MRSA variant in companion animals.
Wieler, Lothar H.
Kopp, Peter A.
|Publication:||Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040|
|Issue:||Date: Dec, 2012 Source Volume: 18 Source Issue: 12|
Worldwide, methicillin-resistant Staphylococcus aureus (MRSA) is a
leading cause of infectious diseases in humans and animals (7). The
staphylococcal cassette chromosome mec (SCCmec) harbors the mecA gene,
which encodes an additional penicillin-binding protein 2a. In the
presence of P-lactam antimicrobial drugs, this transpeptidase
substitutes an essential cross-linking step in the process of cell-wall
building (2). Eleven distinct SCCmec elements have been described (3).
Recent reports of MRSA carrying a novel mecA homologue
([mec[A.sup.LGA251]) of a predicted amino acid identity of 62% with
other mecA allotypes raised awareness about these pathogens, which
possibly remain undetected by conventional PCR approaches (3-5). This
lack of detection might have led to underestimation of the novel MRSA
variant among clinical samples of human and animal origin.
High-level congruence between S. aureus of animal and human lineages has been demonstrated (6), and nearly every sequence type (ST) reported for MRSA associated with infections in companion animals was also commonly found in humans (7). Because previous reports indicated that MRSA harboring [mec.sub.ALGA251] originated from either human or ruminant hosts (3-5), we searched our database for companion animal isolates that displayed a MRSA phenotype but had failed to give a positive PCR result for mecA.
From November 2008 through December 2011, MRSA of companion animal origin was routinely isolated from specimens submitted for diagnostic purposes to Vet Med Labor GmbH in Ludwigsburg, Germany, or to the Institute of Microbiology and Epizootics, Freie Universitat Berlin, in Berlin, Germany. S. aureus was confirmed as described (7) and stored in glycerol stocks at -80[degrees]C.
PCR routinely used to confirm methicillin resistance and species identity had failed to produce a positive signal for mecA in 10 MRSA isolates from companion animals (2 isolates from dogs, 7 from cats, and 1 from a guinea pig) (8). We screened these 10 isolates for the mecA homologue by using the PCR method published by Cuny et al. (5) and sent the amplicons obtained to LGC Genomics GmbH (Berlin, Germany) for sequencing. Automated antimicrobial drug susceptibility testing was performed by using the bioMerieux VITEK 2 system (Nurtingen, Germany) according to the manufacturer's instructions. The following drugs were tested according to Clinical and Laboratory Standards Institute guidelines M31-A3: penicillin, ampicillin-sulbactam, oxacillin, gentamicin, kanamycin, enrofloxacin, marbofloxacin, erythromycin, clindamycin, tetracycline, nitrofurantoin, chloramphenicol, and trimethoprim-sulfamethoxazole, (9). All isolates were further characterized by spa typing, multilocus sequence typing, and microarray hybridization by using the Alere Identibac S. aureus Genotyping chip (Alere Technologies GmbH, Jena, Germany) as described (70-72).
The presence of the mecA homologue was verified for all 10 isolates. All PCR amplicons demonstrated 100% identity with the DNA sequence of [mec.sup.ALGA251] (National Center for Biotechnology Information no. FR821779.1). The strains originated from geographically diverse areas (5 federal states of Germany) and were isolated from different infection sites (Table). All strains were identified as MRSA by the VITEK 2 system (growth in the presence of 6 ug/ mL cefoxitin according to the VITEK 2 Advanced Expert System), although oxacillin MICs were rather low (0.5 ug/ mL) or moderately high ([approximately equals] 4 [micro]g/mL) (Table). Phenotypic resistance toward non-P-lactams was not detected.
As has been described for atypical MRSA, 4 strains belonged to ST130 and 1 strain belonged to ST1945 (differs from ST130 by 1 allele) (3-5). The remaining 5 isolates were assigned to ST599 (differs from ST121 by 2 alleles) (Table). ST599 has been reported for methicillinsusceptible isolates from humans in Europe, Asia, and Africa (www.mlst.net). The Figure shows a minimum spanning tree based on 4,197 entries of the S. aureus multilocus sequence type database (http://saureus.mlst. net/) as of January 19, 2012 (Figure, panel A) and a detailed view of the branches and STs harboring strains with the novel mecA homologue published (Figure, panel B) (3-5).
Microarray hybridization data revealed that the agr type I and capsule type 5 seem to be associated with ST599 and agr III and that clonal complex (CC) 130 isolates harbor the capsule type 8 encoding gene. CC130 and ST599 isolates were positive for the surface-associated proteins clfA, clfB, fnbA, and bbp. All ST599 strains produced a positive hybridization result for 1 of the gene variants encoding the toxin responsible for toxic shock syndrome (tst7 or tst-bov), and all but 1 of them harbored the enterotoxins C (sec) and L (sel), indicating the presence of an S. aureus pathogenicity island that encodes superantigens (73). Positive or ambiguous hybridization signals for ccrB7, ccrA3, and ccrB3 were obtained for 5 isolates, suggesting the presence of the SCCmecXI in those strains, according to the results of Shore et al. (4).
Our findings of CC130 and ST599 MRSA harboring mecALGA251 in several companion animal species suggest that in Germany, the presence of the mecA homologue in MRSA is not exclusively associated with CC130. This finding supports the hypothesis that some, if not all, MRSA strains that harbor the novel mecA variant can cause infections among a broad variety of hosts, as has been shown for MRSA of human, equine, canine, and other companion animal origins (7,7). All currently known mecALGA251 carrying MRSA were observed in a distinct section of the S. aureus population (Figure, panel B). Whether this phylogenetic group possesses the ability to integrate the novel mecA variant needs to be further investigated.
