Isolates found in persons and animals in Germany and Austria show a genetic relationship

Armand-Lefevre

Bens

Birgit Strommenger

Christian Stanek

Christiane Cuny

Cuny

Enright

Grundmann

Johnson

Loeffler

Strommenger

Vandenesch

Weese

Wolfgang Witte

Zhang

Emerging Infectious Diseases

English

PubMed

Crossref

Methicillin-resistant
                    Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus of clonal lineage ST398 that exhibits related spa types and contains SCCmec elements of types IVa or V has been isolated from colonized and infected humans and companion animals (e.g., dog, pig, horse) in Germany and Austria. Of particular concern is the association of these cases with cases of nosocomial ventilator-associated pneumonia

Witte, Wolfgang

Strommenger, Birgit

Stanek, Christian

Cuny, Christiane

Publication server of the Robert Koch Institute

Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe.

Publikationsserver des Robert Koch-Instituts

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007 255Methicillin-resistant Staphylococcus aureus of clonallineage ST398 that exhibits related spa types and containsSCCmec elements of types IVa or V has been isolated fromcolonized and infected humans and companion animals(e.g., dog, pig, horse) in Germany and Austria. Of particu-lar concern is the association of these cases with cases ofnosocomial ventilator-associated pneumonia.Methicillin-resistant Staphylococcus aureus (MRSA)has become an infection control problem in hospitalsworldwide, mainly associated with intrahospital and inter-hospital dissemination of particular epidemic clonal line-ages of the S. aureus population (hMRSA; [1]). MRSAprimarily associated with healthcare facilities may also bedisseminated to the community through colonized medicalstaff or discharged patients. The emergence and spread ofMRSA in the community during the past 5 years, inde-pendent of the healthcare setting and in the absence of typ-ical risk factors for nosocomial MRSA infections, arematters of further concern. These community-acquiredMRSA infections are less broadly resistant to antimicro-bial agents than are healthcare-associated MRSA and oftencontain the determinants lukS-lukF, which code forPanton-Valentine leukocidin (2).Even though MRSA has been known as a nosocomialpathogen for >30 years, its development in companion ani-mals and livestock has been rare (3). Recent reports, how-ever, have documented MRSA infections in animals suchas horses from Canada (4) and Europe (3) and pets (5,6).Of particular interest is whether MRSA may be transmittedbetween animals and humans. MRSA of clonal lineagesequence type (ST) 22 is widely disseminated in humanhospitals in the United Kingdom and Central Europe. Thedemonstration of this lineage among MRSA isolates fromstaff and from pets in a small animal referral hospital inUnited Kingdom suggests transmission between humansand animals (5). Nasal colonization of veterinary staff withMRSA (ST8) from infections in horses in a veterinary hos-pital was frequently observed in Canada (4), and it wasalso recorded in an Austrian university veterinary hospitalwhere horses were affected by MRSA of clonal lineageST254 (3).We report on molecular characterization of MRSA,from sporadic infections in humans and in various animalspecies, that belong to clonal lineage ST398 according tomultilocus sequence typing (MLST). These isolates werefurther characterized by spa-sequence typing (repeat poly-morphism of the X-region of the spa gene) and by PCR forgrouping of staphylococcal cassette chromosome mec(SCCmec) elements, which contain the mecA gene and ofwhich at least 5 basic types have been described.MethodsMRSA isolates from infections in humans and in ani-mals were sent to the National Reference Center forStaphylococci at the Robert Koch Institute, WernigerodeBranch, in Germany, for typing by means of SmaI-macrorestriction pattern as well as spa typing. Selectedisolates also underwent MLST. Four human isolates weregrown from nasal swabs taken from the staff of a veteri-nary practice at Veterinary Analytical Center, Geesthacht,Germany. All isolates were primarily grown on sheepblood agar and confirmed by standard procedures asMethicillin-resistantStaphylococcus aureus ST398 in Humans and Animals,Central EuropeWolfgang Witte,* Birgit Strommenger,* Christian Stanek,† and Christiane Cuny†*Robert Koch Institute, Wernigerode, Germany; and †VeterinaryUniversity, Vienna, AustriaS. aureus. Eleven additional MRSA specimens of lineageST398 (1 isolate per patient affected) were found among4,370 MRSA isolates from patients with recognized infec-tions. These isolates were identified by indigestibility oftheir whole cellular DNA when subjected to SmaI-macrorestriction analysis. Animal isolates were collectedfrom 1 dog and 1 foal at the Veterinary Analytical Center,Geesthacht, Germany; from 1 pig at the diagnostic labora-tory of the Institute for Microbiology and InfectiousDiseases, School of Veterinary Medicine, Hannover,Germany; and from 2 horses at the Department ofOrthopaedics, Veterinary University, Vienna, Austria. Procedures and primers for DNA extraction and PCRdetection of resistance genes were as described previously(6). Macrorestriction patterns were determined by usinglysis of cells, deproteinization and digestion of DNA (hereby SmaI and ApaI), and pulsed-field gel