Characterization of Methicillin-Resistant Staphylococcus aureus StrainsRecovered from a Phase IV Clinical Trial for Linezolid versusVancomycin for Treatment of Nosocomial Pneumonia

Rodrigo E. Mendes,a Lalitagauri M. Deshpande,a Davida S. Smyth,b Bo Shopsin,b David J. Farrell,a and Ronald N. Jonesa,c

JMI Laboratories, North Liberty, Iowa, USAa; Department of Medicine, NYU School of Medicine, New York, New York, USAb; and Tufts University School of Medicine,Boston, Massachusetts, USAc

A total of 434 methicillin-resistant Staphylococcus aureus (MRSA) baseline isolates were collected from subjects enrolled in aprospective, double-blind randomized trial comparing linezolid versus vancomycin for the treatment of nosocomial pneumonia.Isolates were susceptibility tested by broth microdilution, examined for inducible clindamycin resistance by D-test, andscreened for heterogeneous resistance to vancomycin (hVISA) by the Etest macromethod. All strains were subjected to Panton-Valentine leukocidin (PVL) screening, and SCCmec, pulsed-field gel electrophoresis (PFGE), and spa typing. Selected strainswere evaluated by multilocus sequence typing (MLST). Clonal complexes (CCs) were assigned based on the spa and/or MLSTresults. Most strains were CC5 (56.0%), which originated from North America (United States) (CC5-MRSA-SCCmec II/IV;70.0%), Asia (CC5-MRSA-II; 14.0%) and Latin America (CC5-MRSA-I/II; 12.3%). The second- and third-most-prevalent cloneswere CC8-MRSA-IV (23.3%) and CC239-MRSA-III (11.3%), respectively. Furthermore, the CC5-MRSA-I/II clone predominatedin Asia (50.7% within this region) and Latin America (66.7%), followed by CC239-MRSA-III (32.8% and 28.9%, respectively).The European strains were CC8-MRSA-IV (34.5%), CC22-MRSA-IV (18.2%), or CC5-MRSA-I/II/IV (16.4%), while the U.S.MRSA isolates were CC5-MRSA-II/IV (64.4%) or CC8-MRSA-IV (28.8%). Among the U.S. CC8-MRSA-II/IV strains, 73.7%(56/76 [21.2% of all U.S. MRSA strains]) clustered within USA300. One strain from the United States (USA800) was intermediateto vancomycin (MIC, 4 �g/ml). All remaining strains were susceptible to linezolid, daptomycin, vancomycin, and teicoplanin.hVISA strains (14.5%) were predominantly CC5-MRSA-II, from South Korea, and belonged to a single PFGE type. Overall, eachregion had two predominant clones. The USA300 rate corroborates previous reports describing increased prevalence of USA300strains causing invasive infections. The prevalence of hVISA was elevated in Asia, and these strains were associated with CC5.

Staphylococcus aureus remains a leading cause of human bacte-rial infections worldwide, and the incidence of health care-

associated and community-acquired (CA) infections caused bythis organism has increased steadily (7). This species ranks as themain pathogen responsible for nosocomial bloodstream infec-tions (BSI), hospital-acquired bacterial pneumonia (HABP) andventilator-acquired bacterial pneumonia (VABP), and skin andskin structure infections (SSSI) (20, 25). Many infections arecaused by methicillin-resistant S. aureus (MRSA) isolates, and re-cent studies have demonstrated an increased MRSA incidenceover the last decade (31, 34, 43, 51). These facts, along with com-plicating risk factors, comorbidity, and mortality (between 40 and60%), result in extended hospitalizations, escalated health carecosts, and the requirement of potent, broad-spectrum agents of-ten used in combination regimens (7).

The population structure of MRSA strains is constantly evolv-ing. These epidemiologic alterations reflect in changes in the inci-dence and characteristics of MRSA infections in the hospital andcommunity settings. In the United States, the USA300 cloneemerged as important cause of CA-MRSA infections, predomi-nantly SSSI (29). Recently, this clone has also been implicated as acause of health care-associated (HA-MRSA) and invasive infec-tions (22, 39, 45). However, recent studies have demonstrated anoverall increase of noninvasive, community-onset, MRSA infec-tions, while the incidence of HA-MRSA and invasive infectionsdeclined (8, 21, 31, 51). Similar changes in the MRSA epidemiol-ogy among European hospitals have been reported in numerousstudies as well (6, 9, 14, 16). Moreover, CA-MRSA has recently

emerged across Europe (2, 19, 24, 26), and the incidence of HA-MRSA BSI has decreased or remained stable in several countries inEurope (24).

