We performed a hospital - acquired methicillin - resistant Staphylococcus aureus molecular study in Buenos Aires city. Four clones were found harboring the transposable elementsTn4001 and Tn5405 and the erythromycin resistance determinants ermA and mef(E). Of the isolates, 73% belonged to a clone found previously in the city of Córdoba, which showed an epidemic behavior initially attributed to a widely disseminated South American clone.Fil: Jeric, Paola Elba. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Matteo, Mario José. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Ramirez, Maria Soledad. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Couto, Elsa. Hospital Francés; ArgentinaFil: Tokumoto, Marta. Fundación Favaloro; ArgentinaFil: Centron, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

Centron, Daniela

Couto, Elsa

Jeric, Paola Elba

Matteo, Mario José

Ramirez, Maria Soledad

Tokumoto, Marta

English

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

MEDICINA - Volumen 66 - Nº 1, 200636BRIEF COMMUNICATION MEDICINA (Buenos Aires) 2006; 66: 36-39ISSN 0025-7680MOLECULAR CHARACTERIZATION OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUSISOLATES REVEALS THE SPREADING OF A NEW CLONE IN BUENOS AIRES CITYPAOLA E. JERIC1, MARIO MATTEO1, 2, MARIA SOLEDAD RAMIREZ1, ELSA COUTO2,MARTA TOKUMOTO3, DANIELA CENTRON11Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina,Universidad de Buenos Aires; 2Hospital Francés, 3Fundación Favaloro, Buenos AiresAbstract We performed a hospital-acquired methicillin-resistant Staphylococcus aureus molecular study inBuenos Aires city. Four clones were found harboring the transposable elements Tn4001 and Tn5405and the erythromycin resistance determinants ermA and mef(E). Of the isolates, 73% belonged to a clone foundpreviously in the city of Córdoba, which showed an epidemic behavior initially attributed to the widely disseminatedSouth American clone.Key words: methicillin resistance, erythromycin resistance, HA-MRSA, transposable elementsResumen La caracterización molecular de aislamientos de Staphylococcus aureus meticilino-resistenterevela la diseminación de un nuevo clon en la ciudad de Buenos Aires. Realizamos un estudiomolecular en aislamientos de Staphylococcus aureus adquiridos en el ambiente hospitalario de la ciudad deBuenos Aires. Se hallaron cuatro clones que albergaban los elementos transponibles Tn4001 y Tn5405 y losdeterminantes de resistencia a eritromicina, ermA y mef(E). El 73% de los aislamientos pertenecían a un clonhallado previamente en la ciudad de Córdoba con características epidemiológicas atribuidas inicialmente al clonsudamericano ampliamente diseminado en el mundo.Palabras clave: meticilino-resistente, resistencia a eritromicina, HA-MRSA, elementos transponiblesReceived: 11-VIII-2005 Accepted: 15-XI-2005Postal address: Dra Paola E. Jeric, Departamento de Microbiologia,Inmunologia y Parasitologia, Facultad de Medicina, Universidad deBuenos Aires. Paraguay 2155, 1121 Buenos Aires, Argentina.Fax: (54-11) 4964-2554 e-mail: pjeric@fibertel.com.arStaphylococcus aureus (SA) is a microorganism ofremarkable clinical importance due to the wide variety ofinfections observed in nosocomial as non-nosocomialenvironments. In Argentina, the percentage of the hospi-tal-acquired methicillin-resistant SA (HA-MRSA) strainsduring 2002 was 52%, according to a report from theAntibiotic Resistance Informatic System (SIR), a nationalhealth network composed by 22 hospitals. This report alsoshowed that SA was the most prevalent species acquiredin our hospitals (21%).Among all the HA-MRSA epidemic clones describedso far, the Iberian and the South American clones havespread extensively1, 2 and the New York/Japanese clonehas been disseminating in Brazil in the last years3. TheSouth American clone was prevalent among the HA-MRSA isolates in Argentina since the 62.2% of themshowed its characteristic PFGE (pulsed-field gel electro-phoresis) pattern according to a previous report dated in19984. This pattern was related to resistance to a widerange of antibiotics like β-lactams, tetracyclines,aminoglycosides, macrolides, quinolones, trimethoprim-sulfamethoxazole, and chloramphenicol2. Moreover, theemergence of a vancomycin and teicoplanin resistantsubpopulation among the South American