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Journal of Clinical Microbiology, March 2009, p. 704-710, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01626-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Comparative Molecular Analysis Substantiates Zoonotic Potential of Equine Methicillin-Resistant Staphylococcus aureus ^,

Birgit Walther,^1* Stefan Monecke,² Claudia Ruscher,¹ Alexander W. Friedrich,³ Ralf Ehricht,⁴ Peter Slickers,⁴ Alexandra Soba,⁵ Claus-G. Wleklinski,⁵ Lothar H. Wieler,¹ and Antina Lübke-Becker¹

Institute of Microbiology and Epizootics (IMT), Free University Berlin, Philippstraße 13, 10115 Berlin, Germany,¹ Institut for Medical Microbiology and Hygiene, Medical Faculty Carl Gustav Carus, Fiedlerstr. 42, 01307 Dresden, Germany,² Institute for Hygiene, University of Muenster, Robert-Koch-Street 41, 48149 Muenster, Germany,³ Clondiag GmbH, Loebstedter Strasse 103-105, D-07749 Jena, Germany,⁴ Synlab-Vet Augsburg, Leitershofer Str. 25, 86157 Augsburg, Germany⁵

Received 21 August 2008/ Returned for modification 27 October 2008/ Accepted 15 December 2008

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Despite the increasing importance of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary medicine, knowledge about the epidemiology of the pathogen in horses is still poor. The phylogenetic relationship of strains of human and equine origins has been addressed before, usually by analyzing results of common standard classification methods for MRSA. This work intends to go beyond the baseline of typing procedures in order to comparatively characterize equine and human MRSA strains with similar phylogenetic backgrounds. In addition to multilocus sequence typing, pulsed-field gel electrophoresis, spa typing, staphylococcal cassette chromosome mec typing, and a PCR for Panton-Valentine leukocidin gene detection, a microarray analysis of a total of 185 structural, virulence-associated, and resistance loci was applied. The results showed that clonal complex 8 (CC8) was absolutely predominant (16 strains) in 19 investigated equine MSRA strains. Of the CC8 strains, 13 belonged to sequence type 254 (ST254) and the other 3 to ST8. This genotype has been isolated from different equine patients in various regions over several years, substantiating the apparent predominance of CC8 STs in MRSA strains of horses worldwide. Furthermore, comparatively investigated human strains of ST254 displayed molecular-typing results indistinguishable from those for strains of equine origin. Two further equine strains (ST22 and ST1117) showed similarity to ST22 human strains (CC22). One equine strain belonged to ST398, a genotype recently described as being frequently isolated from specimens from pigs and pig farmers. These data provide evidence for the adaptation of certain MRSA genotypes to more than one mammalian species, reflecting their extended host spectra.

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Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known pathogen in human, as well as in veterinary, medicine. First recognized in the mid-1970s as an infective agent in animals, MRSA is now reported frequently from different animal species (5, 13, 27, 30, 36). Apart from studies of MRSA infections in small animals (18, 38) and pigs (34), MRSA also seems to be of particular interest in horses. Equine MRSA infections have been reported from Asia, North America, Australia, and Europe, and strains have been characterized as being important nosocomial pathogens (1, 3, 29, 39).

In regard to potential interspecies transferability, previous studies have reported cases of concurrent MRSA colonization and infection, with strains assigned to indistinguishable genotypes by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), PCR for Panton-Valentine leukocidin (PVL) genes, and staphylococcal cassette chromosome mec (SCCmec) typing in horses and horse personnel (26, 29, 41). Similar to the observed situation in small animals, the predominant loci for MRSA infections in horses seem to be wound and surgical-site infections (8, 16, 37, 38).

To date, there are limited data about the prevalence of MRSA in the horse population in general. Evaluation of nasal swabs taken from 300 healthy horses in Slovenia (2005) in nonclinical facilities showed a complete absence of MRSA from the entire sample population (35). In contrast, another study from Ontario and New York reported a nasal-colonization prevalence of 4.7% in horses on local farms (42). In addition, hospitalization and prophylactic penicillin application were assumed to be risk factors for multidrug-resistant commensal S. aureus in horses (28).