From October 2004 through January 2010, a phase IV random-ized, double-blind, actively controlled study was performed toassess the efficacy and safety of linezolid compared with dose-optimized vancomycin for the treatment of culture-proven MRSAnosocomial pneumonia (NP) in hospitalized adults (53). In thisstudy, a significantly better clinical cure rate was observed withlinezolid (58%) than with vancomycin (47%). Favorable resultswere also obtained for linezolid (58%) in terms of microbiologicalcure rates compared with vancomycin (47%). During this trial, alarge collection of geographically diverse MRSA isolates was ob-tained. This study sought to further characterize this worldwideMRSA population and evaluate possible changing trends of geno-types, molecular characteristics, and antimicrobial susceptibilityprofiles of strains responsible for NP between 2004 and 2010.

Received 31 July 2012 Returned for modification 28 August 2012Accepted 4 September 2012

Published ahead of print 12 September 2012

Address correspondence to Rodrigo E. Mendes, rodrigo-mendes@jmilabs.com.

Supplemental material for this article may be found at http://jcm.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.02024-12

3694 jcm.asm.org Journal of Clinical Microbiology p. 3694–3702 November 2012 Volume 50 Number 11

MATERIALS AND METHODSBacterial strains. A total of 434 microbiologically evaluable baselineMRSA isolates were collected (from October 2004 through January 2010)from hospitalized subjects with clinically documented NP proven to becaused by MRSA. Subjects were required to have a baseline tracheal aspi-rate, bronchoalveolar lavage (BAL) fluid, or high-quality sputum speci-men (defined as having less than 10 squamous epithelial cells and greateror equal to 25 leukocytes per high-power field) positive for MRSA (53).Baseline isolates included in this study originated from patients enrolledin the clinical trial according to preestablished inclusion criteria. There-fore, MRSA isolates included in this study were not consecutively col-lected and did not follow a prevalence mode design (53). Specimens wereprocessed and cultured for bacterial pathogens according to the standardprocedure at each medical center laboratory site. Individual investigatorsforwarded MRSA isolates to the Covance Central Laboratory Service (In-dianapolis, IN) for confirmation of identification and susceptibility test-ing. MRSA isolates were subsequently sent to JMI Laboratories (NorthLiberty, IA) for further studies. Only one strain per patient was included inthis analysis.

The strains included in this study were predominantly collected fromenrolled subjects in the United States (264 [60.8%]), followed by smallernumbers of subjects from the following countries: South Korea (44[10.1%]), Brazil (18 [4.1%]), Belgium (18 [4.1%]) Taiwan (15 [3.5%]),Russia (13 [3.0%]), Mexico (10 [2.3%]), Portugal (8 [1.8%]), Chile (8[1.8%]), France (8 [1.8%]), Malaysia (6 [1.4%]), Puerto Rico (5 [1.2%]),South Africa (3 [0.7%]), Colombia (3 [0.7%]), Spain (2 [0.5%]), andGermany (2 [0.5%]), with 1 (0.2%) strain each from Singapore, Greece,Poland, the United Kingdom, Argentina, Hong Kong, and Turkey.

Epidemiologic typing. Panton-Valentine leukocidin (PVL) (lukF-PVand lukS-PV) screening was performed by using a multiplex real-timePCR (RT-PCR) approach as previously described (30). SCCmec types (Ithrough VI) were characterized using a multiplex PCR strategy (32).Strains showing inconclusive SCCmec typing results were subjected to asecondary strategy proposed by Oliveira et al. (35). Typing of the agroperon (groups I through IV) was assessed using multiplex RT-PCR aspreviously described by Strommenger et al. (49).

Bacterial chromosomal DNA was digested with SmaI and subjected topulsed-field gel electrophoresis (PFGE). PFGE types were assigned ac-cording to the origin of the isolates (United States, Europe [includingRussia and Turkey], Latin America, the Asian-Pacific [APAC] region, andSouth Africa), followed by a capital letter (PFGE type) and a number(PFGE subtype). Gel pattern analysis was performed using the GelCompar IIsoftware (Applied Math, Kortrijk, Belgium), and the patterns obtained werecompared to those of the major U.S. and international clones, which wereprovided by the Network on Antimicrobial Resistance in S. aureus (NARSA;www.narsa.net). Percent similarities were identified on a dendrogram de-rived by the unweighted-pair group method using arithmetic averages andbased on Dice coefficients. Band position tolerance and optimization were setat 1.3 and 0.5%, respectively. Isolates showing similarity coefficient at �80%were considered genetically related (same PFGE type), while those with asimilarity coefficient at �95% were assigned the same PFGE subtype (29).

