JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2006, p. 266 270 Vol. 44, No. 1 0095-1137/06/$08.00 0 doi:10.1128/jcm.44.1.266 270.2006 Copyright 2006, American Society for Microbiology. All Rights Reserved. Prevalence and Evolution of Methicillin-Resistant Staphylococcus aureus in Spanish Hospitals between 1996 and 2002 A. Vindel, 1 * P. Trincado, 1 E. Gómez, 1 R. Cabrera, 1 T. Boquete, 1 C. Solá, 2 S. Valdezate, 1 and J. A. Saez-Nieto 1 Nosocomial Infectious Diseases Laboratory, Servicio de Bacteriología, Instituto de Salud Carlos III, Centro Nacional de Microbiología, 28220 Majadahonda, Madrid, Spain, 1 and Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clinica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, Pabellon Argentina, 5000 Córdoba, Argentina 2 Received 26 August 2005/Returned for modification 27 September 2005/Accepted 7 October 2005 Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV. * Corresponding author. Mailing address: Nosocomial Infectious Diseases Laboratory, Instituto de Salud Carlos III, Centro Nacional de Microbiología, 28220 Majadahonda, Madrid, Spain. Phone: 34-91- 8223666. Fax: 34-91-5097966. E-mail: avindel@isciii.es. Staphylococcus aureus is a major cause of nosocomial infections, and methicillin-resistant S. aureus (MRSA) have spread around the world (30, 37). There are considerable variations in the prevalence of MRSA in different geographic areas. In Spain, the prevalence of MRSA has increased from 1.5% in 1986 to 31.2% in 2002 (5, 8). In the 1980s, the first Spanish epidemic MRSA strain, called E1 in our pulsed-field gel electrophoresis (PFGE) scheme and known as the Iberian clone, spread throughout the country (1, 13). This clone presented two subtypes, E1a and E1b, and belonged to phage groups I and III at 100 RTD (routine test dilution) and phage group III at the RTD, respectively (3). The two subtypes presented only one-band difference in the restriction patterns by pulsed-field gel electrophoresis (PFGE) (41). This multiresistant clone has been isolated in other countries and accounted for a large proportion of MRSA isolates until 1995 (10, 11, 22, 23, 25). To determine which clones are circulating in Spain and whether the strains had spread between hospitals, in this study we examined 2,144 MRSA isolates selected from all known MRSA strains and two MRSA strains representative of Iberian clone isolates during the beginning of the epidemic period (1989 to 1990). We studied the MRSA isolates by PFGE as previously described (25). The presence of the mec gene was confirmed by detection of the mec gene by multiplex PCR (18). The selected strains were from sporadic cases, and the strains were isolated from patients in 110 Spanish hospitals representing all Spanish Autonomous Communities. As initial markers, the phenotypic methods, phage typing and antibiotic profiling, were used to improve the selection of strains for further molecular epidemiology studies. For this reason, the screening was based on differences in: (i) the phage types obtained with the 23 phages of the Basic International Set at 100 RTD and 1,000 RTD and with the Specific Set of MRSA (3, 34, 40); (ii) the patterns of susceptibility to oxacillin, gentamicin, ciprofloxacin, rifampin, teicoplanin, and vancomycin, determined by the agar dilution method and interpreted according to CLSI (formerly NCCLS) guidelines (26); and (iii) the geographic location. The most frequent phage types isolated from MRSA isolates were the nontypeable strains (60%), followed by groups III (20%) and I and III (8%). When the MRSA strains were retested at 1,000 RTD, the nontypeable percentage was reduced to 40%. The antimicrobial resistance profiles showed a strong decrease in gentamicin-resistant (Gen r ) strains from 60.8% in 1996 to 11% in 2001 and a subsequent increase in 2002 (24.2%). Gentamicin-sensitive (Gen s ) strains emerged in Belgium (10) and France (14, 17, 19, 20). A decrease in rifampinresistant strains was detected; 34.8% of the strains were rifampin-resistant strains in 1996, but the value fell to 5.2% in 2002. However, the high rates of ciprofloxacin-resistant strains at the beginning of the study (84.2%) continued to increase to 92%, while no vancomycin- and teicoplanin-resistant strains were detected. We identified the 17 most common PFGE types (Fig. 1) (including several subtypes); each of the most common PFGE types was isolated with the same pattern 10 times in a year. We designated each PFGE type with the letter E, followed by a number that correlated with the date of isolation, and the subtypes were designated by a letter (the letter of the main pulsotype to which it was closest). A dendrogram (Fig. 2) of all predominant genomic groups was constructed by using the Dice coefficient, and gel findings were interpreted on the basis of standard criteria (39). Four branches were identified. The E1 clone (Iberian clone) and three subtypes of E1 and the E4, E5, E6, and E9 clones grouped together into one branch. Profiles E15, E16, and E17 grouped in another branch. There is a third branch, which contains some of the predominant clones isolated recently (E3, E7, E8, E10, and E11). Finally, the rest of the profiles with low similarity values (E2, E12, E13, and E14) grouped in the fourth branch. Before 1996, the Iberian clone (E1) was the predominant strain, but it decreased (40.1% to 11.4%) from 1996 to 1998 266
VOL. 44, 2006 NOTES 267 FIG. 1. PFGE patterns of representative predominant MRSA clones identified from 2,144 nosocomial MRSA strains during a 7-year period (1996 to 2002). The clones, including Iberian clone (E1) subtypes (E1a to E1d), E5 (Brazilian clone), E7 subtypes (E7a to E7c), E8 subtypes (E8a and E8 b) and molecular markers are shown above the lanes. Downloaded from http://jcm.asm.org/ on January 6, 2019 by guest FIG. 2. Dendrogram showing the genetic relationships among the 17 PFGE profiles identified from 2,144 Spanish MRSA strains and the SSCmec detected.
