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Molecular and Phenotypic Characteristics of Methicillin-resistant Staphylococcus aureus Strains Isolated From Hospitalized Patients in Transplantation Wards
Molecular and Phenotypic Characteristics of Methicillin-resistant Staphylococcus aureus Strains Isolated From Hospitalized Patients in Transplantation Wards A. Mlynarczyka, K. Szymanek-Majchrzakb, W. Grzybowskac, M. Durlikd, D. Deborska-Materkowskad, L. Paczeke, A. Chmuraf, E. Swoboda-Kopeca, S. Tyskic,g, and G. Mlynarczykb,* a Department of Dental Microbiology, Medical University of Warsaw, Warsaw, Poland; bDepartment of Medical Microbiology, Medical University of Warsaw, Warsaw, Poland; cDepartment of Antibiotics and Microbiology, National Medicines Institute, Warsaw, Poland; d Department of Transplant Medicine and Nephrology, Transplantation Institute, Medical University of Warsaw, Warsaw, Poland; e Department of Immunology, Transplantology and Internal Diseases, Transplantation Institute, Medical University of Warsaw, Warsaw, Poland; fDepartment of General and Transplantation Surgery, Transplantation Institute, Medical University of Warsaw, Warsaw, Poland; and gDepartment of Pharmaceutical Microbiology, Medical University of Warsaw, Warsaw, Poland
ABSTRACT Objectives. Hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) frequently causes therapeutic problems and provides information about the epidemiological condition of the ward. Materials and Methods. HA-MRSA isolated from patients on transplantation wards in 1991, 1994, 1996, and from 2005 to 2007 were compared using molecular methods such as restriction fragment length polymorphismepulse field gel electrophoresis, multilocus sequence typing (MLST), multiplex polymerase chain reaction (PCR) for detection type of staphylococcal chromosomal cassette mec, and PCR for detection. Results. The analysis covered HA-MRSA strains, each from a different patient. All organisms were typed using molecular methods. MLST results were compared with an international base. The examined strains belonged to five different worldwide known clonal complexes: CC8 (78%), CC5 (12%), CC1 (4%), CC30 (2%), and CC51 (4%). All could be recognized as representatives of a clonal complex CC8 clones: ST239-III (sequence type 239 and SCCmec type III named EMRSA-1, -4, -11, Brasilian, Hungarian) occurred with a frequency of 35.9%, ST254-IV (EMRSA-10, Hannover) occurred in 33.3%, ST247-I (EMRSA-5,-7, Iberian) occurred in 20.5%, ST241-III (Finland-UK) occurred in 5.15%, and ST8-IV (EMRSA-2,-6) occurred in 5.15%. Conclusion. The predomination of different clones of HA-MRSA in the particular years was observed. In 1991, the EMRSA-10 (Hannover) clone predominated (53.3%). The Brasilian-Hungarian (EMRSA-1, -4, -11) clone predominated in 1994 (50%) as well as from 2005 to 2007 (41.3%), whereas in 1996 the Iberian clone was most frequent (53.9%).
T
HE METHICILLIN-RESISTANT Staphylococcus aureus (MRSA) shortly after emergence became a serious therapeutic and epidemiological problem [1]. This intensified the necessity of epidemiological typing of staphylococci. Epidemiological typing of bacterial strains usually aims to detect a nosocomial epidemic or to determine evolutionary relationship of same-species bacterial strains. The selection of a suitable typing method depends on predetermined goals and the type of bacteria. In the case of S aureus, the most
common methods include multilocus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) Supported by the Medical University of Warsaw, Warsaw, Poland (1M20/N/2012-2013). *Address correspondence to Grazyna Mlynarczyk, Department of Medical Microbiology, Medical University of Warsaw, T. Chalubinskiego 5 Str., 02-004 Warsaw. E-mail: grazyna.mlynarczyk@ wum.edu.pl
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.08.026
Transplantation Proceedings, 46, 2579e2582 (2014)
2579
2580
typing, sequence analysis of the variable number tandem repeat (VNTR) in S aureus protein A gene (spa typing), multilocus VNTR analysis, restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA fragments after SmaI digestion, etc. The worldwide use of various typing methods over many years has yielded the identification of certain easily transmitted strains of S aureus. The individual epidemiologic clones of MRSA differ from one another in terms of multiple features such as colonization site (nasal cavity or groins), colonization ability, virulence, spread, as well as multiple genes responsible for pathogenicity [2] and drugresistance [3e5]. Recent years have shown that the type of SCCmec may play a crucial role in the course (acute versus chronic) of the infection. SCCmecs type II, III, and VIII encode the transcription factor psm-mec that inhibits translation of the global regulator agr, which results in eliminating the production of multiple factors of pathogenicity, including cytotoxins (hemolysins, leukotoxins, phenol soluble modulin [PSM] peptide toxins) on one hand, and stimulating surface protein synthesis and biofilm production on the other. Other SCCmec types (eg, types I or IV) do not contain psm-mec [6].The purpose of this study was to determine which of the international MRSA clones could be isolated from transplantation patients and if the same clones would persist over the study period.