In the past, [mec.sub.ALGA251]-carrying MRSA could have been misidentified as methicillin sensitive by routine PCR. However, all isolates were correctly identified as MRSA by the VITEK 2 system, as reported (4).
Of the 10 isolates, 7 were found in specimens from cats. A recent study identified cats as a potential natural reservoir for S. aureus of CC133, a genetic lineage that has also been reported for S. aureus of ruminant origin (74). Moreover, we have identified a CC130 strain (MSSAST2024, t8403) from a wild rat (IMT21250; ID4035) (www.mlst.net). In addition, CC130 MRSA containing the mecA homologue has only recently been reported for isolates from humans in Germany (5).
Although many investigators focus on livestock-associated MRSA, and because particular companion animal lineages of MRSA seem to be lacking, transmission of MRSA between companion animals and human family members in close proximity might be underestimated, especially in cases of recurrent infection (75). The emergence of MRSA harboring the novel mecA homologue has consequences for the verification methods for MRSA used in veterinary medicine; implementation of new methods will be inevitable. Their supposed restriction to only a few genetic lineages and the potential risk for interspecies transmission of atypical MRSA between companion animals and their owners in household environments needs further elucidation.
We thank Christel Simon and Sylwia Marquardt for their excellent technical assistance and Matthias Stange and Mario Heising for help with rat sampling.
This work was conducted at the Freie Universitat Berlin, Institute of Microbiology and Epizootics and Vet Med Labor GmbH, Ludwigsburg, Germany. It was supported by grants of the German Federal Ministry of Education and Research, MedVetStaph grant no. 01KI1014F and FBI-Zoo grant no. 01KI1012A, and by the German Research Foundation, GRK1673. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Dr Walther is a veterinarian holding a postdoctoral position at the Institute of Microbiology and Epizootics in the Infectious Disease Diagnostics and Molecular Epidemiology Working Group. Her primary research interests focus on molecular epidemiology of MRSA and methicillin-resistant S. pseudintermedius in companion animals.
Author affiliations: Freie Universitat Berlin, Berlin, Germany (B. Walther, L. H. Wieler, S. Vincze, E.-M. Antao, B. Kohn, T. Semmler, A. Lubke-Becker); and Vet Med Labor GmbH, Ludwigsburg, Germany (A. Brandenburg, I. Stamm, P.A. Kopp)
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Address for correspondence: Birgit Walther, Institute of Microbiology and Epizootics, Freie Universitat Berlin, Philippstr. 13, 10115 Berlin, PO Box 040225, 10061 Berlin, Germany; email: firstname.lastname@example.org
Table. Characteristics of 10 methicillin-resistant Staphylococcus aureus isolates harboring mecALGA251 obtained from companion animals, Germany, 2008-2011 * Year IMT no. Original no. isolated 17403 VB 999987 2008 21135 VB 964992 2010 21231 VB 971931 2010 24068 VB 961584 2010 25044 VB 969929 2010 25147 VB 969572-2 2010 25470 VB 972406 2010 25715 VB 969935 2010 28299 VB 952042 2011 28429 IMT 2272/11 2011 IMT no. Host Site 17403 Cat Eye 21135 Cat Wound 21231 Cat Skin 24068 Cat Tachea 25044 Dog Abscess 25147 Cat Wound 25470 Dog Eye 25715 Guinea Fistula 28299 pig Cat Phlegmon 28429 Cat Abscess IMT no. Clinical signs 17403 Purulent infection 21135 Lymphadenitis 21231 Dermatitis 24068 Stridor 25044 Tumor, dolor 25147 Suture dehiscence 25470 Purulent infection 25715 Purulent infection 28299 Dermatitis 28429 Fever Free-ranging IMT no. animal 17403 Yes 21135 Yes 21231 Unknown 24068 Yes 25044 No 25147 Yes 25470 No 25715 No 28299 Yes 28429 Yes IMT no. Federal state 17403 Rhineland- Palatinate 21135 Bavaria 21231 Hessia 24068 Hessia 25044 Bavaria 25147 North Rhine- Westphalia 25470 Bavaria 25715 Hessia 28299 Bavaria 28429 Berlin OXA IMT no. MIC ([dagger]) 17403 [greater than or equal to] 4 21135 [greater than or equal to] 4 21231 1 24068 0.5 25044 2 25147 2 25470 2 25715 2 28299 1 28429 [greater than or equal to] 4 spa IMT no. type ([double dagger]) 17403 t10033 21135 t843 21231 t1773 24068 t10006 25044 t1694 25147 t278 25470 t1694 25715 t843 28299 t278 28429 t10009 IMT no. ST ([section]) 17403 1945 21135 130 21231 130 24068 599 25044 599 25147 599 25470 599 25715 130 28299 599 28429 130 * IMT, Institute of Microbiology and Epizootics, Freie Universitat Berlin, Berlin, Germany; OXA, oxacillin; ST, sequence type; VB, Vet Med Labor GmbH, Ludwigsburg, Germany. All isolates were positive for nuc and negative for mecA according to PCR to detect MRSA (8), and all were positive for mecALGA251 according to PCR to detect the novel mecA homologue (5). ([dagger]) Detected by the VITEK 2 system (bioMerieux, Nurtingen, Germany). ([double dagger]) Spa repeats and spa type determined according to Harmsen et al. (11) and the Ridom SpaServer (http://spaserver.ridom.de). ([section]) Determined according to Enright et al. (10).
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