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JAC Advance Access originally published online on December 19, 2007
Journal of Antimicrobial Chemotherapy 2008 61(2):282-285; doi:10.1093/jac/dkm487
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius detected in the BfT-GermVet monitoring programme 2004–2006 in Germany

Stefan Schwarz1,*, Kristina Kadlec1 and Birgit Strommenger2

1 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institute (FLI), Höltystr. 10, 31535 Neustadt-Mariensee, Germany 2 Robert Koch Institute, Wernigerode Branch, D-38855 Wernigerode, Germany

* Corresponding author. Tel: +49-5034-871-241; Fax: +49-5034-871-246; E-mail: stefan.schwarz{at}fal.de

Received 13 September 2007; returned 25 October 2007; revised 8 November 2007; accepted 26 November 2007


   Abstract
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Objectives: During recent years, methicillin-resistant Staphylococcus aureus (MRSA) strains from animals have become the focus of various studies. In the present study, coagulase-positive staphylococci obtained from pigs, dogs and cats suffering from acute infections, which had been collected in the BfT-GermVet monitoring programme in Germany and phenotypically identified as oxacillin-resistant, were characterized.

Methods: The staphylococci were comparatively investigated for their resistance phenotypes and genotypes. Resistance genes were identified by PCR. MRSA strains were further characterized by SmaI macrorestriction analysis and spa typing to assess their genomic relationships.

Results: Among the 248 strains tested, 7 strains (5 porcine S. aureus and 2 canine Staphylococcus pseudintermedius) carried the resistance gene mecA. Gentamicin resistance was based on the presence of the gene aacA/aphD while three different tetracycline resistance genes, tet(K), tet(L) and tet(M), alone or in combinations, were detected. The single macrolide/lincosamide-resistant strain carried an erm(A) gene. All MRSA strains proved to be non-typeable by SmaI macrorestriction analysis and exhibited the spa types t011 (four strains) or t034 (one strain).

Conclusions: Based on their spa types and their non-respondence to SmaI digestion, all five porcine MRSA strains resembled MRSA strains of multilocus sequence type ST398, previously detected among pigs in neighbouring countries such as The Netherlands or Denmark. The results of this study indicate that such strains are also involved in defined disease conditions of pigs from various parts of Germany.

Keywords: MRSA , pigs , spa typing , MLST type 398


   Introduction
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Two antimicrobial resistance monitoring programmes on veterinary pathogens, GERM-Vet and BfT-GermVet, have been conducted during recent years in Germany. The monitoring programme GERM-Vet, which has been operating since 2001 on an annual basis and is conducted by the Federal Office for Consumer Protection and Food Safety in Berlin, concentrates on the economically most relevant bacterial pathogens associated with selected disease conditions of food-producing animals. Since there was a lack of data on the susceptibility status of bacterial pathogens from pet and companion animals, the Federation for Animal Health [Bundesverband für Tiergesundheit (BfT) e.V.] decided in 2003 to start another nationwide monitoring programme, which should complement the GERM-Vet programme. This second programme included mainly bacterial pathogens from horses, dogs and cats, but also bacteria from disease conditions of cattle and pigs not tested in the GERM-Vet programme. During a 27 month period between January 2004 and March 2006, a total of 1632 bacterial strains—including 85 strains from cattle, 322 strains from pigs, 326 strains from horses and 899 strains from dogs/cats—obtained from 31 defined disease conditions were sampled and investigated for their in vitro susceptibility to 22 single antimicrobial agents and two combinations of antimicrobial agents. Details of the structure and organization of this programme as well as on the sampling plan, the bacteria sampled and the disease conditions from which they were obtained have recently been published.1

Among the bacteria sampled, a total of 248 coagulase-positive and coagulase-variable staphylococci were investigated from two disease conditions in pigs and dogs/cats. These included 46 staphylococcal strains from infections of the urinary-genital tract including the mastitis metritis agalactia (MMA) syndrome of pigs, 44 strains from skin infections of pigs, 57 strains from respiratory tract infections of dogs/cats and 101 strains from infections of skin/ear/mouth of dogs/cats.2 Initial in vitro susceptibility testing revealed that 8 (3.2%) of these 248 staphylococcal strains were considered as oxacillin-resistant with MIC values ranging between 8 and ≥32 mg/L.2 These strains were further investigated in the present study.