MATERIALS AND METHODS Bacteria The study involved an analysis of a total of 50 MRSA strains isolated from various biological samples from patients hospitalized in transplantation wards of one of the hospitals in Warsaw over the study period: 15 strains in 1991, 10 strains in 1994, 13 strains in 1996, and 12 strains from 2005 to 2007. The evaluated MRSA strains were cultured from the following biological samples: wound swab (37), pus (7), bile (2), catheter (1), drain (1), tracheostomy tube (1), and fistula (1). The strains identified as MRSA were frozen and stored at 70 C for further analyses. Only one isolate per patient was considered.
MLST Seven housekeeping gene fragments (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) of each of the evaluated strains were amplified. Polymerase chain reaction (PCR) products were purified with Clean Up AX (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s instructions. DNA sequencing was conducted with a modified Sanger method, with the use of DYEnamic Energy Transfer Terminator Cycle Sequencing Kit (GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, United Kingdom) and the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, United States). The obtained sequences were compared with data collected from international databases [7].
SCCmec Typing The SCCmec type was determined by multiplex PCR as described by [8,9].
MLYNARCZYK, SZYMANEK-MAJCHRZAK, GRZYBOWSKA ET AL
RFLP-PFGE We used the previously described method [10]. Fragment separation was conducted with the clamped homogeneous electric fields system with dynamic regulation (CHEF-DR II; Bio-Rad, United States).
Detection of Antibiotic-resistance Genes The strains were examined via PCR for the presence of ermA, ermB, ermC, msrA/B (macrolide, lincosamide, streptogramine B resistance genes) [11], aacA-aphD, aadD, aph(3”)-IIIa (aminoglycoside resistance genes) [12], and vanA, vanB (glycopeptide resistance genes). The method and primers used have been described in our earlier study [13].
Antibiotic Sensitivity Testing Minimum inhibitory concentration (MIC) was determined via antibiotic dilution in solid medium MH2. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods and criteria from 2012 were used.
RESULTS
Figure 1 shows the relationship of the MRSA strains evaluated with the use of RLFP-PFGE, MLST, and SCCmec typing. Eighteen different PFGE types, labeled from A to T, were obtained via the RLFP-PFGE technique. Each strain was classified into the appropriate clonal complex (CC) and, within these complexes, into appropriate international clones, based on the sequence type (ST) determined via MLST and the SCCmec type. Of the 50 MRSA strains, the most numerous, in decreasing order, were: CC8 (78%), followed by CC5 (12%), CC51 (ST120-I; 4%), CC1 (ST1-IV; 4%), and CC30 (ST30-III; 2%). CC8 included five international clones, such as Brazilian/Hungarian (35.9%), Hannover (33.3%), Iberian (20.5%), Finland-UK (5.15%), and EMRSA-2, -6 (5.15%). CC5 included two clones: ST461-I (75%) and the Pediatric clone ST5-IV (25%). The population of evaluated strains contained the following antibiotic-resistance genes: ermA (82%, absent in ST461-I and ST5-IV), ermC (8%), msrA/B (4%), aacA-aphD (60%, absent in ST254-IV, ST461-I, ST5-IV), aadD (32%), and aph(3”)IIIa (40%). Genes ermB, lnuA, vanA, and vanB were not detected. The strains exhibited resistance to macrolides and clindamycin (82%), gentamicin and tobramycin (60%, but all representatives of Hannover clone were susceptible), amikacin (46%), netilmicin (30%, but all representatives of Hannover clone were susceptible), doxycycline (44%, but all representatives of Iberian clone were susceptible), ciprofloxacin (44%), levofloxacin (20%), co-trimoxazole (6%), teicoplanin (18%, MIC ¼ 4e8 mg/L, resistant according to EUCAST, susceptible according to Clinical and Laboratory Standarts Institute), and vancomycin (2%, ST1-IV, MIC ¼ 4 mg/L, resistant according to EUCAST, intermediately susceptible according to CLSI). No resistance to linezolid or mupirocin was observed.
