [6/10], P = 0.03) and internal medicine (55.6% [5/9], P = 0.04) areas than in the horses held in the obstetrics department (9.1% [1/11]). The risks of a horse testing positive were 15 (95% CI 1.3, 167.6) and 12.5 (95% CI 1.1, 143.4) times higher for animals held in the internal medicine/surgical, than in the obstetrics area, respectively. Strain typing, influence of swab type and sequence of swabbing A selected 19 isolates were strain typed. The spa type t011 was the most frequently identified (11/19), whereas type t1451 was only found on horses held in the surgery department, where it was the dominant type (5/7). Three isolates did not have the classic SCCmec IVa cassette, and were classified as non-typable. These isolates appeared to carry a ccr complex of type A2/B2. Although the difference in the percentage of positives between the classic (10.8%) and ESwab (7.4%) swabs was not significant (P = 0.18), agreement between the results obtained from both swab types was only moderate (j = 0.44). Only 37.9% of the sampled anatomical locations that tested positive using classic swabs also tested positive when ESwabs were used, and only 57.9% of sampled sites positive using ESwabs were also positive using the classic alternative. Furthermore, whether a sample was taken first or second did not make a significant difference to the MRSA detection rate (P = 0.66), and agreement between the first and second samples was only moderate (j = 0.44). Analysis per swab type indicated that the classic swab and ESwab cultured positive more frequently when taken first (P = 0.01) and second (P = 0.02), respectively. Discussion This is the first study to screen for the presence of MRSA on the skin of horses hospitalised long-term. Its findings have important implications for the design of effective screening and containment protocols for this pathogen in equine clinics. Substantial MRSA reservoirs were detected both on the skin and in the nasal chambers of such horses, and animals tested positive from skin locations frequently handled by hospital personnel. MRSA can readily spread from carrier horses to other animals, to humans and to the wider environment (van Duijkeren et al., 2010; Weese, 2010b; Weese and van Duijkeren, 2010). Similar to the situation in humans (Eveillard et al., 2008), positive horses hospitalised long-term could initiate and/or contribute to the substantial environmental contamination reported in several equine studies (van Duijkeren et al., 2010; Weese et al., 2004). It is thus essential that personnel working in equine clinics strictly adhere to the hand-hygiene
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A. Van den Eede et al. / The Veterinary Journal 193 (2012) 408–411
Fig. 2. Diagram illustrating the extent of isolation of MRSA from the nasal chambers and skin of the 12/30 positive horses.
guidelines that are central to the control of nosocomial MRSA infections (Kampf and Kramer, 2004; Anderson et al., 2008; Tschudin-Sutter et al., 2010). Our results also support the hypothesis that different management practices in different areas of an equine hospital can influence MRSA isolation rates (Weese and Rousseau, 2005; van Duijkeren et al., 2010). Further research is required to determine which factors (e.g. movement of staff, students, equipment and patients) are important in this context. As described in humans (Bradley, 2007; CLSI, 2010), there was not always a correlation between the isolation of MRSA from the skin and the nasal chambers and isolation rates from the skin varied with anatomical location. Thus, MRSA screening protocols in equine hospitals would benefit from sampling at least one targeted skin site in addition to the nasal chambers, similar to the procedures followed in humans (Combined Working Party of the BSAC, 1998; Wanten et al., 1998; Lautenbach et al., 2009; Bitterman et al., 2010). Since the current study only sampled animals hospitalised long-term (>6 months), further work will be required to confirm the validity of the selected ‘high-contact’ locations for the screening of various patient populations. Successful screening programmes require good sampling and isolation techniques (Songer and Post, 2005; Quinn et al., 2007). Until now, a limited number of screening protocols, based largely on experiences with humans and other animal species, have been used in horses but have not been subject to critical assessment (Anzai et al., 1996; Weese et al., 2005, 2006; Busscher et al., 2006; Cuny et al., 2006; Van den Eede et al., 2009; van Duijkeren et al., 2010). The establishment of a ‘gold standard’ for screening for MRSA in horses remains an important goal of further research. In