MRSA variant in companion animals.
Subject: Staphylococcus aureus
Authors: Walther, Birgit
Wieler, Lothar H.
Vincze, Szilvia
Antao, Esther-Maria
Brandenburg, Anja
Stamm, Ivonne
Kopp, Peter A.
Kohn, Barbara
Semmler, Torsten
Lubke-Becker, Antina
Pub Date: 12/01/2012
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
Accession Number: 313345652
Full Text: 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.

The Study

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).

[FIGURE OMITTED]

Conclusions

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.

Acknowledgments

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)

DOI: 10.3201/eid1812.120238

References

(1.) Wieler LH, Ewers C, Guenther S, Walther B, Lubke-Becker A. Methicillin-resistant staphylococci (MRS) and extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae in companion animals: nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples. Int J Med Microbiol. 2011;301:635-41. http://dx.doi. org/10.1016/j.ijmm.2011.09.009

(2.) Pinho MG, de Lencastre H, Tomasz A. An acquired and a native penicillin-binding protein cooperate in building the cell wall of drug resistant staphylococci. Proc Natl Acad Sci U S A. 2001;98:1088691. http://dx.doi.org/10.1073/pnas.191260798

(3.) Garcia-Alvarez L, Holden MT, Lindsay H, Webb CR, Brown DF, Curran MD, et al. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study. Lancet Infect Dis. 2011;11:595603. http://dx.doi.org/10.1016/S1473-3099(11)70126-8

(4.) Shore AC, Deasy EC, Slickers P, Brennan G, O'Connell B, Monecke S, et al. Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA, mecI, mecR1, blaZ, and ccr genes in human clinical isolates of clonal complex 130 methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2011;55:3765-73. http://dx.doi.org/10.1128/AAC.00187-11

(5.) Cuny C, Layer F, Strommenger B, Witte W. Rare occurrence of methicillin-resistant Staphylococcus aureus CC130 with a novel mecA homologue in humans in Germany. PLoS ONE. 2011;6:e24360. http://dx.doi.org/10.1371/journal.pone.0024360

(6.) Sung JM, Lloyd DH, Lindsay JA. Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray. Microbiology. 2008;154:1949-59. http:// dx.doi.org/10.1099/mic.0.2007/015289-0

(7.) Walther B, Monecke S, Ruscher C, Friedrich AW, Ehricht R, Slickers P, et al. Comparative molecular analysis substantiates a zoonotic potential of equine methicillin- resistant Staphylococcus aureus (MRSA). J Clin Microbiol. 2009;47:704-10. http://dx.doi. org/10.1128/JCM.01626-08

(8.) Merlino J, Watson J, Rose B, Beard-Pegler M, Gottlieb T, Bradbury R, et al. Detection and expression of methicillin/oxacillin resistance in multidrug-resistant and non-multidrug-resistant Staphylococcus aureus in central Sydney, Australia. J Antimicrob Chemother. 2002;49:793-801. http://dx.doi.org/10.1093/jac/dkf021

(9.) Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals. Approved standard. Wayne (PA); 2008.

(10.) Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol. 2000;38:1008-15.

(11.) Harmsen D, Claus H, Witte W, Rothganger J, Turnwald D, Vogel U. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol. 2003;41:5442-8. http://dx.doi.org/10.1128/JCM.41.12.5442-5448.2003

(12.) Monecke S, Kuhnert P, Hotzel H, Slickers P, Ehricht R. Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle. Vet Microbiol. 2007;125:128-40. http://dx.doi.org/10.1016/j. vetmic.2007.05.016

(13.) Fitzgerald JR, Monday SR, Foster TJ, Bohach GA, Hartigan PJ, Meaney WJ, et al. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J Bacteriol. 2001;183:63-70. http://dx.doi. org/10.1128/JB.183.1.63-70.2001

(14.) Sasaki T, Tsubakishita S, Tanaka Y, Ohtsuka M, Hongo I, Fukata T, et al. Population genetic structures of Staphylococcus aureus isolates from cats and dogs in Japan. J Clin Microbiol. 2012;50:2152-5.

(15.) Rutland BE, Weese JS, Bolin C, Au J, Malani AN. Human-todog transmission of methicillin-resistant Staphylococcus aureus. Emerg Infect Dis. 2009;15:1328-30. http://dx.doi.org/10.3201/ eid1508.081635

Address for correspondence: Birgit Walther, Institute of Microbiology and Epizootics, Freie Universitat Berlin, Philippstr. 13, 10115 Berlin, PO Box 040225, 10061 Berlin, Germany; email: walther.birgit@vetmed.fu-berlin.de
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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