CHARACTERISTICS OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
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Fig 1. Dendrogram presenting the relationship of the MRSA strains evaluated with the use of RLFP-PFGE, MLST, and SCCmec typing. Abbreviation: CC, clonal complex.
DISCUSSION
In our study, five different globally important clonal complexes (CCs) among the hospital-acquiredeMRSA
(HA-MRSA) were found in transplantation wards of a single hospital over a period of 15 years. The most frequently reported clones within CC5 were: ST5-I (EMRSA-3), ST5-II (New-York-Japan), ST5-IV (Pediatric), ST5/ST225-II
2582
(Rhine Hesse), and ST228-I (Southern Germany). CC8, which was the most prevalent clonal complex in our study, has been also prevalent in other countries, with especially high prevalence of ST239-III (EMRSA-1,-4,-11; Brazilian, Hungarian, Brazilian-Hungarian, Portugal, Vienna) and ST247-I (EMRSA-5,-17; Iberian) clones. ST-22-IV (EMRSA-15, Barnim) is the most prevalent clone of CC22. ST36-II (EMRSA-16) is the most prevalent clone of CC30. ST45-IV (Berlin) is the most prevalent clone of CC45 [14e16]. Until 2005, the following HA-MRSA clones had been reported in Poland: ST239-III, ST247-I, ST5-IV, and ST45-IV [17]. With the exception of the considerable dominance of CC8 (78%), our study results are consistent with the data from other European countries. We found the following three CC8 clones to be the most prevalent: EMRSA-1, -4, -11 (35.9%), EMRSA10 (33.3%), and EMRSA-5,-7 (20.5%). In the different years of the study period, various HA-MRSA clones were shown to dominate in the evaluated hospital, although most of them persisted throughout the study. In 1991, EMRSA-10 (53.3%) and EMRSA-1,-4,-11 (20%) were the most prevalent. In 1994, EMRSA-1, -4, -11 (50%), EMRSA-10 (30%), and EMRSA-5,-7 (10%) were the most prevalent. In 1996, the most prevalent clones were EMRSA-5,-7 (53.9%) and ST120I/CC51 (15.4%). From 2005 to 2007, once more the most prevalent clones were EMRSA-1,-4,-11 (41.7%) and EMRSA-10 (16.7%), with an emergence of EMRSA-2,-6 (16.7%). In summary, during the 15-year period covered by the study, the same MRSA clones persisted in patients from transplantation wards of the evaluated hospital with different clones dominating in different years. All dominant clones were representatives of the epidemic clones widely distributed all over the world. REFERENCES [1] Jeljaszewicz J, Mlynarczyk G, Mlynarczyk A. Present and future problems of resistance in Gram-positive cocci. Infection 1998;26:1e6. [2] Argudín MA, Mendoza MC, Méndez FJ, et al. Clonal complexes and diversity of exotoxin gene profiles in methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from patients in a Spanish hospital. J Clin Microbiol 2009;47:2097e105. [3] Mlynarczyk A, Mlynarczyk B, Kmera-Muszynska M, et al. Mechanisms of the resistance and tolerance to beta-lactam and glycopeptide antibiotics in pathogenic Gram-positive cocci. Mini Rev Med Chem 2009;9:1527e37.