   Materials and methods
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The oxacillin-resistant strains were re-tested by broth microdilution according to the recommendations given in the document M31-A2 of the CLSI for phenotypic expression of resistance. The two Staphylococcus intermedius strains were biochemically re-investigated for correct assignment to the species S. intermedius or the recently published new species Staphylococcus pseudintermedius.3,4 This was done by using the ID32 Staph system (bioMérieux, Nürtingen, Germany) and an additional home-made bouillon to detect utilization of mannitol under anaerobic conditions.

PCR-directed detection of the mecA gene followed previously described specifications.5 Analysis of further resistance genes was conducted by multiplex or specific PCR assays as previously described.5–7 SmaI macrorestriction analysis and spa typing followed the specifications given by Strommenger et al.5 For spa typing, the polymorphic X-region of the protein A gene (spa) was PCR amplified and sequenced as described.5 The spa types were determined by using the software Ridom StaphType (Ridom GmbH, Würzburg, Germany).


   Results and discussion
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Re-testing of the presumably oxacillin-resistant strains confirmed phenotypic oxacillin resistance in seven cases. These included five porcine Staphylococcus aureus strains, two from skin infections, two from urinary-genital tract infections and one from the MMA syndrome, all revealing oxacillin MICs of 8 to ≥32 mg/L. Moreover, two oxacillin-resistant canine strains, tentatively identified as S. intermedius, one from a respiratory tract infection and the other from a case of canine pyoderma, exhibited oxacillin resistance with MICs of ≥32 or 8 mg/L, respectively. Analysis of the biochemical capacities of these latter two strains showed that they were identified as S. intermedius by the ID32 Staph system. However, their ability to produce the enzyme arginine dihydrolase and their inability to utilize D-mannitol under anaerobic conditions strongly suggested that these two strains are most likely S. pseudintermedius.3 In this context, it should be noted that members of this new species are not yet included in the databases of the currently available identification kits and, thus, may be misidentified as S. intermedius.

All phenotypically oxacillin-resistant strains carried the gene mecA. Further analysis of the antimicrobial susceptibility of these seven strains identified them as additionally resistant to tetracyclines, gentamicin, erythromycin, clindamycin, chloramphenicol and/or sulfamethoxazole/trimethoprim (Table 1). Analysis of the resistance genes identified the gene aacA/aphD in all gentamicin-resistant strains, a constitutively expressed erm(A) gene in the single erythromycin/clindamycin-resistant strain and the genes tet(K), tet(L) and/or tet(M) alone or in different combinations (Table 1). No cat genes were detected in the single chloramphenicol-resistant S. pseudintermedius and all sulfamethoxazole/trimethoprim-resistant strains were negative in a specific PCR for the Tn4003-associated dfrA gene.


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  		Table 1. Characteristics of the methicillin-resistant staphylococci detected in the BfT-GermVet programme 2004–2006 in Germany

 
All five porcine MRSA strains were non-typeable by SmaI macrorestriction analysis, but were typeable by spa typing. The resulting spa types were t034 in one strain from a skin infection and t011 in the remaining four strains (Table 1). These two types are among those spa types most frequently associated with the multilocus sequence type 398 as demonstrated in recent studies.8–11 Moreover, the non-typeability by SmaI macrorestriction analysis is another characteristic feature of ST398 MRSA strains.9–11 In addition, the observed multi-resistance phenotypes are also in good agreement with those recently reported for MRSA strains of spa type t011 from pigs and pig farmers in The Netherlands.10,11