the present study, the comparison of the results obtained using the two types of swab and the sequence of usage have raised pertinent questions: although we expected the ESwab to be superior to the conventional swab in terms of organism recovery, this was not found to be the case, with a difference in detection rate of 7.4% vs. 10.8% (P = 0.18) in favour of the classic swab (Drake et al., 2005; Van Horn et al., 2008; Fontana et al., 2009; Smismans et al., 2009). In addition, although the sampling sequence did not seem to affect overall bacterial detection, isolation was influenced by whether a particular swab type was used first or second, the explanation of which remains unclear. All spa-typed MRSA isolates belong to the livestock-associated ST398 clone which has been identified most commonly in equine clinics in Belgium and The Netherlands (Hermans et al., 2008; Van den Eede et al., 2009; van Duijkeren et al., 2010). Although this clone is not the most virulent, it is zoonotic and could possibly become more pathogenic through the acquisition of virulence genes (Catry et al., 2010; Vanderhaeghen et al., 2010b). In this study, the ST398 clone has been detected at sites with which equine hospital personnel have frequent contact. Thus, all hospital staff should be correctly informed of its zoonotic potential so that raised
awareness can result in improved hygiene regulation compliance and thus decreased chances of transmission to humans and animals (Scheithauer et al., 2010). Conclusions Containment strategies for MRSA at equine clinics should be cognisant of potential MRSA reservoirs in the nasal chambers and on the skin of horses hospitalised long-term. Our findings indicate that, as in man, the sampling of at least one skin site, in addition to the nose, should be considered when devising screening protocols. Conflict of interest statement None of the authors of this paper has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper. References Anderson, M.E., Lefebvre, S.L., Weese, J.S., 2008. Evaluation of prevalence and risk factors for methicillin-resistant Staphylococcus aureus colonization in veterinary personnel attending an international equine veterinary conference. Veterinary Microbiology 129, 410–417. Anzai, T., Kamada, M., Kanemaru, T., Sugita, S., Shimizu, A., Higuchi, T., 1996. Isolation of methicillin-resistant Staphylococcus aureus (MRSA) from mares with metritis and its zooepidemiology. Journal of Equine Science 7, 7–11. Bitterman, Y., Laor, A., Itzhaki, S., Weber, G., 2010. Characterization of the best anatomical sites in screening for methicillin-resistant Staphylococcus aureus colonization. European Journal of Clinical Microbiology and Infectious Diseases 29, 391–397. Bradley, S.F., 2007. Eradication or decolonization of methicillin-resistant Staphylococcus aureus carriage: What are we doing and why are we doing it? Clinical Infectious Diseases 44, 186–189. Busscher, J.F., van Duijkeren, E., van Oldruitenborgh-Oosterbaan, M.M.S., 2006. The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands. Veterinary Microbiology 113, 131–136. Catry, B., van Duijkeren, E., Pomba, M.C., Greko, C., Moreno, M.A., Pyörälä, S., Ruzauskas, M., Sanders, P., Threlfall, E.J., Ungemach, F., Törneke, K., MunozMadero, C., Torren-Edo, J., 2010. Reflection paper on MRSA in food-producing and companion animals: Epidemiology and control options for human and animal health. Epidemiology and Infection 138, 626–644. Cimolai, N., 2008. MRSA and the environment: Implications for comprehensive control measures. European Journal of Clinical Microbiology and Infectious Diseases 27, 481–493. CLSI, 2010. Laboratory Methods for Detecting Methicillin-Resistant Staphylococcus aureus in Samples. CLSI Document X07-R. Surveillance for Methicillin-Resistant Staphylococcus aureus: Principles, Practices and Challenges: A Report. Clinical and Laboratory Standards Institute, Wayne, PA, USA, pp. 8–11. Combined working party of the BSAC, H.I., 1998. Revised guidelines for the control of methicillin-resistant Staphylococcus aureus infection in hospitals. Journal of Hospital Infection 39, 253–290. Cuny, C., Kuemmerle, J., Stanek, C., Willey, B., Strommenger, B., Witte, W., 2006. Emergence of MRSA infections in horses in a veterinary hospital: strain characterisation and comparison with MRSA from humans. Eurosurveillance 11, 44–47. Drake, C., Barenfanger, J., Lawhorn, J., Verhulst, S., 2005. Comparison of easyflow Copan Liquid Stuart’s and Starplex swab transport systems for recovery
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Contents lists available at SciVerse ScienceDirect
The Veterinary Journal journal homepage: www.elsevier.com/locate/tvjl