MLYNARCZYK, SZYMANEK-MAJCHRZAK, GRZYBOWSKA ET AL [4] Mlynarczyk B, Mlynarczyk A, Kmera-Muszynska M, et al. Mechanisms of resistance to antimicrobial drugs in pathogenic Gram-positive cocci. Mini Rev Med Chem 2010;10:928e37. [5] Turlej A, Hryniewicz W, Empel J. Staphylococcal cassette chromosome mec (SCCmec) classification and typing methods: an overview. Pol J Microbiol 2011;60:95e103. [6] Kaito C, Saito Y, Ikuo M, et al. Mobile genetic element SCCmec-encoded psm-mec RNA suppresses translation of agrA and attenuates MRSA virulence. PLoS Pathog 2013;9: e1003269. [7] Enright MC, Day NP, Davies CE, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillinsusceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38:1008e15. [8] Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin- resistant Staphylococcus aureus. Antimicrob Agents Chemother 2002;46:2155e61. [9] Okuma K, Iwakawa K, Turnidge JD, et al. Dissemination of new methicillin resistant Staphylococcus aureus clones in the community. J Clin Microbiol 2002;40:4289e94. [10] Mlynarczyk G, Rosdahl VT, Skov R, et al. Epidemiology of methicillin resistant Staphylococcus aureus in a Warsaw hospital. Occurrence of sporadic as well as outbreak strains. J Hosp Infect 1996;34:151e60. [11] Sutcliffe J, Grebe T, Tait-Kamradt A, et al. Detection of erythromycin resistant determinants by PCR. Antimicrob Agents Chemother 1996;40:2562e6. [12] Ida T, Okamoto R, Shimauchi C, et al. Identification of aminoglycoside-modifying enzymes by susceptibility testing: epidemiology of methicillin-resistant Staphylococcus aureus in Japan. J Clin Microbiol 2001;39:3115e21. [13] Mlynarczyk G, Grzybowska W, Mlynarczyk A, et al. The significant increase of glicopeptide resistant enterococci from patients hospitalised in the transplant surgery wand in 2004-2005. Transplant Proc 2007;39:2883e5. [14] Cookson BD, Robinson DA, Monk AB, et al. Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection. J Clin Microbiol 2007;45:1830e7. [15] Grundmann H, Aanensen DM, van den Wijngaard CC, et al. Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. PLoS Med 2010;7:e1000215. [16] Friedrich AW, Witte W, de Lencastre H, et al. A European laboratory network for sequence-based typing of methicillinresistant Staphylococcus aureus (MRSA) as a communication platform between human and veterinary medicine an update on SeqNet.org. Euro Surveill 2008;13:1e5. [17] Luczak-Kadłubowska A, Sulikowska A, Empel J, et al. Countrywide molecular survey of methicillin-resistant Staphylococcus aureus strains in Poland. J Clin Microbiol 2008;46:2930e7.
ABSTRACT Objectives. Hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) frequently causes therapeutic problems and provides information about the epidemiological condition of the ward. Materials and Methods. HA-MRSA isolated from patients on transplantation wards in 1991, 1994, 1996, and from 2005 to 2007 were compared using molecular methods such as restriction fragment length polymorphismepulse field gel electrophoresis, multilocus sequence typing (MLST), multiplex polymerase chain reaction (PCR) for detection type of staphylococcal chromosomal cassette mec, and PCR for detection. Results. The analysis covered HA-MRSA strains, each from a different patient. All organisms were typed using molecular methods. MLST results were compared with an international base. The examined strains belonged to five different worldwide known clonal complexes: CC8 (78%), CC5 (12%), CC1 (4%), CC30 (2%), and CC51 (4%). All could be recognized as representatives of a clonal complex CC8 clones: ST239-III (sequence type 239 and SCCmec type III named EMRSA-1, -4, -11, Brasilian, Hungarian) occurred with a frequency of 35.9%, ST254-IV (EMRSA-10, Hannover) occurred in 33.3%, ST247-I (EMRSA-5,-7, Iberian) occurred in 20.5%, ST241-III (Finland-UK) occurred in 5.15%, and ST8-IV (EMRSA-2,-6) occurred in 5.15%. Conclusion. The predomination of different clones of HA-MRSA in the particular years was observed. In 1991, the EMRSA-10 (Hannover) clone predominated (53.3%). The Brasilian-Hungarian (EMRSA-1, -4, -11) clone predominated in 1994 (50%) as well as from 2005 to 2007 (41.3%), whereas in 1996 the Iberian clone was most frequent (53.9%).