Recent reports from neighbouring countries such as The Netherlands revealed a rather high prevalence of MRSA in pigs of up to 39%.10,11 These MRSA strains were detected in the nares of apparently healthy slaughter pigs. In contrast, the prevalence of MRSA among porcine staphylococci associated with defined disease conditions in Germany—as detected in the BfT-GermVet programme—was rather low at 5.6%.2 This might at least in part be due to the criteria for strains to become included in the different studies. In the BfT-GermVet programme, only bacterial strains from animals suffering from acute clinical infections were included.1 Moreover, the animals, from which the strains originated, must not have been pre-treated with antimicrobial agents in the 4 weeks prior to probe sampling.1 Since most MRSA strains exhibit a multi-resistance phenotype, it is anticipated that these strains have developed the respective resistance phenotype as a consequence of the selective pressure to which they had been exposed. Thus, it is assumed that MRSA strains might be found at higher frequencies in animals pre-treated with antimicrobial agents and as a consequence that the prevalence of MRSA in the BfT-GermVet programme might have been understated due to the study design. However, regardless of the true prevalence, the results of this study showed that MRSA strains of the same spa type as previously identified in pigs and/or pig farmers in The Netherlands and Denmark8,10,11 are also present in pigs from four different regions in Germany (Lower Saxony, Bavaria, Mecklenburg-Western Pomerania and Brandenburg). Thus, this observation supplements the data recently published on the presence of MRSA of clonal lineage ST398 in humans and animals in Central Europe.9


   Funding
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Funding
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This study was financially supported by the Bundesverband für Tiergesundheit (BfT) e.V.


   Transparency declarations
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None to declare.


   Acknowledgements
 
We thank Roswitha Becker and Kerstin Meyer for their excellent technical assistance.


   References
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1 Schwarz S, Alesík E, Grobbel M, et al. The BfT-GermVet monitoring program—aims and basics. Berl Münch Tierärztl Wochenschr (2007) 120:357–62.[Web of Science][Medline]

2 Schwarz S, Alesík E, Werckenthin C, et al. Antimicrobial susceptibility of coagulase-positive and coagulase-variable staphylococci from various indications of swine, dogs and cats as determined in the BfT-GermVet monitoring program 2004–2006. Berl Münch Tierärztl Wochenschr (2007) 120:372–9.[Web of Science][Medline]

3 Sasaki T, Kikuchi K, Tanaka Y, et al. Reclassification of phenotypically identified Staphylococcus intermedius. J Clin Microbiol (2007) 45:2770–8.[Abstract/Free Full Text]

4 Bannoehr J, Ben Zakour NL, Waller AS, et al. Population genetic structure of the Staphylococcus intermedius group: insights into agr diversification and the emergence of methicillin-resistant strains. J Bacteriol (2007) 189:8685–92.[Abstract/Free Full Text]

5 Strommenger B, Kehrenberg C, Kettlitz C, et al. Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and their relationship to human isolates. J Antimicrob Chemother (2006) 57:461–5.[Abstract/Free Full Text]

6 Strommenger B, Kettlitz C, Werner G, et al. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol (2003) 41:4089–94.[Abstract/Free Full Text]

7 Lüthje P, Schwarz S. Molecular basis of resistance to macrolides and lincosamides among staphylococci and streptococci from various animal sources collected in the resistance monitoring program BfT-GermVet. Int J Antimicrob Agents (2007) 29:528–35.[CrossRef][Web of Science][Medline]

8 Guardabassi L, Stegger M, Skov R. Retrospective detection of methicillin resistant and susceptible Staphylococcus aureus ST398 in Danish slaughter pigs. Vet Microbiol (2007) 122:384–6.[CrossRef][Web of Science][Medline]

9 Witte W, Strommenger B, Stanek C, et al. Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe. Emerg Infect Dis (2007) 13:255–8.[Web of Science][Medline]

10 van Duijkeren E, Ikawaty R, Broekhuizen-Stins MJ, et al. Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms. Vet Microbiol (2007) 126:383–9.[Medline]

11 de Neeling AJ, van den Broek MJ, Spalburg EC, et al. High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet Microbiol (2007) 122:366–72.[CrossRef][Web of Science][Medline]


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