268 NOTES J. CLIN. MICROBIOL. FIG. 3. Evolution of predominant nosocomial MRSA clones between 1996 and 2002 in Spain. (Fig. 3), while clone E6 was isolated more frequently (23.3%). Clones E1 and E6 were the predominant clones, but over time E7 and E8 clones became the predominant clones. In 1996, E8 was isolated for the first time. The frequency of E8 increased from 2.4% to 40.0% from 1996 to 2000 and subsequently decreased to 26.5%. Unlike the other clones, the frequency of clone E7 continued to increase from 4.1% to 32.3%. The frequencies of the other clones remained the same from 1998 to 2002. The geographic distributions of all identified clones were examined, but no special geographic distribution was revealed. For further epidemiological studies, a representative sample set of all MRSA strains studied were considered in order to determine the type of staphylococcal chromosomal cassette (SSCmec) (n 136) according to the genotypes observed and year of isolation (31). Also, a representative sample set (n 34) of the defined genotypes was characterized by multilocus sequence typing (MLST) (15) in order to determine the origin and evolution of the genotypes. Table 1 and Fig. 2 show the distribution of the SSCmec PFGE type complex and MLST types in correlation with the previous name given in order to identify correctly the MRSA clones circulating in Spain. We found several clones that harbored SSCmec type I (E1, E6, E9, E15, E16, and E17), belonging to ST228 and exhibiting a multiresistant profile. This sequence type (ST) had been previously described in Italy (6). SSCmec type II includes the E12 clone, with a pattern similar to that of EMRSA-16 (24) and the same allelic profile as ST36. The E12 clone was involved in outbreaks in England, and it had been reported to be a major clone in a Spanish hospital (32), but in our study, it represented only 1.8% of all MRSA strains analyzed. The E4 (Brazilian clone) and E5 clones (33, 38) belonged to SSCmec type IIIA. Both clones display multiresistant profiles, but they are scarce in Spain. These clones clustered in the same lineage as ST239. SSCmec type IV was initially found in community-acquired MRSA isolates (7, 12, 28, 36) and is one of the most frequently acquired elements in hospital MRSA infections in other countries (16, 21, 27, 29, 35). In our study, type IV was the predominant type found recently and was found in the eight clusters in Spain (Fig. 1). SSCmec type IV was identified the first time in clone E3, which had been lysed by phage 95 (9), and it shares the same allelic profile as ST146 in E10 and E11 clones isolated in a hospital in Tenerife, Spain (32). The E13 clone contains ST22, which is related to the epidemic EMRSA-15 (24), isolated in several countries, while the E2 clone has ST45, which was identified in several European countries (15). ST125 was found in Spain for the first time in 2001 (32). However, in our study, we demonstrated that ST125 emerged during 1996, and at present, it is responsible for more than 50% of the infections caused by nosocomial MRSA strains in Spain. ST125-IV has diverse patterns and includes E7, E8, and E11 clones. This ST also displays different gentamicin susceptibilities and contains Gen s and Gen r strains. Some authors TABLE 1. Details of predominant MRSA PFGE profiles and genetic background obtained by MLST and SSCmec typing Frequency (%) ST-MRSA-SSCmec Allelic profile a Current name Geographic location E1 6 ST247-MRSA-I 3-3-1-12-4-4-16 Iberian E2 0.6 ST45-MRSA-IV 10-14-8-6-10-3-2 Berlin, Germany E3 2 ST146-MRSA-IV 1-43-1-4-12-1-10 Tenerife, Spain E10 4.7 E4 1.2 ST239-MRSA-III 2-3-1-1-4-4-3 Brazilian Brazil E5 1.7 E6 11.5 ST228-MRSA-I 1-4-1-4-12-24-29 Southern Germany E9 1.8 E15 1.1 E16 1.9 E17 2 E7 27 ST125-MRSA-IV 1-4-1-4-12-1-54 Norway; Tenerife, Spain E8 31.6 E11 3.3 E12 1.9 ST36-MRSA-II 2-2-2-2-2-3-2 EMRSA-16 United Kingdom E13 0.7 ST22-MRSA-IV 7-6-1-5-8-8-6 EMRSA-15, Barnim United Kingdom E14 0.9 a The allelic profile shows the allele numbers of the seven housekeeping genes in the following order: arcc-aroe-glp-gmk-pta-tpi-yqil.