T
HE METHICILLIN-RESISTANT Staphylococcus aureus (MRSA) shortly after emergence became a serious therapeutic and epidemiological problem [1]. This intensified the necessity of epidemiological typing of staphylococci. Epidemiological typing of bacterial strains usually aims to detect a nosocomial epidemic or to determine evolutionary relationship of same-species bacterial strains. The selection of a suitable typing method depends on predetermined goals and the type of bacteria. In the case of S aureus, the most
common methods include multilocus sequence typing (MLST), staphylococcal chromosomal cassette mec (SCCmec) Supported by the Medical University of Warsaw, Warsaw, Poland (1M20/N/2012-2013). *Address correspondence to Grazyna Mlynarczyk, Department of Medical Microbiology, Medical University of Warsaw, T. Chalubinskiego 5 Str., 02-004 Warsaw. E-mail: grazyna.mlynarczyk@ wum.edu.pl
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.08.026
Transplantation Proceedings, 46, 2579e2582 (2014)
2579
2580
typing, sequence analysis of the variable number tandem repeat (VNTR) in S aureus protein A gene (spa typing), multilocus VNTR analysis, restriction fragment length polymorphism (RFLP) analysis by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA fragments after SmaI digestion, etc. The worldwide use of various typing methods over many years has yielded the identification of certain easily transmitted strains of S aureus. The individual epidemiologic clones of MRSA differ from one another in terms of multiple features such as colonization site (nasal cavity or groins), colonization ability, virulence, spread, as well as multiple genes responsible for pathogenicity [2] and drugresistance [3e5]. Recent years have shown that the type of SCCmec may play a crucial role in the course (acute versus chronic) of the infection. SCCmecs type II, III, and VIII encode the transcription factor psm-mec that inhibits translation of the global regulator agr, which results in eliminating the production of multiple factors of pathogenicity, including cytotoxins (hemolysins, leukotoxins, phenol soluble modulin [PSM] peptide toxins) on one hand, and stimulating surface protein synthesis and biofilm production on the other. Other SCCmec types (eg, types I or IV) do not contain psm-mec [6].The purpose of this study was to determine which of the international MRSA clones could be isolated from transplantation patients and if the same clones would persist over the study period.
MATERIALS AND METHODS Bacteria The study involved an analysis of a total of 50 MRSA strains isolated from various biological samples from patients hospitalized in transplantation wards of one of the hospitals in Warsaw over the study period: 15 strains in 1991, 10 strains in 1994, 13 strains in 1996, and 12 strains from 2005 to 2007. The evaluated MRSA strains were cultured from the following biological samples: wound swab (37), pus (7), bile (2), catheter (1), drain (1), tracheostomy tube (1), and fistula (1). The strains identified as MRSA were frozen and stored at 70 C for further analyses. Only one isolate per patient was considered.
MLST Seven housekeeping gene fragments (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) of each of the evaluated strains were amplified. Polymerase chain reaction (PCR) products were purified with Clean Up AX (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s instructions. DNA sequencing was conducted with a modified Sanger method, with the use of DYEnamic Energy Transfer Terminator Cycle Sequencing Kit (GE Healthcare, Life Sciences, Little Chalfont, Buckinghamshire, United Kingdom) and the ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, California, United States). The obtained sequences were compared with data collected from international databases [7].
SCCmec Typing The SCCmec type was determined by multiplex PCR as described by [8,9].
MLYNARCZYK, SZYMANEK-MAJCHRZAK, GRZYBOWSKA ET AL
RFLP-PFGE We used the previously described method [10]. Fragment separation was conducted with the clamped homogeneous electric fields system with dynamic regulation (CHEF-DR II; Bio-Rad, United States).
Detection of Antibiotic-resistance Genes The strains were examined via PCR for the presence of ermA, ermB, ermC, msrA/B (macrolide, lincosamide, streptogramine B resistance genes) [11], aacA-aphD, aadD, aph(3”)-IIIa (aminoglycoside resistance genes) [12], and vanA, vanB (glycopeptide resistance genes). The method and primers used have been described in our earlier study [13].
Antibiotic Sensitivity Testing Minimum inhibitory concentration (MIC) was determined via antibiotic dilution in solid medium MH2. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods and criteria from 2012 were used.