All strains were subjected to spa typing (46). Clonal complexes (CCs)were assigned based on the spa typing results using the multilocus se-quence typing (MLST) mapping database (http://spa.ridom.de/mlst) orpeer-reviewed reports. Strains with new spa typing denominations andpreviously unknown MLST associations, but clustering within PFGEtypes containing strains with similar patterns of spa repeat sequences andknown CC results, were assigned the same CCs. MLST was performed(36) for a given strain showing a spa type with an unknown MLST asso-ciation and a unique PFGE pattern. See the supplemental material foradditional information related to the strains and molecular testing results(including heterogeneous vancomycin-intermediate S. aureus [hVISA],SCCmec, PVL, agr, spa [Ridom and Kreiswirth nomenclatures]), MLST,and CCs generated during this study.

Antimicrobial susceptibility profile. Isolates were tested for suscep-tibility by broth microdilution in cation-adjusted Mueller-Hinton me-dium according to the Clinical and Laboratory Standards Institute (CLSI)recommendations (12). Quality assurance was performed by concurrenttesting of CLSI-recommended (M100-S22) strains: Enterococcus faecalisATCC 29212 and S. aureus ATCC 29213. Interpretation of MIC resultswas in accordance with published CLSI criteria (13).

Inducible clindamycin resistance was detected using the D-test diskdiffusion method according to CLSI (13). Briefly, a 2-�g clindamycin diskwas placed 15 mm from the edge of a 15-�g erythromycin disk. Followingincubation, isolates that showed flattening of the clindamycin zone on theedge adjacent to the erythromycin disk were considered D-test positive.Screening for heterogeneous vancomycin-intermediate S. aureus (hVISA)was performed using the Etest (bioMérieux, Marcy l’Etoile, France) mac-romethod as previously described (30).

RESULTSEpidemiologic typing. MRSA strains will be referred to hereinaccording to the CC and SCCmecA typing results. Therefore,CC5-MRSA-II indicates that a particular strain or group of strainsare associated with CC5 and carried SCCmec type II. The mostfrequent clone identified in this study was CC5-MRSA-I/II/IV(56.0% [243/434]), followed by CC8-MRSA-IV (23.3% [101/434]) and CC239-MRSA-III (11.3% [49/434]), while another 10CCs detected were each represented by �3.5% of the total strainsincluded (Table 1). MRSA strains associated with CC5 (56.0%)originated mostly from the United States (CC5-MRSA-II/IV;70.0% [170/243]), Asia (CC5-MRSA-II; 14.0% [34/243]), andLatin America (CC5-MRSA-I/II; 12.3% [30/243]). CC8 MRSAstrains were most commonly observed in the United States (75.2%[76/101]) and Europe (18.8% [19/101]) (Table 1), while the third-most-observed MRSA lineage (CC239-MRSA-III) was more com-monly noted in Asia (44.9% [22/49]) and Latin America (26.5%[13/49]), where these strains represented the second-most-com-mon clone (Table 1).

The majority (64.4% [170/264]) of clinical trial strains col-lected from the United States were CC5-MRSA-II/IV, agr type 2and PVL negative (Table 2). Among these strains, 159 and 11carried SCCmec types II and IV, respectively. While most CC5-MRSA-II strains (69.2% [110/159]) clustered within a PFGE type(USA-B) with profiles similar to or indistinguishable from that ofthe USA100 strain, the other CC5-MRSA-II isolates were distrib-uted among 14 clusters (PFGE types). All 11 CC5-MRSA-IVstrains clustered within USA-N, which showed PFGE patternssimilar to or indistinguishable from that of NRS387, a represen-tative of USA800. The second major cluster detected in the UnitedStates was CC8-MRSA-IV (28.8% [76/264]), among which 73.7%(56/76 [21.2% of all U.S. strains]) (Table 2) were PVL positive andclustered within USA-A (USA300 pattern) (Table 2). CC8-MRSA-IV, which was PVL negative, from the United Statesgrouped into nine PFGE types (six spa types), including isolateswith PFGE patterns similar to USA500 (USA-Q) (Table 2).

Figure 1 displays the percentages of CC8-MRSA-IV (USA300)compared to CC5-MRSA-II strains (agr type 2, USA100 and asso-ciated ancestors) recovered during each year of the study period.Only one and three strains included in the study were collectedduring 2004 and 2010, respectively (data not shown). Thus, these2-year periods were excluded from this analysis. A total of 64.0%(32/50) of the USA100 strains were recovered from subjects in-cluded in 2005 versus 10.0% (5/50) of USA300 strains. However,45.1% (23/51) of USA100 strains were observed in 2009 against an

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increasing 33.3% (17/51) of USA300 MRSA isolates (P � 0.0049;odds ratio [OR] � 0.21 [range, 0.06 to 0.73]).