electrophoresis (7).The polymorphic X-region of the protein A gene (spa)was amplified and sequenced according to the RidomStaphType standard protocol (www.ridom.org). The result-ing spa-types were assigned by using the RidomStaphType software package (Ridom GmbH, Würzburg,Germany). The BURP algorithm, implemented in the mostrecent Ridom StaphType software version, was used forcluster analysis of spa types (7).Primers used for MLST correspond to the protocol asdescribed previously (8), with the exception of the forwardprimer for tpi; we used the sequence tpif 5′-GCATTAGCAGATTTAGGCGT-3′. Antimicrobial susceptibilitytesting was performed by broth microdilution, performedaccording to DIN 58940, Deutsches Institut für Normung(9). SCCmec elements of types I to IV were characterizedby using a PCR approach, including a combination of dif-ferent PCRs (6). To demonstrate SCCmec-elements of typeV, we used primers type VF/type VR, as described byZhang et al. (10), as well primer pair ccrC9f 5′-CACT-TAATCCATGTACACAG-3′ and ccrC-R (10).The following set of primers was used for PCR forvirulence-associated genes: tst, sea, seb, sec, sed, see, asdescribed by Johnson et al. (11); for lukS-lukF, forward 5′-ATCATTAGGTAAAATGTCTGGACATGATCCA-3′,reverse 5′-GCATCAAGTGTATTGGATAGCAAAAGC-3′; for cna, forward 5′-CGGTTCCCCCATAAAAGTGAAG-3′, reverse 5′-CCCATAGCCTTGTGGATTTG-3′.Annealing temperature was 55° C; cyclic scheme and fur-ther conditions were as reported previously (6).Specimen collection, characterization of the isolates,data processing, and exchange of data were performedwithin the framework of German public health activitiesfor infection control and prevention of MRSA dissemina-tion. Ethical approval was obtained within this frameworkas well.ResultsCharacteristics of the 20 MRSA isolates investigatedare shown in the Table. All isolates share MLST ST398with the allelic profile 3-35-19-2-20-26-39. Three differentspa-types are obviously related (Figure). Types t11 and t34may have been derived from each other by either deletionor duplication of 2 repeats; t1197 and t11 differ by a singlenucleotide polymorphism. BURP analysis of these spa-types groups them as a separate cluster unrelated to otherBURP clusters (7). A peculiarity of S. aureus of clonal lin-eage ST398 is the indigestibility of whole cellular DNA byrestriction enzyme SmaI. Therefore, SmaI macrorestrictionpatterns generate only 1 large fragment because of protec-tion by a novel DNA methylation enzyme (12). We alsofound poor digestion by the isoschizomeric enzyme XmaI.However, digestion by enzyme ApaI generated similarfragment patterns that differed at most by 3 fragmentsindependent of spa types.The 2 horse isolates from the Vienna veterinary uni-versity contained SCCmec elements of group IVa. For allother isolates investigated, PCR indicated SCCmecV.These findings suggest that MRSA of ST398 from horsesare unrelated to the other isolates and probably haveevolved independently by acquisition of a differentSCCmec element.In addition to mecA, all investigated isolates containedtetM; isolates from animals and humans from LowerSaxony also contained ermA. The nosocomial human andhorse isolates contained ermC; in the horse isolates, aph2″-aac6′–mediating aminoglycoside resistance was demon-strated. PCR was negative for virulence-associated genesand for lukS-lukF (coding for Panton-Valentine leuko-cidin), tst, sea, seb, sec, and sed, as well as for cna (colla-gen-binding protein).DiscussionIsolates of clonal lineage ST398 seem not to be fre-quently represented among the S. aureus population. Theywere not recorded by Grundmann et al. (13) among a pop-ulation sample of nasal colonizers in the Nottingham areain the United Kingdom and were not found among 108 iso-lates from carriers in a rural territory in northern Germany(S. Holtfreter et al., unpub. data). Only 2 notations ofST398 are found in the S. aureus MLST database(www.mlst.net), 1 from the Netherlands and 1 from theCape Verde Islands.Among 11,250 isolates of various origin (colonizationand infections in hospitals as well in the community inhumans from all Germany) typed from 1992 through 2003,no isolates refractory to SmaI macrorestrition analysiswere seen. Therefore, a rather recent emergence of MRSAST398 among humans seems likely. However, MRSA oflineage ST398 had been reported from infections in pigsRESEARCH256 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007and from nasal colonization in pig farmers in France (14).A more recent report from the Netherlands describesMRSA of ST398 (spa t108, which is in the same BURPcluster as t11 and t34) in pigs and in humans who had con-tact with pigs (15). A comparison of the allelic profile ofST398 by means of the MLST database does not indicateany relationship to profiles of prevalent clonal complexesof methicillin-susceptible S. aureus (13), of epidemichealthcare-associated MRSA, or of lukS-lukF–containingcommunity-associated MRSA from Europe.ConclusionsMRSA exhibiting ST398 may colonize and causeinfections in humans and in certain animal species such asdogs, horses, and pigs. The isolation of MRSA ST398showing the same characteristics from a wound infectionin a dog and from nasal colonization of the staff of a vet-erinary practice where this dog had been treated suggeststhat interspecies transmission may occur. The differencesin spa types between the isolates containing