VOL. 44, 2006 NOTES 269 support the hypothesis that Gen s strains emerged from a Gen r population (2, 4). Moreover, other authors suggest that ST125 and ST146 were derived from ST5 (pediatric clone), because they differ from ST5 in only one allele. ST125 has a different sequence in yqil (acetyl coenzyme A acetyltransferase), and ST146 differs in aroe (shikimate dehydrogenase) (32). In conclusion, the use of PFGE in combination with SSCmec and MLST typing has identified the clones causing the majority of MRSA infections in Spain. The epidemiological background of characterized Spanish MRSA strains show an increased prevalence of SSCmec type IV and the dissemination of a relatively few clones. Moreover, we found in our retrospective study that ST125, which mainly includes E7 and E8 clones, emerged for the first time in 1996 and has been isolated more often since then. This ST is responsible for more than half of nosocomial MRSA infections, with a significant decrease in the level of gentamicin resistance. REFERENCES 1. Aparicio, P., J. Richardson, S. Martin, A. Vindel, R. R. Marples, and B. D. Cookson. 1992. An epidemic methicillin-resistant strain of Staphylococcus aureus in Spain. Epidemiol. Infect. 108:287 298. 2. Baba, T., F. Takeuchi, M. Kuroda, H. Yuzawa, K. Aoki, A. Oguchi, Y. Nagai, N. Iwama, K. Asano, T. Naimi, H. Kuroda, L. Cui, K. Yamamoto, and K. Hiramatsu. 2002. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359:1819 1827. 3. Blair, J. E., and R. E. O. Williams. 1961. Phage typing of staphylococci. Bull. W. H. O. 41:771 784. 4. Blanc, D. S., P. Francioli, A. Le Coustumier, L. Gazagne, E. Lecaillon, P. Gueudet, F. Vandenesch, and J. Etienne. 2001. Reemergence of gentamicinsusceptible strains of methicillin-resistant Staphylococcus aureus in France: a phylogenetic approach. J. Clin. Microbiol. 39:2287 2290. 5. Bouza, E., J. Martinez-Beltran, and Grupo de Trabajo para el Estudio de Estafilococos. 1988. Estudio multicéntrico sobre la prevalencia de estafilococos en España. Enferm. Infecc. Microbiol. Clin. 6:68 79. 6. Cassone, M., F. Campanile, A. Pantosti, M. Venditti, and S. Stefani. 2004. Identification of a variant Rome clone of methicillin-resistant Staphylococcus aureus with decreased susceptibility to vancomycin, responsible for an outbreak in an intensive care unit. Microb. Drug Resist. 10:43 49. 7. Centers for Disease Control and Prevention. 1999. Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus Minnesota and North Dakota 1997 1999. Morb. Mortal. Wkly. Rep. 48:707 710. 8. Cuevas, O., E. Cercenado, A. Vindel, J. Guinea, M. Sanchez-Conde, M. Sanchez-Somolinos, and E. Bouza. 2004. Evolution of the antimicrobial resistance of Staphylococcus spp. in Spain: five prevalence studies, 1986 to 2002. Antimicrob. Agents Chemother. 48:4240 4245. 9. del Valle, O., P. Trincado, M. T. Martin, E. Gomez, A. Cano, and A. Vindel. 1999. The prevalence of methicillin-resistant Staphylococcus aureus phagotype 95 in the Hospitales Vall d Hebron of Barcelona. Enferm. Infecc. Microbiol. Clin. 17:498 505. (In Spanish.) 10. Denis, O., A. Deplano, C. Nonhoff, R. De Ryck, R. de Mendonca, S. Rottiers, R. Vanhoof, and M. J. Struelens. 2004. National surveillance of methicillinresistant Staphylococcus aureus in Belgian hospitals indicates rapid diversification of epidemic clones. Antimicrob. Agents Chemother. 48:3625 3629. 11. De Sousa, M. A., and H. de Lencastre. 2004. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus. FEMS Immunol. Med. Microbiol. 40:101 111. 