As mentioned above, the epidemiology of MRSA in horses is poorly understood, and there is also a lack of information regarding the virulence and resistance determinants associated with MRSA strains from clinical specimens from equine patients. MRSA epidemiology may follow other rules than those that are known in human medicine, and molecular epidemiologic studies must address such significant contrasts (40). As a matter of fact, employment of distinct genetic-typing techniques allows a deep insight into the molecular compositions of MRSA strains from different host species.

Therefore, the aim of the present study was to provide a broad spectrum of genetic characteristics of equine MRSA strains in order to allow comparative analysis of the study collection, as well as to assess the relationship to epidemic human strains by means of a large number of molecular-typing results.

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Bacterial strains. A total of 19 MRSA strains were collected from 2003 to 2007 and stored at the Institute of Microbiology and Epizootics (IMT), Free University Berlin, which provides MRSA confirmation and typing for MRSA strains of animal origin. The strains were initially collected from routine microbiological specimens at the IMT, the Synlab-Vet laboratories (Augsburg, Bavaria, Germany), the Animal Health Services Bavaria (Tiergesundheitsdienst Bayern e.V), and the Institute for Hygiene and Infectious Diseases of Animals (Justus-Liebig University Giessen, Hesse, Germany). On receipt at the IMT, the strains were confirmed as MRSA by classical diagnostic and genotyping methods (38).

Further information regarding the geographical distributions, dates of isolation, and body sites of origin of specimens is given in Fig. 1.

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FIG. 1. Dendrogram (percent similarity) showing DNA restriction patterns after digestion with SmaI for 18 of 19 equine MRSA PFGE profiles (A, BE-2, H and H-1, and G and G-1) of this study in direct comparison to human epidemic strains (GelComparII by the unweighted-pair group method using average linkages). Dice coefficient, 1.2% tolerance and 0.5% optimization. One equine isolate (IMT-12553; ST398) was nontypeable (n.t.) using endonuclease SmaI and was therefore excluded from PFGE analysis. spa, spa type; a, German epidemic MRSA strain; b, representative PFGE type IMT-A, a pattern frequently associated with MRSA isolates of small- and exotic-animal origin (38); c, according to the MLST home page (http://www.mlst.net) and previously published data.

For comparative analysis, we used the human epidemic MRSA (EMRSA) strains Hannover (1000/93), Vienna (635/93), South German (131/98), Northern German (134/93), Rhine-Hesse (2387/00), and Barnim (BE) (1678/96) and a representative strain for PFGE type IMT-A (IMT-818-04) (38).

Typing of equine MRSA strains. (i) PFGE, MLST, PVL, and spa typing. MRSA strains were investigated by PFGE analysis using the restriction endonuclease SmaI and were resolved on a Chef DR II apparatus (Bio-Rad Laboratories) according to the method of Mulvey et al. (25). The relatedness of PFGE types was determined by visual comparison of gels and by employing the software GelCompar II (Applied Math, Kortrijk, Belgium) for the percent similarity calculation (Dice coefficient; tolerance, 1.2%; optimization, 0.5%). MRSA strains were named according to previously described principles (33) with an IMT prefix. The control strain S. aureus NCTC 8325 was included for normalizing band distances. PCR detection of PVL genes (lukS-PV and lukF-PV) was carried out as previously described by Lina et al. (17). For all MRSA strains, spa types were determined according to the method of Harmsen and coworkers (12). MLST was performed on representative strains of each PFGE spa type as described by Enright and coworkers (6). The allelic profile was obtained by sequencing internal fragments of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) using software provided on the MLST home page (http://www.mlst.net).

(ii) Microarray hybridization. The DNA microarray used in this study has been published previously (21). Briefly, it covers probes for 185 distinct genes and 300 alleles, including species-specific controls, accessory gene regulator (agr) alleles, genes encoding virulence factors and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), and capsule type-specific genes, as well as antimicrobial resistance determinants. These probes were synthetic, amino-modified oligonucleotides with an average length of 28 bases which were spotted on surface-modified glass slides mounted on the bottoms of standard reaction vials (AT system; Clondiag, Jena, Germany).

Staphylococcal cultures were grown in Columbia blood broth. Culture material was harvested and lysed using a buffer containing lysostaphin, lysozyme, and RNase A. This was followed by digestion with proteinase K and DNA purification employing an automated kit (Qiagen EZ1, tissue lysis protocol).