clone-relatedMRSA strains, has been noticed in Brazil recently5.In 2002, researchers from Córdoba City (located at798 km from Buenos Aires city), found the South Ameri-can epidemic clone only in 34% of the isolates and iden-tified a novel prevalent clone also with epidemic featuresin  38% of the isolates6. This new clone differed from theMRSA South American clone in that was susceptible tominocycline and trimethoprim-sulfamethoxazole6.In regard of the mechanisms of resistance, a limitednumber of aminoglycoside, and macrolide resistance geneshave been described in Gram positive cocci: aac(6')-aph(2’), aph(3’)-IIIa, aadE7, 8, erm genes, msrA, mef(A),vgaA and vgaB9 frequently carried in transposons such asTn4001 or Tn5405 (aac(6')-aph(2’), aph(3’)-IIIa, aadE)7,10.The purpose of this study was to perform a molecularsurveillance of HA-MRSA clones from five hospitals ofBuenos Aires city with the aim to analyze the spreadingof prevalent multiple resistant genotypes. We also deter-METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS 37TABLE 1.– MRSA isolates collected for this study at H1 hospital in August 2000Isolate n0 Antibiotype1 PCR Antibiotic treatment PFGE6Resistance- patterntype21 a 1 1.Ciprofloxacin2.Vancomicin A12 b 1 Vancomicin C13 a 1 Vancomicin A34 b 2 Vancomicin plus gentamicin D153 a 1 1.Ciprofloxacin plus gentamicin2.Vancomicin A46 a 1 1.Cefalotin plus gentamicin2.Teicoplanin A17 a 1 Not determined. A58 b 1 Teicoplanin C29 b 1 Teicoplanin B10 a 1 1.Ceftazidime, amikacin and metronidazol2.Vancomicin A2113 b 2 Vancomicin plus rifampicin D21 a: Rifampicin, trimethoprim/sulfamethoxazole, tetracycline, minocycline, vancomycin and teicoplanin susceptible; oxacillin, gentamicin,erythromycin, ciprofloxacin, and clindamicin resistant; b: vancomycin and teicoplanin susceptible; oxacillin, gentamicin, erythromycin,ciprofloxacin, minocycline, rifampin, trimethoprim/sulfamethoxazole, clindamicin and tetracycline resistant.2 1: aac(6’)-aph(2’’); aph(3’)-IIIa; aadE; erm(A); sat4; mecA and mef(E); 2: idem 1 but absence of  mef(E);3 Patients dead by sepsis due to staphylococcal infection.4 Outpatient 7 was hospitalized before in H1a.5 1: corresponds to the first antimicrobial treatment choice, and 2 corresponds to the second antimicrobial treatment choice.6 PFGE: pulsed-field gel electrophoresis.TABLE 2.– Characteristics of MRSA isolates collectedin September 2003.Isolate n0 Hospital Antibiotype1 PFGE2pattern12 H1 c A113,  14, 16, 17 H1 a A115 H1 a A118-20 H1 a A321-25 H2 a A126-27 H3 a A128,29 H3 b B30 H4 a A231 H4 a A132 H4 b C233 H4 b B34 H5 a A335 H5 a A336-37 H5 a A31 a: Rifampicin, trimethoprim/sulfamethoxazole, tetracycline, minocycline,vancomycin and teicoplanin susceptible; oxacillin, gentamicin,erythromycin, ciprofloxacin, and clindamicin resistant; b: vancomycin andteicoplanin susceptible; oxacillin, gentamicin, erythromycin, minocycline,ciprofloxacin, rifampicin, trimethoprim/sulfametoxazole, clindamicin andtetracycline resistant; c: trimethoprim/sulfamethoxazole, minocycline,vancomycin and teicoplanin susceptible; oxacillin, gentamicin, rifampicin,erythromycin, ciprofloxacin, clindamicin and tetracycline resistant.2 PFGE: pulsed-field gel electrophoresismined the mechanisms of resistance involved in some ofthe MRSA isolated from hospital H1 in the first windowperiod (Table 1).We performed two one-month molecular surveillancesof HA-MRSA epidemiologically unrelated isolates duringAugust 2000 (n=11) from hospital H1 and September 2003(n=26) from five hospitals including H1 (n=9) (Tables 1and 2) in Buenos Aires city. All MRSA isolates (n=37) werecollected from inpatients with different types of infections(Tables 1 and 2). In the hospital H1, the samples wereisolated from two buildings that are separated 3 miles fromeach other (H1a and H1b) that share several nurses andmedical staff as well as patients. However, the patientsfrom our study have never been at the two hospital centersbefore this study. All strains collected were grown in brain-heart infusion agar with 5% sheep blood and incubated at37 °C during 24 hs in a 5% CO2 atmosphere.The susceptibility to a wide variety of antibiotics wasdetermined by the Kirby & Bauer method according tothe NCCLS guidelines11 using S. aureus strain 25923 ascontrol10. Genomic DNA was extracted by the