Methicillin-resistant Staphylococcus aureus (MRSA) on the skin of long-term hospitalised horses A. Van den Eede a,⇑, K. Hermans b, A. Van den Abeele c, K. Floré d, J. Dewulf e, W. Vanderhaeghen f, F. Crombé f, P. Butaye b,f, F. Gasthuys a, F. Haesebrouck b, A. Martens a a
Department of Surgery and Anaesthesiology of Domestic Animals, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Department of Medical Microbiology, AZ St.-Lucas, Groenebriel 1, 9000 Ghent, Belgium d Department of Medical Microbiology, AZ St.-Lucas, St.-Lucaslaan 29, 8310 Bruges, Belgium e Veterinary Epidemiology Unit, Department of Obstetrics, Reproduction and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium f Veterinary and Agrochemical Research Centre, CODA-CERVA-VAR, Groeselenberg 99, 1180 Ukkel, Belgium b c
a r t i c l e
i n f o
Article history: Accepted 14 December 2011
Keywords: MRSA Bacterial screening Skin locations Horse Hospitalisation
a b s t r a c t Given the significance of methicillin-resistant Staphylococcus aureus (MRSA) infections for both horses and staff in equine veterinary hospitals, protocols are required to minimise the risk of nosocomial transmission, including the screening of the skin and nasal chambers of equine patients for evidence of infection. The objective of this study was to clarify the potential existence and extent of MRSA on the skin of horses requiring long-term hospitalisation (P6 months). Thirty such horses were sampled at eight different locations on their skin and from their nasal chambers. MRSA was isolated from 12 animals (40%), with all sample sites testing positive on at least one occasion. Organisms were most frequently detected in the nasal chambers (relative sensitivity, 83.3%; 34.5% positive horses; isolation rate 33.3%). Skin presence was found in 30% of animals with the highest isolation rates found at the carpus (16.7%), neck, withers and croup (13.3% each). To achieve a relative screening sensitivity of >90%, at least one skin site was required in addition to nasal sampling. This evidence of skin as well as nasal reservoirs of MRSA in long-term hospitalised horses should facilitate the design of effective screening and containment protocols. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction The emergence of methicillin-resistant Staphylococcus aureus (MRSA) in equine hospitals has highlighted the need for the implementation of containment strategies that minimise the risk of nosocomial transmission (Tschudin-Sutter et al., 2010; van Duijkeren et al., 2010; Weese, 2010b), which can result in the infection of both hospital personnel and patients (van Duijkeren et al., 2010; Weese, 2010a; Weese and van Duijkeren, 2010). To facilitate the introduction of evidence-based, effective MRSA containment protocols (Kampf and Kramer, 2004; Lukas et al., 2010; Tschudin-Sutter et al., 2010), the factors that contribute to nosocomial transmission require examination and analysis. Sources of MRSA contamination in equine hospitals include the environment, humans and horses (Weese et al., 2004; Van den Eede, 2008; Van den Eede et al., 2009). Up to 14% of hospital personnel can be carriers of MRSA (Weese and van Duijkeren, 2010)
⇑ Corresponding author. Tel.: +32 9 264 76 18. E-mail address: [email protected] (A. Van den Eede). 1090-0233/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tvjl.2011.12.004
and equine clinic environments can be extensively contaminated (Weese et al., 2004; Cimolai, 2008; van Duijkeren et al., 2010). Hospitalised horses can also form a substantial infection reservoir with nasal detection rates of up to 10.9% and 42% on admission and during hospitalisation, respectively (Weese et al., 2006; Van den Eede et al., 2009; van Duijkeren et al., 2010). It is likely that S. aureus colonises the nasal mucous membrane and skin of animals in a manner analogous to humans (Williams, 1963; Quinn et al., 2007; Leonard and Markey, 2008; Bitterman et al., 2010; Weese and van Duijkeren, 2010). Although the presence of MRSA on the skin of horses in the absence of clinical signs has not been demonstrated (Busscher et al., 2006; van Duijkeren et al., 2010; Weese and van Duijkeren, 2010), such an occurrence would be important in nosocomial transmission, especially when hospitalised horses are frequently handled (Kampf and Kramer, 2004; Anderson et al., 2008; Tschudin-Sutter et al., 2010). The objective of the present study was to determine if MRSA is resident on the healthy skin as well as on the nasal mucosa of hospitalised horses. Animals were sampled at several skin sites on the body and two different types of swab were used in order to optimise detection.