Repeated linear primer elongation was employed to amplify and to label all targets simultaneously using one primer per target in a mixture comprising all these primers. In this step, amplicons were also labeled by incorporation of biotin-16-dUTP. The labeled sample was denatured, chilled on ice, and hybridized to the array. After the washing steps and incubation with a blocking reagent, horseradish peroxidase-streptavidin conjugate was added to the array. This was followed by incubation and washing. Then, Seramun Green precipitating dye (Seramun, Heidesee, Germany) was added. After 5 min, a picture of the array was recorded using a dedicated reader (ATR 01; Clondiag) and analyzed.

More detailed protocols and procedures have also been described previously (19, 20).

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MLST, PFGE, PVL, and spa typing. The 19 MRSA strains derived from horses belonged to five sequence types clustering into three different genetic groups: clonal complex 8 (CC8), including sequence type 8 (ST8) and ST254; CC22, containing ST22 and ST1117; and ST398 (Fig. 1).

CC8 was found to be the predominant clonal background of the equine MRSA strains investigated in this study (16/19). PFGE types IMT-G (8) and IMT-G-1 (5) belonged to ST254, whereas ST8 was associated with a PFGE pattern assigned to IMT-H/IMT-H-1 (3). Two equine strains were found to share a CC22 background: one strain was assigned to PFGE type IMT-BE-2 (ST22), and a second showed PFGE type IMT-A, which had been identified previously as a common PFGE pattern for strains of small-animal origin (38). MLST analysis showed a single nucleotide substitution compared to the ST22 sequence of the pta gene for the equine IMT-A strain (IMT-10652). Therefore, it was accepted as a new sequence type, ST1117 (http://www.mlst.net).

One further strain (IMT-12553), assigned to ST398 (see below), was nontypeable by using endonuclease SmaI, a finding previously reported for strains of porcine and human origin with an ST398 background (34).

Cluster analysis of PFGE patterns led to the dendrogram shown in Fig. 1. In addition, the figure includes six representative human epidemic strains and one representative strain for PFGE pattern IMT-A. This comparison revealed a close relatedness of MRSA strains from horses from different geographic regions. In particular, the PFGE types designated IMT-G and IMT-G-1 were the most frequently occurring patterns and were associated with strains from horses located in Hesse, Berlin, Brandenburg, and Bavaria. Furthermore, strains displaying PFGE types IMT-G and IMT-G-1 seemed to be genetically related to the human epidemic strain Hannover (Fig. 1). PFGE patterns IMT-H/IMT-H-1 were found in strains derived from equine patients in Hesse and Brandenburg, respectively. PFGE type IMT-BE-2 was considered to be related to the human epidemic strain Barnim (Fig. 1). None of the 19 equine MRSA strains showed a positive result for the PVL PCR.

IMT-G and IMT-G-1 were associated with spa t036 and t009, IMT-H with spa t064, IMT-BE-2 with t032, IMT-A with t022, and the ST398 strain with spa t011. A summary overview is provided in Fig. 1.

Microarray analysis. Array data concerning resistance genes and virulence determinants are summarized in Fig. 2 and 3. Detailed additional data concerning agr allelic variants, staphylococcal superantigen-like or exotoxin-like genes, capsule types, biofilm, and MSCRAMM genes are provided in Fig. S1 and S2 in the supplemental material. For the sake of conciseness, some species markers that always yielded positive results (e.g., nuc and femA), most gene probe genes that were absent from all studied strains, and allelic markers without relevance to the studied strains were omitted. Some reference strains (typed and kindly provided by W. Witte, Robert Koch Institut, Wernigerode, Germany) and some isolates sampled from human patients at the University Hospital Dresden, Dredsen, Saxony, Germany, were included for comparison.

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FIG. 2. Microarray hybridization results for resistance genes. The strain characteristics in the leftmost column are followed by the numbers of isolates, which showed identical hybridization profiles. Black squares, positive; divided squares, variable results within one strain; gray squares, ambiguous results. Reference strains and human isolates (shaded background) are included for comparison.

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FIG. 3. Microarray hybridization results for virulence determinants. The strain characteristics in the leftmost column are followed by the numbers of isolates, which showed identical hybridization profiles. Black squares, positive; divided squares, variable results within one strain; gray squares, ambiguous results, usually cross-reacting probes for other allelic variants of the same gene. Reference strains and human isolates (shaded background) are included for comparison.