the same PCRresults for SCCmec can be explained by a single geneticevent. Because isolates taken at the same time from nasalcolonization in veterinary staff of the same practice exhib-it either spa-type t011 or t034, this difference does not jus-tify discrimination between the two types. Of particularconcern was the subsequent detection of MRSA ST398 notonly in outpatients but also in inpatients with ventilator-associated pneumonia in the same hospital unit at about thesame time (Table).Future recording of MRSA ST398 from infected andcolonized humans (especially when detected by screeningat admission to hospitals) will require a thorough analysiswith respect to association with animals and routes oftransmission. Tracing MRSA carriers among contactsshould also include pet animals, horses, and other live-stock. Because of the time and labor needed to completeMLST, spa-typing combined with BURP analysis of typesis an efficient tool for recognizing this clonal lineage.Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007 257Figure. Repeats of the X-region in methicillin-resistantStaphylococcus aureus of clonal lineage ST398.MRSA ST398, Central EuropeRESEARCH258 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 2, February 2007Furthermore, detection of MRSA by appropriate methodsshould be implemented into antimicrobial resistance sur-veillance programs in veterinary medicine.Prof Witte is head of the laboratory for nosocomial infec-tions at the Robert Koch Institute, Wernigerode Branch. He alsois a professor on the medical faculty of Magdeburg University.References1. Enright MC, Robinson DA, Randle G, Feil EJ, Grundmann H,Spratt BG. The evolutionary history of methicillin-resistantStaphylococcus aureus (MRSA). Proc Natl Acad Sci U S A.2002;99:7687–92.2. Vandenesch F, Naimi T, Enright MC, Lina G, Nimmo GR,Heffernan H, et al. Community acquired methicillin-resistantStaphylococcus aureus carrying Panton-Valentin leukocidin genes:worldwide emergence. Emerg Infect Dis. 2003;9:978–84.3. Cuny C, Kuemmerle J, Stanek C, Willey B, Strommenger B, WitteW. Emergence of MRSA infections in horses in a veterinary hospi-tal: strain characterization and comparison with MRSA fromhumans. Euro Surveill. 2006;11:44–7.4. Weese JS, Archambault M, Willey BM, Hearn P, Kreiswirth BN,Said-Salim B, et al. Methicillin-resistant Staphylococcus aureus inhorses and horse personnel, 2000–2002. Emerg Infect Dis.2005;11:430–5.5. Loeffler A, Boag AK, Sung J, Lindsay JA, Guardabassi L,Dalsgaard A, et al. Prevalence of methicillin-resistantStaphylococcus aureus among staff and pets in a small animal refer-ral hospital in the UK. J Antimicrob Chemother. 2005;56:692–7.6. Strommenger B, Kehrenberg C, Kettlitz C, Cuny C, Verspohl J,Witte W, et al. Molecular characterization of methicillin-resistantStaphylococcus aureus strains from pet animals and their relation-ship to human isolates. J Antimicrob Chemother. 2006;57:461–5.7. Strommenger B, Kettlitz C, Weniger T, Harmsen D, Friedrich AW,Witte W. Assignment of Staphylococcus aureus isolates to groupsby spa-typing, SmaI-macrorestriction analysis, and multilocussequence typing. J Clin Microbiol. 2006;44:2533–40.8. Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG.Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcusaureus. J Clin Microbiol. 2000;38:1008–15.9. German Institute for Standardization. Methods for susceptibilitytesting of bacterial pathogens (besides mycobacteria) againstchemotherapeutics. DIN 58940, Part 8. Microdilution. In: Medicalmicrobiology and immunology: diagnostic procedures. Vienna:Barth Publishing; 2000. p. 215–27. 10. Zhang K, McClure JA, Elsayed S, Louie T, Conley JM. Novel mul-tiplex PCR assay for characterization and concomitant subtyping ofstaphylococcal cassette chromosome mec types I to V in methi-cillin-resistant Staphylococcus aureus. J Clin Microbiol.2005;43:5026–33.11. Johnson WM, Tyler SD, Ewan EP, Aston FE, Pollard DR, KozeeKR. Detection of genes for enterotoxins, exfoliative toxins, andtoxic shock syndrome toxin 1 in Staphylococcus aureus by the poly-merase chain reaction. J Clin Microbiol. 1991;29:426–30.12. Bens CC, Voss A, Klaassen CH. Presence of a novel DNA methyla-tion enzyme in methicillin-resistant Staphylococcus aureus associ-ated with pig farming leads to uninterpretable results in standardpulsed-field gel electrophoresis analysis. J Clin Microbiol.2006;44:1875–6.13. Grundmann H, Hori S, Enright MC, Webster C, Tami A, Feil EJ, etal. Determining the genetic structure of the natural population ofStaphylococcus aureus: a comparison of multilocus sequence typ-ing with pulsed-field gel electrophoresis, randomly amplified poly-morphic DNA analysis, and phage typing. J Clin Microbiol.2002;40:4544–6.14. Armand-Lefevre L. Ruimy R, Andremont A. Clonal comparison ofStaphylococcus aureus isolates from healthy pig farmers, humancontrols, and pigs. Emerg Infect Dis. 2005;11:711–4.15. van Diijke B, Kopgen H, Wannet W, Huisdens X, Neeling H, VossA. Methicillin-resistant Staphylococcus aureus and pig farming[abstract]. 16th European Congress of Clinical Microbiology andInfectious Diseases. 2006  April 1–4; Nice, France [cited 2007 Jan10]. Available from http://www.blackwellpublishing.com/eccmid16/abstract.asp?id=49270Address for correspondence: Wolfgang Witte, Robert Koch Institute,Wernigerode Br, Burgstraße 37, 38855 Wernigerode, Germany; email:wittew@rki.de