guanidiniumthiocyanate method12. The presence of mecA, aac(6’)-aph(2’’), aph(3’)-IIIa, ant(4’)-Ia, aadA, aadE, mef(A),mef(E), msrA, erm(A), erm(B), erm(C), erm(M), erm(TR),Tn4001, Tn5405 and the insertion sequences IS256,IS1181and IS1182, was determined by the standard PCRtechnique. All the PCRs were carried out as previouslyMEDICINA - Volumen 66 - Nº 1, 200638described8, 13 using primers designed in our laboratory orby others7, 13 and automated nucleotide sequence analy-sis was performed.PFGE with enzyme SmaI was used for the clonal char-acterization of the 37 MRSA isolates using the CHEF DR-III system (BioRad, Hercules, Calif.). Percent similaritywas estimated by the simple matching coefficient, andthe matrix was clustered by the unweight pair groupmethod (UPMGA). In this study, the analysis was per-formed as previously described13; an 80% similarity levelwas considered, corresponding to differences in 7 bands.The 37 MRSA isolates showed 10 different patterns,which were grouped in 4 clusters (A, B, C, and D, Figures1A and B). The difference in the number of bands amongthe clusters varied between 8 and 12 bands and between2 and 5 within the same cluster. The prevalent cluster(A), was identified as the Córdoba city epidemic clone(73% of the isolates) that showed the antibiotype a withthe exception of one isolate that showed antibiotype c(isolate 12, table 2), while the other clones B, C and D(the first recognized as the South American clone)harbored a more resistant antibiotype (antibiotype b, Ta-ble 1). The antibiotype a showed susceptibility to rifampin,trimethoprim-sulfamethoxazole, tetracycline, minocycline,vancomycin and teicoplanin, the antibiotype b was onlysusceptible to vancomycin and teicoplanin and theantibiotype c was susceptible to rifampin, minocycline,trimethoprim-sulfamethoxazole, vancomycin and teico-planin. The molecular resistance profile analysis of theCórdoba city clone, showed the presence of the lociaac(6’)-aph(2’’), aph(3’)-IIIa, aadE and sat4 (resistanceto streptothricin). Two PCR products of 1.5 kb each (rightand left junctions) and one of 1.1 kb (IS256) confirmedthe presence of Tn4001 and the DNA sequence showedFig. 1A.– PFGE patterns of SmaI digested genomic DNA of the 11 MRSA strainsisolated from H1 in 2000. L: phage lambda concatemers used as molecular marker.The dendogram shows the four clones or clusters in the MRSA population. Thearrow indicates the cut off used to differ the clusters.Fig. 1B.– PFGE patterns of SmaI digested genomic DNA of the three clones andsubtypes. The dendogram shows the three clones or clusters of the MRSA strainsisolated from H1, H2, H3, H4 and H5 during the period 2003. The arrow indicatesthe cut off used to differ the clusters. See also Table 2.METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS 39100% homology with this element. The PCR to detectIS1181, IS1182, aadE and aph(3’)-IIIa sequences ren-dered amplification products of 1.2 kb (IS1181), 1.1 kb(IS1182), 3.6 kb (IS1182-aadE), 5.7 kb (IS1182-aph(3’)-IIIa) and 2.2 kb (aadE-aph(3’)-IIIa) that, by nucleotidesequence analysis, belonged to the complete structureof the Tn5405. Other resistance determinants detectedby PCR in these clones were erm(A)(645pb), mef(A)(316pb) and mef(E)(513 pb) (Table 1).The percentage of isolation of MRSA strains in ourhospitals remained the same (55%), since 1996 whenthe South American clone was predominant4. Our resultssuggest that since 2000, the South American clone is nothaving an epidemic behavior in Buenos Aires city, andthat the clone from Córdoba city is taking its niche. In Ar-gentina, the dissemination of resistance genes has beenincreasing in the last decade4, 14. Although several workshave focused on epidemiological surveys of MRSA2, 15, toour knowledge, this is the first study where the antimicro-bial determinants as well as the clonal spreading have beenevaluated at the same time in a molecular surveillance.The emergence of a novel epidemic clone of MRSA inour hospitals denotes that although the spreading of aMRSA clone may be limited, it does not imply the eradi-cation of this species in the hospital environment. In re-gard of MRSA, the molecular identification of virulencefactors could be helpful to identify the reason of the pre-dominance of this clone over another because the