Microarray hybridization results for human and equine MRSA isolates were in accordance with the results of MLST and PFGE analysis: ST8 and ST254 strains of equine origin closely resembled CC8 strains of human origin, and especially the Hannover EMRSA strain (21) (Fig. 2 and 3; see Fig. S1 and S2 in the supplemental material).

Notable variability was observed to affect antibiotic resistance determinants. The most striking feature was the presence of different variants of recombinant, or mosaic-structured, SCCmec elements in 13 strains, all belonging to ST254/spa t009 or t036. These strains harbored mecA, mecR, ugpQ, and the dcs region, but not the recombinase gene ccrA2 or ccrB2. However, a weak and irregular reactivity with ccrA4 probes was observed. Eleven out of these 13 strains also harbored the mercury resistance operon (merA merB merR merT). Both the absence of ccrA and ccrB2 and the presence of the mer operon were also observed in some Hannover EMRSA of human origin (Fig. 2).

The three strains belonging to ST8/spa t064 harbored regular SCCmec type IV elements. All CC8 equine MRSA strains carried aacA-aphD (gentamicin resistance), tetM (tetracycline resistance), and fosB (fosfomycin resistance) (Fig. 2).

Fourteen CC8 strains harbored the beta-lactamase operon (blaZ blaI blaR), 13 were positive for both aphA3 (neomycin/kanamycin resistance) and sat (streptothricin resistance), 4 carried dfrA (trimethoprim resistance), and 2 were positive for the macrolide/lincosamide resistance gene ermC.

In contrast to the majority of Hannover human EMRSA strains, all equine MRSA strains lacked the genes for staphylokinase (sak) and enterotoxin A (sea). Some equine strains yielded weak signals for the gene encoding the staphylococcal complement inhibitor (scn), and some lacked it. In regard to the detection of other virulence factors (including the enterotoxin genes seb, sek, and seq and the leucocidin genes lukD and lukE) (Fig. 3), as well as the set and ssl genes (see Fig. S1 in the supplemental material), ST254 equine and human strains showed identical results.

The presence of most genes encoding MSCRAMMs of the host was similar to that in other CC8 strains, including Hannover EMRSA (see Fig. S2 in the supplemental material), COL (GenBank accession number CP000046.1), NCTC 8325 (CP000253.1), and USA300 (CP000255.1 and CP000730.1) (21). Like these strains, the equine MRSA strains belonged to capsule type 5 (see Fig. S2 in the supplemental material).

The gene for the bone-binding protein (bbp) was absent in all ST254/spa t009 or t036 strains, as well as in the sequenced strain COL (GenBank accession number CP000046.1) and human strains of the Hannover EMRSA. However, the gene was present in three equine strains belonging to ST8/spa t064. One ST254/spa t036 strain (variant 4 in Fig. 2 and 3; see Fig. S1 and S2 in the supplemental material) lacked the genes encoding clumping factor A (clfA) and the von Willebrand factor binding protein (vwb).

The single ST398 strain carried a SCCmec type V element and the beta-lactamase operon, as well as the tetracycline resistance determinants tetK and tetM. It did not harbor any known enterotoxin genes (except for a ubiquitous homologue), and the protease genes splA, splB, and splE were not detectable. Neither probes for hemolysin beta nor those for genes carried by hemolysin beta-converting phages (sea, sak, chp, and scn) yielded positive hybridization results. The capsule type of this strain was 5. The gene for collagen-binding adhesin (cna) was present, but sasG (S. aureus surface protein G) was not detectable.

Two strains belonged to CC22 (ST22 and the novel ST1117). Essentially they showed results identical to those for human MRSA of the Barnim EMRSA type (21). Both strains harbored the egc locus comprising the enterotoxin genes seg, sei, sem, sen, seo, seu, and sey. Other enterotoxin genes were absent, although the PFGE reference strain 1678/96 was positive for the enterotoxin genes sec and sel (which also occur in many, but not all, human strains of that genotype [21]). Similar to other ST22 strains, both strains yielded atypical ssl and set gene patterns, showing cross-reactivity between probes for different allelic variants of a given gene, indicating the presence of yet-unidentified alleles. Similarly, probes based on CC1, -5, -8, and -30 sequences of hemolysin gamma/leukocidin loci (lukF, lukS, and hlgA) gave no signals, while a specific probe for a deviant lukS allele (based on GenBank EF672356 [23]) yielded a positive signal in ST22 and ST45. The protease genes splA, splB, and splE were not detectable. The capsule type was 5. Both strains had a phage insertion in the hemolysin beta gene, carrying sak, chp, and scn. In addition, they carried SCCmec type IV elements, as well as the beta-lactamase operon.