12. De Sousa, M. A., and H. de Lencastre. 2003. Evolution of sporadic isolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community-acquired MRSA. J. Clin. Microbiol. 41:3806 3815. 13. Dominguez, M. A., H. de Lencastre, J. Linares, and A. Tomasz. 1994. Spread and maintenance of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone during an outbreak of MRSA disease in a Spanish hospital. J. Clin. Microbiol. 32:2081 2087. 14. Donnio, P. Y., L. Preney, A. L. Gautier-Lerestif, J. L. Avri, and N. Lafforgue. 2004. Changes in staphylococcal cassette chromosome type and antibiotic resistance profile in methicillin-resistant Staphylococcus aureus isolates from a French hospital over an 11 year period. J. Antimicrob. Chemother. 53: 808 813. 15. Enright, M. C., N. P. Day, C. E. Davies, S. J. Peacock, and B. G. Spratt. 2000. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J. Clin. Microbiol. 38:1008 1015. 16. Fey, P. D., B. Said-Salim, M. E. Rupp, S. H. Hinrichs, D. J. Boxrud, C. C. Davis, B. N. Kreiswirth, and P. M. Schlievert. 2003. Comparative molecular analysis of community- or hospital-acquired methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 47:196 203. 17. Galdbart, J. O., A. Morvan, and N. El Solh. 1997. Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in France from 1995 to 1997. J. Clin. Microbiol. 38:185 190. 18. Geha, D. J., J. R. Uhl, C. A. Gustaferro, and D. H. Persing. 1994. Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory. J. Clin. Microbiol. 32:1768 1772. 19. Laurent, F., H. Lelievre, M. Cornu, F. Vandenesch, G. Carret, J. Etienne, and J. P. Flandrois. 2001. Fitness and competitive growth advantage of new gentamicin-susceptible MRSA clones spreading in French hospitals. J. Antimicrob. Chemother. 47:277 283. 20. Lelievre, H., G. Lina, M. E. Jones, C. Olive, F. Forey, M. Roussel-Delvallez, M. H. Nicolas-Chanoine, C. M. Bebear, V. Jarlier, A. Andremont, F. Vandenesch, and J. Etienne. 1999. Emergence and spread in French hospitals of methicillin-resistant Staphylococcus aureus with increasing susceptibility to gentamicin and other antibiotics. J. Clin. Microbiol. 37:3452 3457. 21. Mangeney, N., K. Drollee, V. Cloitre, M. Bordes, E. Faubert, and C. Dupeyron. 2002. Comparative pulsed-field gel electrophoresis typing of gentamicin-resistant and -susceptible methicillin-resistant Staphylococcus aureus strains isolated in France between 1991 and 1998. Changes in antibiotic susceptibility. J. Hosp. Infect. 51:262 268. 22. Mato, R., I. Santos Sanches, M. Venditti, D. J. Platt, A. Brown, M. Chung, and H. de Lencastre. 1998. Spread of the multiresistant Iberian clone of methicillin-resistant Staphylococcus aureus (MRSA) to Italy and Scotland. Microb. Drug Resist. 4:107 112. 23. Melter, O., M. Aires de Sousa, P. Urbaskova, V. Jakubu, H. Zemlickova, and H. de Lencastre. 2003. Update on the major clonal types of methicillinresistant Staphylococcus aureus in the Czech Republic. J. Clin. Microbiol. 41:4998 5005. 24. Moore, P. C., and J. A. Lindsay. 2002. Molecular characterisation of the dominant UK methicillin-resistant Staphylococcus aureus strains, EMRSA-15 and EMRSA-16. J. Med. Microbiol. 51:516 521. 25. Murchan, S., M. E. Kaufmann, A. Deplano, R. de Ryck, M. Struelens, C. E. Zinn, V. Fussing, S. Salmenlinna, J. Vuopio-Varkila, N. El Solh, C. Cuny, W. Witte, P. T. Tassios, N. Legakis, W. van Leeuwen, A. van Belkum, A. Vindel, I. Laconcha, J. Garaizar, S. Haeggman, B. Olsson-Liljequist, U. Ransjo, G. Coombes, and B. Cookson. 2003. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. J. Clin. Microbiol. 