RESULTS
Figure 1 shows the relationship of the MRSA strains evaluated with the use of RLFP-PFGE, MLST, and SCCmec typing. Eighteen different PFGE types, labeled from A to T, were obtained via the RLFP-PFGE technique. Each strain was classified into the appropriate clonal complex (CC) and, within these complexes, into appropriate international clones, based on the sequence type (ST) determined via MLST and the SCCmec type. Of the 50 MRSA strains, the most numerous, in decreasing order, were: CC8 (78%), followed by CC5 (12%), CC51 (ST120-I; 4%), CC1 (ST1-IV; 4%), and CC30 (ST30-III; 2%). CC8 included five international clones, such as Brazilian/Hungarian (35.9%), Hannover (33.3%), Iberian (20.5%), Finland-UK (5.15%), and EMRSA-2, -6 (5.15%). CC5 included two clones: ST461-I (75%) and the Pediatric clone ST5-IV (25%). The population of evaluated strains contained the following antibiotic-resistance genes: ermA (82%, absent in ST461-I and ST5-IV), ermC (8%), msrA/B (4%), aacA-aphD (60%, absent in ST254-IV, ST461-I, ST5-IV), aadD (32%), and aph(3”)IIIa (40%). Genes ermB, lnuA, vanA, and vanB were not detected. The strains exhibited resistance to macrolides and clindamycin (82%), gentamicin and tobramycin (60%, but all representatives of Hannover clone were susceptible), amikacin (46%), netilmicin (30%, but all representatives of Hannover clone were susceptible), doxycycline (44%, but all representatives of Iberian clone were susceptible), ciprofloxacin (44%), levofloxacin (20%), co-trimoxazole (6%), teicoplanin (18%, MIC ¼ 4e8 mg/L, resistant according to EUCAST, susceptible according to Clinical and Laboratory Standarts Institute), and vancomycin (2%, ST1-IV, MIC ¼ 4 mg/L, resistant according to EUCAST, intermediately susceptible according to CLSI). No resistance to linezolid or mupirocin was observed.
CHARACTERISTICS OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS
2581
Fig 1. Dendrogram presenting the relationship of the MRSA strains evaluated with the use of RLFP-PFGE, MLST, and SCCmec typing. Abbreviation: CC, clonal complex.
DISCUSSION
In our study, five different globally important clonal complexes (CCs) among the hospital-acquiredeMRSA
(HA-MRSA) were found in transplantation wards of a single hospital over a period of 15 years. The most frequently reported clones within CC5 were: ST5-I (EMRSA-3), ST5-II (New-York-Japan), ST5-IV (Pediatric), ST5/ST225-II
2582
(Rhine Hesse), and ST228-I (Southern Germany). CC8, which was the most prevalent clonal complex in our study, has been also prevalent in other countries, with especially high prevalence of ST239-III (EMRSA-1,-4,-11; Brazilian, Hungarian, Brazilian-Hungarian, Portugal, Vienna) and ST247-I (EMRSA-5,-17; Iberian) clones. ST-22-IV (EMRSA-15, Barnim) is the most prevalent clone of CC22. ST36-II (EMRSA-16) is the most prevalent clone of CC30. ST45-IV (Berlin) is the most prevalent clone of CC45 [14e16]. Until 2005, the following HA-MRSA clones had been reported in Poland: ST239-III, ST247-I, ST5-IV, and ST45-IV [17]. With the exception of the considerable dominance of CC8 (78%), our study results are consistent with the data from other European countries. We found the following three CC8 clones to be the most prevalent: EMRSA-1, -4, -11 (35.9%), EMRSA10 (33.3%), and EMRSA-5,-7 (20.5%). In the different years of the study period, various HA-MRSA clones were shown to dominate in the evaluated hospital, although most of them persisted throughout the study. In 1991, EMRSA-10 (53.3%) and EMRSA-1,-4,-11 (20%) were the most prevalent. In 1994, EMRSA-1, -4, -11 (50%), EMRSA-10 (30%), and EMRSA-5,-7 (10%) were the most prevalent. In 1996, the most prevalent clones were EMRSA-5,-7 (53.9%) and ST120I/CC51 (15.4%). From 2005 to 2007, once more the most prevalent clones were EMRSA-1,-4,-11 (41.7%) and EMRSA-10 (16.7%), with an emergence of EMRSA-2,-6 (16.7%). In summary, during the 15-year period covered by the study, the same MRSA clones persisted in patients from transplantation wards of the evaluated hospital with different clones dominating in different years. All dominant clones were representatives of the epidemic clones widely distributed all over the world. REFERENCES [1] Jeljaszewicz J, Mlynarczyk G, Mlynarczyk A. Present and future problems of resistance in Gram-positive cocci. Infection 1998;26:1e6. [2] Argudín MA, Mendoza MC, Méndez FJ, et al. Clonal complexes and diversity of exotoxin gene profiles in methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from patients in a Spanish hospital. J Clin Microbiol 2009;47:2097e105. [3] Mlynarczyk A, Mlynarczyk B, Kmera-Muszynska M, et al. Mechanisms of the resistance and tolerance to beta-lactam and glycopeptide antibiotics in pathogenic Gram-positive cocci. Mini Rev Med Chem 2009;9:1527e37.