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We have present detailed insight into the molecular composition of MRSA strains from clinically infected horses in comparison to well-known human epidemic strains by assessment of more than 185 genetic markers, MLST, PFGE, and spa-, PVL-, and SCCmec-typing procedures. In light of the emerging importance of this veterinary and zoonotic pathogen (36, 40), we found interesting similarities in the genetic compositions of MRSA strains from human and equine origin. Our data indicate a close molecular relationship of MRSA strains from human and equine origins, providing evidence for potential interspecies transferability and highlighting the zoonotic character of certain MRSA genotype lineages.

With one exception (the new ST1117), we found STs (ST254, ST22, ST8, and ST398) and associated spa types (t009, t036, t064, t011, t022, and t032) that had already been reported for MRSA of human and horse origins (3, 7, 15, 41, 43).

The vast majority (16/19) of strains studied belonged to CC8 (ST254 and ST8). Interestingly, equine strains belonging to CC8 were found to be widely distributed in Germany, i.e., in Hesse, Berlin, Brandenburg, and Bavaria. This finding substantiated the limited clonality of equine MRSA strains from different geographical regions that had been observed previously, e.g., in Europe and on the North American continent. Strains belonging to a CC8 background were reported from studies in Austria and Ireland (3, 24). Weese and coworkers investigated MRSA strains of equine origin from Ontario, Canada, and New York state (41) and revealed an absolute predominance of subtypes of (human) Canadian epidemic MRSA 5 (also known as USA500), which also belongs to ST8 (10). It is notable that ST254 and ST8 are very closely related; in fact, they differ only by a single nucleotide change (a single-locus variant [slv]) in gene aroE.

Furthermore, the equine ST254 strains investigated in this study are very similar to the human Hannover EMRSA (1000/93; clinical strains), which was highly prevalent in Germany in the 1990s (PFGE analysis is shown in Fig. 1, and microarray hybridization results are shown in Fig. 2 and 3; see Fig. S1 and S2 in the supplemental material).

Thus, MRSA strains belonging to CC8 seem to be well adapted, not only to the human host and environment, but also to the equine host, reflecting the extended host spectrum of the genotype.

In contrast to the wide range of investigated molecular markers, a remarkably low number of significant differences between strains of human and equine origins sharing the CC8 background could be determined in this study.

In regard to the virulence-associated factors investigated (Fig. 3), we found that all equine, but only one human, CC8 MRSA strains lacked the staphylococcal virulence factor staphylokinase (sak), which is a plasminogen-activator protein. Interestingly, the sensitivities of plasminogens to staphylokinase activation differ significantly between mammalian species and are highest in the plasminogens of humans and dogs (2). The staphylokinase-encoding gene sak is located on bacteriophages of serotype F (phl3 and ph42D) and serotype B (phC). Most of these not only harbor sak, but also sea and the chemotaxis-inhibitory protein (CHIP) genes, as well as genes coding for staphylococcal complement inhibitor (SKIN). As the activities of these accessory virulence factors are specific against human target molecules, a tight host-pathogen coevolution has been assumed (9).

The majority of the human CC8 isolates harbored sea, which was absent in equine isolates. Some equine isolates also lacked the SKIN gene (scn) (Fig. 3). Thus, we assume that harboring sea, scn, and sak might confer a selective advantage on MRSA only in the human host and that there is no need for S. aureus to maintain these genes in an equine environment. Absence of sea and sak has also been observed in strains belonging to various clonal groups that have been sampled in cattle (22). Furthermore, a recently published study by Sung and coworkers (32) investigating S. aureus host specificity found genes carried on mobile genetic elements, such as chp, scn, and sak, to be less common in strains of animal origin.