41:1574 1585. 26. National Committee for Clinical Laboratory Standards. 2004. Performance standards for antimicrobial susceptibility testing. Fourteenth informational supplement. M100 S14. National Committee for Clinical Laboratory Standards, Wayne, Pa. 27. O Brien, F. G., T. T. Lim, F. N. Chong, G. W. Coombs, M. C. Enright, D. A. Robinson, A. Monk, B. Saïd-Salim, B. N. Kreiswirth, and W. B. Grubb. 2004. Diversity among community isolates of methicillin-resistant Staphylococcus aureus in Australia. J. Clin. Microbiol. 42:3185 3190. 28. Okuma, K., K. Iwakawa, J. D. Turnidge, W. B. Grubb, J. M. Bell, F. G. O Brien, G. W. Coombs, J. W. Pearman, F. C. Tenover, M. Kapi, C. Tiensasitorn, T. Ito, and K. Hiramatsu. 2002. Dissemination of new methicillinresistant Staphylococcus aureus clones in the community. J. Clin. Microbiol. 40:4289 4294. 29. Oliveira, D. C., A. Tomasz, and H. de Lencastre. 2001. The evolution of pandemic clones of methicillin-resistant Staphylococcus aureus: identification of two ancestral genetic backgrounds and the associated mec elements. Microb. Drug Resist. 7:349 361. 30. Oliveira, D. C., A. Tomasz, and H. de Lencastre. 2002. Secrets of success of a human pathogen: molecular evolution of pandemic clones of methicillinresistant Staphylococcus aureus. Lancet Infect. Dis. 2:180 189. 31. Oliveira, D. C., and H. de Lencastre. 2002. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 46: 2155 2161. 32. Perez-Roth, E., F. Lorenzo-Diaz, N. Batista, A. Moreno, and S. Mendez- Alvarez. 2004. Tracking methicillin-resistant Staphylococcus aureus clones during a 5-year period (1998 to 2002) in a Spanish hospital. J. Clin. Microbiol. 42:4649 4656. 33. Ramos, R. L., L. A. Teixeira, L. R. Ormonde, P. L. Siqueira, M. S. Santos, D. Marangoni, and A. M. Figueiredo. 1999. Emergence of mupirocin resistance in multiresistant Staphylococcus aureus clinical isolates belonging to Brazilian epidemic clone III::B:A. J. Med. Microbiol. 48:303 307. 34. Richardson, J. F., V. T. Rosdahl, W. J. van Leeuwen, A. M. Vickery, A. Vindel, and W. Witte. 1999. Phages for methicillin-resistant Staphylococcus aureus: an international trial. Epidemiol. Infect. 122:227 233.
270 NOTES J. CLIN. MICROBIOL. 35. Robinson, D. A., and M. C. Enright. 2003. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob. Agents Chemother. 47:3926 3934. 36. Said-Salim, B., B. Mathema, and B. N. Kreiswirth. 2003. Community-acquired methicillin-resistant Staphylococcus aureus: an emerging pathogen. Infect. Control Hosp. Epidemiol. 24:451 455. 37. Stefani, S., and P. E. Varaldo. 2003. Epidemiology of methicillin-resistant staphylococci in Europe. Clin. Microbiol. Infect. 9:1179 1186. 38. Teixeira, L. A., M. C. Lourenco, and A. M. Figueiredo. 1996. Emergence of a methicillin-resistant Staphylococcus aureus clone related to the Brazilian epidemic clone III::B:A causing invasive disease among AIDS patients in a Brazilian hospital. Microb. Drug Resist. 2:393 399. 39. Tenover, F. C., R. D. Arbeit, R. V. Goering, P. A. Mickelsen, B. E. Murray, D. H. Persing, and B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:2233 2239. 40. Vindel, A., C. Martin-Bourgon, and J. A. Saez-Nieto. 1987. Characterization of non-typable strains of Staphylococcus aureus from cases of hospital infection. Epidemiol. Infect. 99:191 200. 41. Vindel, A. 1995. Epidemiologia molecular de cepas de Staphylococcus aureus resistentes a meticilina productoras de infección nosocomial. Ph.D. thesis. University of Madrid, Madrid, Spain.