Directory of Open Access Journals

Methicillin-resistant Staphylococcus aureus ST398 in Humans and Animals, Central Europe

Assignment of Staphylococcus aureus isolates to groups by spa-typing, SmaI-macrorestriction analysis, and multilocus sequence typing.

Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs. Emerg Infect Dis.

Community acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentin leukocidin genes: worldwide emergence. Emerg Infect Dis.

Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.

Determining the genetic structure of the natural population of Staphylococcus aureus: a comparison of multilocus sequence typing with pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing.

Emergence of MRSA infections in horses in a veterinary hospital: strain characterization and comparison with MRSA from humans. Euro Surveill.

for Standardization. Methods for susceptibility testing of bacterial pathogens (besides mycobacteria) against chemotherapeutics. DIN 58940, Part 8. Microdilution. In: Medical microbiology and immunology: diagnostic procedures.

Methicillin-resistant Staphylococcus aureus and pig farming [abstract]. 16th European Congress of Clinical Microbiology and Infectious Diseases.

Methicillin-resistant Staphylococcus aureus in horses and horse personnel, 2000–2002. Emerg Infect Dis.

Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and their relationship to human isolates.

Multilocus sequence typing for characterization of methicillinresistant and methicillin-susceptible clones of Staphylococcus aureus.

Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus.

Presence of a novel DNAmethylation enzyme in methicillin-resistant Staphylococcus aureus associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis.

Prevalence of methicillin-resistant Staphylococcus aureus among staff and pets in a small animal referral hospital in the UK.

The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA).

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2725865

Methicillin-resistant Staphylococcus aureus ST398 in Humans and Animals, Central Europe

Abstract

Similar works

Full text

Available Versions

Crossref

Publication server of the Robert Koch Institute

Publikationsserver des Robert Koch-Instituts

Directory of Open Access Journals