Minor differences between human and equine isolates were found in the SCCmec composition of CC8 MRSA. All equine ST254 isolates gave negative hybridization results for the site-specific recombinase genes, especially for ccrAB2, the recombinase system associated with a majority of human ST254 strains (SCCmec IV). However, some human strains of ST254 showed a similar recombinant, or mosaic, structure (Fig. 2). In addition, components of the mer operon were detected inconsistently. Whether equine and some of the human strains have lost ccrAB2 or harbored unknown allelic variants can only be answered by complete sequencing of these SCC elements (11). Some of the strains investigated also differed slightly in genetic composition, e.g., occurrence of the beta-lactamase operon, the tobramycin resistance-encoding gene (aadD), and the clumping factor A encoding-gene, clfA.

These minor differences in SCCmec composition and other genetic markers between some of the equine ST254 strains may reflect independent acquisition of unknown SCCmec variants from coagulase-negative staphylococci in equine ST254 strains, as has been assumed for human MRSA (7). Previously, Vengust and coworkers found a rate of 42% methicillin-resistant coagulase-negative staphylococci in nasal swabs of clinically healthy horses (35).

Alternatively, the good adaptation of ST254 isolates to both the human and the equine host may have increased the transferability of human MRSA isolates to horses. Human-to-horse transmissions may have happened several times in different geographic areas and countries. It has been reported recently that horses were much more likely to be infected with S. aureus isolates from human lineages than were cows (32).

Strains belonging to CC22, ST22-MRSA IV (PFGE type IMT-BE-2), and ST1117-MRSA IV (PFGE IMT-A) share a genetic background highly similar to that of the human epidemic strain Barnim (BE), or EMRSA-15, as it is referred to in the United Kingdom. It should be mentioned that (the novel) ST1117 showed the presence of an slv in the housekeeping gene pta, which therefore distinguishes this sequence type from ST22. ST22-MRSA IV is frequently observed in small animals in veterinary settings (31, 38), and it is also widely disseminated in medical hospitals in the United Kingdom and Central Europe (43). In contrast to the common occurrence of strains belonging to ST22 in strains of small-animal origin, this genotype seems to appear rarely in strains of equine origin.

One further strain has been assigned to ST398-MRSA V, a genetic background that has been reported for horses only recently (43). This genotype has been detected in strains of human origin, as well as in a variety of domestic animals, including pigs, cattle, and horses (3, 4, 14, 19, 21). Altogether, the differences detected between the human and equine MRSA strains investigated in this study were minimal. While some of the differences (slv of MLST genes and variations in SCCmec composition) are probably accidental, originating from random mutations or deletions, others (the prevalence of sak, scn, and sea) might be related to differences in the pathophysiologies of different host species. In conclusion, further studies must address potential host-specific factors in S. aureus. Therefore, data from fully sequenced MRSA and MSSA strains of equine origin, as well as functional studies of differences in pathogenetic pathways of S. aureus infections in different hosts, are needed. Generally, there is an unfortunate lack of knowledge concerning MSSA frequency, virulence factor carriage, and the abundances of clonal groups of S. aureus in horses. Therefore it is not yet possible to draw meaningful comparisons between the clonal structures of equine MRSA and MSSA populations.

As mentioned previously, accumulation of microbiological characteristics does not replace proper epidemiological investigation (40) for conclusions regarding the (species) origin of infection or interspecies transferability. Nonetheless, the extensive molecular pathogen profiling of MRSA strains of human and animal origins presented here may provide evidence for the possible ease of interspecies transferability of certain MRSA genotypes (here, CC8, CC22, and ST398) and may contribute to clarifying the question of why some genotypes seem to be easily transferable between different hosts. They are obviously adapted to more one than one mammalian species, and we propose to call these genotypes "extended-host-spectrum genotypes" (EHSG).

 
We thank W. Witte (Robert Koch Institut, Wernigerode, Germany) for providing all human EMRSA strains used in this study. We also thank Esther-Maria Antao for critical reading of the manuscript.

 
* Corresponding author. Mailing address: Institute of Microbiology and Epizootics (IMT), Free University Berlin, P.O. Box 040225, 10061 Berlin, Germany. Phone: 49 30 2093 6153. Fax: 49 30 2093 6067. E-mail: walther.birgit{at}vetmed.fu-berlin.de

Published ahead of print on 24 December 2008.

Supplemental material for this article may be found at http://jcm.asm.org/.

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Journal of Clinical Microbiology, March 2009, p. 704-710, Vol. 47, No. 3
0095-1137/09/$08.00+0     doi:10.1128/JCM.01626-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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