MLYNARCZYK, SZYMANEK-MAJCHRZAK, GRZYBOWSKA ET AL [4] Mlynarczyk B, Mlynarczyk A, Kmera-Muszynska M, et al. Mechanisms of resistance to antimicrobial drugs in pathogenic Gram-positive cocci. Mini Rev Med Chem 2010;10:928e37. [5] Turlej A, Hryniewicz W, Empel J. Staphylococcal cassette chromosome mec (SCCmec) classification and typing methods: an overview. Pol J Microbiol 2011;60:95e103. [6] Kaito C, Saito Y, Ikuo M, et al. Mobile genetic element SCCmec-encoded psm-mec RNA suppresses translation of agrA and attenuates MRSA virulence. PLoS Pathog 2013;9: e1003269. [7] Enright MC, Day NP, Davies CE, et al. Multilocus sequence typing for characterization of methicillin-resistant and methicillinsusceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38:1008e15. [8] Oliveira DC, de Lencastre H. Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin- resistant Staphylococcus aureus. Antimicrob Agents Chemother 2002;46:2155e61. [9] Okuma K, Iwakawa K, Turnidge JD, et al. Dissemination of new methicillin resistant Staphylococcus aureus clones in the community. J Clin Microbiol 2002;40:4289e94. [10] Mlynarczyk G, Rosdahl VT, Skov R, et al. Epidemiology of methicillin resistant Staphylococcus aureus in a Warsaw hospital. Occurrence of sporadic as well as outbreak strains. J Hosp Infect 1996;34:151e60. [11] Sutcliffe J, Grebe T, Tait-Kamradt A, et al. Detection of erythromycin resistant determinants by PCR. Antimicrob Agents Chemother 1996;40:2562e6. [12] Ida T, Okamoto R, Shimauchi C, et al. Identification of aminoglycoside-modifying enzymes by susceptibility testing: epidemiology of methicillin-resistant Staphylococcus aureus in Japan. J Clin Microbiol 2001;39:3115e21. [13] Mlynarczyk G, Grzybowska W, Mlynarczyk A, et al. The significant increase of glicopeptide resistant enterococci from patients hospitalised in the transplant surgery wand in 2004-2005. Transplant Proc 2007;39:2883e5. [14] Cookson BD, Robinson DA, Monk AB, et al. Evaluation of molecular typing methods in characterizing a European collection of epidemic methicillin-resistant Staphylococcus aureus strains: the HARMONY collection. J Clin Microbiol 2007;45:1830e7. [15] Grundmann H, Aanensen DM, van den Wijngaard CC, et al. Geographic distribution of Staphylococcus aureus causing invasive infections in Europe: a molecular-epidemiological analysis. PLoS Med 2010;7:e1000215. [16] Friedrich AW, Witte W, de Lencastre H, et al. A European laboratory network for sequence-based typing of methicillinresistant Staphylococcus aureus (MRSA) as a communication platform between human and veterinary medicine an update on SeqNet.org. Euro Surveill 2008;13:1e5. [17] Luczak-Kadłubowska A, Sulikowska A, Empel J, et al. Countrywide molecular survey of methicillin-resistant Staphylococcus aureus strains in Poland. J Clin Microbiol 2008;46:2930e7.