spread-ing of an epidemic clone can be explained not only byselective antibiotic pressure but also by its ability to sur-vive within the nosocomial environment and colonize pa-tients and hospital personnel.It can be concluded that it is necessary to perform acontinuously molecular and epidemiological surveillancesystem to detect rapid changes that modify the genotypeand the resistance profile of HA-MRSA clones with epi-demic behavior. Such studies would help to evaluatecontrol infection policies with the aim to reduce the inci-dence of infections caused by MRSA isolates.Acknowledgements: This study was supported by thegrant from the “Agencia Nacional para la Promoción de la Cien-cia y la Tecnología” (ANPCyT)-(grant PICT 13431), to D.C.REFERENCES1. Aubry-Damon H, Soussy CJ. Methicillin-resistant Staphy-lococcus aureus: factors responsible for its incidence.Rev  Med  Int 2000; 21: 344-52.2. Aires de Sousa M, Miragaia M, Santos Sanchez I et al.Three- year assessment of methicillin-resistant Staphylo-coccus aureus clones in Latin America from 1996 to1998. J Clin  Microbiol 2001; 39: 2197-205.3. Melo MC, Silva-Carvalho MC, Ferreira RL et al. Detectionand molecular characterization of a gentamicin-suscep-tible, methicillin-resistant Staphylococcus aureus (MRSA)clone in Rio de Janeiro that resembles the New York/Japanese clone. J Hosp Infect 2004; 58: 276-85.4. Corso A, Santos Sanchez I, Aires de Sousa M, Rossi A,de Lencastre H. Spread of a methicillin-resistant andmultiresistant epidemic clone of Staphylococcus aureusin Argentina. Microb Drug Resist 1998; 4: 277-88.5. Dos Santos Soares MJ, da Silva-Carvalho MC, Ferreira-Carvalho BT, Figueiredo AM. Spread of methicillin-resistant Staphylococcus aureus belonging to theBrazilian epidemic clone in a general hospital andemergence of heterogenous resistance to glycopeptideantibiotics among these isolates. J Hosp Infect 2000; 44:301-8.6. Sola C, Gribaudo G, Vindel A, Patrito L, Bocco JL andMRSA Collaborative Study Group. Identification of anovel methicillin - resistant Staphylococcus aureusepidemic clone in Córdoba, Argentina, involved in noso-comial infections. J Clin  Microbiol 2002; 40: 1427-35.7. Derbise A, Aubert S, el Sohl N. Mapping the regionscarrying the three contiguous antibiotic resistance genesaadE, sat4, and aphA – 3 in the genomes of staphylo-cocci. Antimicrob Agents Chemother 1997; 41: 1024-32.8. Sutcliffe J, Grebe T, Tait-Kamradt A, Wondrack L.Detection of erythromycin–resistant determinants byPCR. Antimicrob Agents Chemother 1996; 40: 2562-66.9. Roberts MC, Sutcliffe J, Courvalin P, Jensen LB, RoodJ, Seppala H. Nomenclature for macrolide and macrolide-lincosamide-streptogramin B resistance determinants.Antimicrob Agents Chemother 1999; 43: 2823-30.10. Gillespie MT, Lyon BR, Messerotti M, Skurray RA.Chromosome and plasmid-mediated gentamicin resis-tance in Staphylococcus aureus encoded by Tn4001. JMed. Microb 1987; 24: 139-44.11. National Committee for Clinical Laboratory Standards.Performance Standards for Antimicrobial Disk Suscep-tibility Test; Approved standard. 2000; 7th ed. M2-A7.NCCLS, Wayne, PA. USA.12. Pitcher DG, Saunders NA, Owen RJ. Rapid extraction ofbacterial genomic DNA with guanidinium thiocyanate.Lett Applied Microbiol 1989; 8: 151-56.13. Jeric PE, Lopardo H, Vidal P et al. Multicenter study onspreading of the tet(M) gene in tetracycline-resistantStreptococcus group G and C isolates in Argentina.Antimicrob Agents Chemother 2002; 46: 239-41.14. Rossi A, Tokumoto M, Galas M, Soloaga R, Corso A.Monitoring antibiotic resistance in Argentina. TheWHONET program, 1995-1996. Rev Panam  Salud Pub1999; 6: 234-41.15. Eveillard M, Schmidt JL, Biendo M et al. Evaluation ofthe efficacy of a multiresistant bacteria control programin a teaching hospital, studying the evolution of methicillin- resistant Staphylococcus aureus incidence. Pathol  Biol2002; 50: 538-43.

La caracterización molecular de aislamientos de Staphylococcus aureus meticilino-resistente revela la diseminación de un nuevo clon en la ciudad de Buenos Aires

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La caracterización molecular de aislamientos de Staphylococcus aureus meticilino-resistente revela la diseminación de un nuevo clon en la ciudad de Buenos Aires

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