A. Van den Eede et al. / The Veterinary Journal 193 (2012) 408–411 Materials and methods Sample collection During February 2009, 30 clinically normal horses resident at the large animal hospital of Ghent University (Belgium) for P6 months were screened for MRSA. These animals, used for both educational and research purposes, were being accommodated at three separate locations within the hospital (surgery, internal medicine and obstetrics), and had been permanently indoors for >1 month prior to sampling. All were swabbed at nine body locations (Fig. 1) and nasal samples were taken from 10 cm within the left ventral meatus as previously described (Weese et al., 2005, 2006; Van den Eede et al., 2009). The skin sites chosen for sampling were those frequently touched by human hands and by the noses of horse during individual and mutual grooming. Since horses are conventionally handled from the left, skin locations on this side were favoured (Fig. 1). Swabs were stroked, whilst being rotated, at least 10 times over a 5 10 cm area of skin, which had been neither cleaned nor disinfected prior to sampling. Two different types of swab were used consecutively at each location: a ‘classic’ cotton-tipped swab embedded in solid Stuart’s medium (swab A) (UniTer Amies CLR, MEUS); and a nylon-flocked elution swab (ESwab, Copan) immersed in liquid Amies medium (swab B). The sampling sequence (swab A–swab B, swab B– swab A) was alternated randomly between horses and all specimens were held at 4 °C overnight prior to further processing. Isolation, identification and typing of MRSA isolates Either the classic swab or a volume of 50 lL of the ESwab transport medium was transferred to a 0.001% colistin and nalidixic acid containing brain heart infusion (BHI) enrichment broth. Further isolation, phenotypic identification and confirmation of oxacillin resistance were performed as previously described (Van den Eede et al., 2009), with the modification of inoculating 50 lL (instead of 1 lL) of the enrichment broth onto the ChromID MRSA plates (Biomérieux). This modification was designed to improve the bacterial detection limit. A triplex PCR targeting the 16S rRNA, mecA and nuc genes (Maes et al., 2002), was used to confirm the presence of MRSA. When available, a nasal and randomly chosen skin isolate were selected from each positive horse for spa (Harmsen et al., 2003) and SCCmec (Vanderhaeghen et al., 2010a) typing. Non-typable SCCmec cassettes were further determined according to Kondo et al. (2007). Statistical analysis The relative sensitivity (RS) and 95% confidence interval (CI) for MRSA detection was calculated for each skin site using the ‘gold standard’ of isolation at any location from at least one swab type (Lautenbach et al., 2009). The same procedure was followed for all combinations of two or three locations. The isolation rate/location (IR) was calculated as the proportion of animals testing positive at a particular location when the results of both swab types were pooled. Differences in IRs, swab types, sequences of sampling and hospital location were examined using logistic regression including an odds ratio calculation with a 95% CI. The SPSS Statistics 17.0 package (IBM Corporation) was used and a significance level (a) of 0.05 was applied. To quantify any agreement between the different swab types and sequence of sampling, kappa values (j) were also calculated and interpreted according to the scale proposed by Viera and Garrett (2005).
Results Culture-positive horses and sampling sites A total of 538 samples (269 classic and 269 ESwabs) were taken as nasal samples could not be obtained from one of the 30 horses.
Fig. 1. Diagram highlighting the selected sampling locations on the left side of the body: within the nasal chambers and at eight positions on the skin surface.
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Twelve animals (40%) tested positive for at least one anatomical location. MRSA was detected in the nasal chambers and on the skin of 34.5% (10/29) and 30% (9/30) of horses respectively and two of the skin-positive animals had negative nasal samples (Fig. 2). All skin locations sampled had at least one positive result although isolation rates (IR) varied between these sites. Bacteria were most commonly recovered from nasal samples (IR, 33.3%), whereas the highest IRs for the sampled skin regions were found at the carpus (16.7%), and neck, withers and croup (13.3% in each case). Other skin locations such as the perineum and pastern (10%, P = 0.04), and flank and thigh (6.7%, P = 0.02) tested positive significantly less frequently than the nasal chambers. The likelihood of detecting a positive animal by screening a single location was highest for the nasal chambers (RS = 83.3%, 95% CI 69.8–96.9). Combining nasal sampling with samples from either the carpus, neck, withers, pastern or perineum increased the likelihood of detection (RS = 91.7%, 95% CI 81.6–100), whereas additional sampling of the croup, flank or thigh did not. The proportion of positive animals was significantly higher in those hospitalised in the surgery areas (60% [6/10], P = 0.03) and internal medicine (55.6% [5/9], P = 0.04) areas than in the horses held in the obstetrics department (9.1% [1/11]). The risks of a horse testing positive were 15 (95% CI 1.3, 167.6) and 12.5 (95% CI 1.1, 143.4) times higher for animals held in the internal medicine/surgical, than in the obstetrics area, respectively. Strain typing, influence of swab type and sequence of swabbing A selected 19 isolates were strain typed. The spa type t011 was the most frequently identified (11/19), whereas type t1451 was only found on horses held in the surgery department, where it was the dominant type (5/7). Three isolates did not have the classic SCCmec IVa cassette, and were classified as non-typable. These isolates appeared to carry a ccr complex of type A2/B2. Although the difference in the percentage of positives between the classic (10.8%) and ESwab (7.4%) swabs was not significant (P = 0.18), agreement between the results obtained from both swab types was only moderate (j = 0.44). Only 37.9% of the sampled anatomical locations that tested positive using classic swabs also tested positive when ESwabs were used, and only 57.9% of sampled sites positive using ESwabs were also positive using the classic alternative. Furthermore, whether a sample was taken first or second did not make a significant difference to the MRSA detection rate (P = 0.66), and agreement between the first and second samples was only moderate (j = 0.44). Analysis per swab type indicated that the classic swab and ESwab cultured positive more frequently when taken first (P = 0.01) and second (P = 0.02), respectively. Discussion This is the first study to screen for the presence of MRSA on the skin of horses hospitalised long-term. Its findings have important implications for the design of effective screening and containment protocols for this pathogen in equine clinics. Substantial MRSA reservoirs were detected both on the skin and in the nasal chambers of such horses, and animals tested positive from skin locations frequently handled by hospital personnel. MRSA can readily spread from carrier horses to other animals, to humans and to the wider environment (van Duijkeren et al., 2010; Weese, 2010b; Weese and van Duijkeren, 2010). Similar to the situation in humans (Eveillard et al., 2008), positive horses hospitalised long-term could initiate and/or contribute to the substantial environmental contamination reported in several equine studies (van Duijkeren et al., 2010; Weese et al., 2004). It is thus essential that personnel working in equine clinics strictly adhere to the hand-hygiene
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Fig. 2. Diagram illustrating the extent of isolation of MRSA from the nasal chambers and skin of the 12/30 positive horses.
guidelines that are central to the control of nosocomial MRSA infections (Kampf and Kramer, 2004; Anderson et al., 2008; Tschudin-Sutter et al., 2010). Our results also support the hypothesis that different management practices in different areas of an equine hospital can influence MRSA isolation rates (Weese and Rousseau, 2005; van Duijkeren et al., 2010). Further research is required to determine which factors (e.g. movement of staff, students, equipment and patients) are important in this context. As described in humans (Bradley, 2007; CLSI, 2010), there was not always a correlation between the isolation of MRSA from the skin and the nasal chambers and isolation rates from the skin varied with anatomical location. Thus, MRSA screening protocols in equine hospitals would benefit from sampling at least one targeted skin site in addition to the nasal chambers, similar to the procedures followed in humans (Combined Working Party of the BSAC, 1998; Wanten et al., 1998; Lautenbach et al., 2009; Bitterman et al., 2010). Since the current study only sampled animals hospitalised long-term (>6 months), further work will be required to confirm the validity of the selected ‘high-contact’ locations for the screening of various patient populations. Successful screening programmes require good sampling and isolation techniques (Songer and Post, 2005; Quinn et al., 2007). Until now, a limited number of screening protocols, based largely on experiences with humans and other animal species, have been used in horses but have not been subject to critical assessment (Anzai et al., 1996; Weese et al., 2005, 2006; Busscher et al., 2006; Cuny et al., 2006; Van den Eede et al., 2009; van Duijkeren et al., 2010). The establishment of a ‘gold standard’ for screening for MRSA in horses remains an important goal of further research. In the present study, the comparison of the results obtained using the two types of swab and the sequence of usage have raised pertinent questions: although we expected the ESwab to be superior to the conventional swab in terms of organism recovery, this was not found to be the case, with a difference in detection rate of 7.4% vs. 10.8% (P = 0.18) in favour of the classic swab (Drake et al., 2005; Van Horn et al., 2008; Fontana et al., 2009; Smismans et al., 2009). In addition, although the sampling sequence did not seem to affect overall bacterial detection, isolation was influenced by whether a particular swab type was used first or second, the explanation of which remains unclear. All spa-typed MRSA isolates belong to the livestock-associated ST398 clone which has been identified most commonly in equine clinics in Belgium and The Netherlands (Hermans et al., 2008; Van den Eede et al., 2009; van Duijkeren et al., 2010). Although this clone is not the most virulent, it is zoonotic and could possibly become more pathogenic through the acquisition of virulence genes (Catry et al., 2010; Vanderhaeghen et al., 2010b). In this study, the ST398 clone has been detected at sites with which equine hospital personnel have frequent contact. Thus, all hospital staff should be correctly informed of its zoonotic potential so that raised
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