A greater genetic diversity was observed among strains fromEuropean countries, including Russia and Turkey. Isolates weremostly CC8-MRSA-IV (34.5% [19/55]) or CC22-MRSA-IV(18.2% [10/55]) (Tables 1 and 2). CC8-MRSA-IV strains clusteredwithin three PFGE types (EUR-D, -E, and -F), among whichEUR-D (from Belgium, France, and Russia) and -E (all from Bel-gium) were also similar to the EMRSA-6 and USA500 patterns,respectively (Table 2). All CC22-MRSA-IV strains clusteredwithin EUR-B, also known as EMRSA-15, and originated fromPortugal (70.0% [7/10]), Belgium (10.0% [1/10]), Germany(10.0% [1/10]), and the United Kingdom (10.0% [1/10]). MRSAisolates from Europe associated with CC5 possessed SCCmec typesI, II, or IV. CC5-MRSA-I isolates grouped within EUR-I and orig-inated from Poland and Spain (Table 2). Four CC5-MRSA-IIstrains from Belgium showed a unique PFGE pattern (EUR-G; spatype 437), which matched that of NRS382 (USA100), while oneCC5-MRSA-IV strain each from Belgium (EUR-H; spa type 2)and France (EUR-M; spa type 1480) was detected (Table 2). Otherless prevalent lineages noted in Europe are as follows: CC398-MRSA-IV (one strain from Belgium), CC239-MRSA-III (onestrain each from France and Turkey and four isolates from Rus-sia), CC80-MRSA-IV (one strain from Greece), CC96-MRSA-III(one strain from Russia), CC45-MRSA-IV (five strains from Bel-gium and one from Germany), and CC30-MRSA-II (one strainfrom Portugal) (Table 1).

The most prevalent clone found in Latin America was CC5-MRSA-I/II (66.7% [30/45]), followed by CC239-MRSA-III(28.9% [13/45]) (Table 1). CC5-MRSA-I isolates were repre-

sented by the LAT-C (84.6% [11/13]), LAT-D (7.7% [1/13]), andLAT-E (7.7% [1/13]) PFGE types, among which LAT-C showedprofiles that matched that from a representative of the Cordobes/Chilean clone (Tables 1 and 2). LAT-C strains originated fromChile (72.7% [8/11]) and Colombia (27.3% [3/11]). Those strainsassociated with CC5-MRSA-II clustered within LAT-A (47.0% [8/17]), LAT-B (35.3% [6/17]), LAT-L (5.9% [1/17]), and LAT-N(11.8% [2/17]). All LAT-A strains were collected from Mexico,while LAT-B MRSA isolates were from Puerto Rico (50.0% [3/6]),Brazil (33.3% [2/6]), or Mexico (16.7% [1/6]). All CC239-MRSA-III strains (28.9% [13/45]) observed from Latin America origi-nated from a single country (Brazil). Other minor clones wereCC97-MRSA-IV (2.2% [1/45]) and CC8-MRSA-IV (2.2% [1/45]). The latter strain originated from Puerto Rico and demon-strated molecular and PFGE profiles (LAT-F) similar to those ofthe USA300 clone (Table 2).

Overall, strains from the APAC region were CC5-MRSA-II(50.7% [34/67]) or CC239-MRSA-III (32.8% [22/67]), PVL neg-ative, and agr types 2 and 1, respectively (Tables 1 and 2). Five andthree isolates were CC8-MRSA-IV and CC59-MRSA-IV, whichwere from South Korea (ASI-D) and Taiwan (ASI-K and -L), re-spectively (Table 3). One strain from Taiwan was CC398-MRSA-III, agr type 1, and PVL negative, while a single strain collectedfrom Hong Kong was CC45-MRSA-III, agr type 1, and PVL neg-ative (Table 2). Regarding PFGE patterns, the majority of APACMRSA strains clustered within ASI-A (26/67 [38.8%]) or -I (15/67[22.4%]). Isolates showing the ASI-A PFGE type were CC5-MRSA-II, spa type 2 or related types, and agr type 2, also known asthe New York/Japan clone, whereas those belonging to ASI-I wereCC239-MRSA-III, spa type 3, and agr type 1 (Hungarian/Brazilian

TABLE 1 Clonal distribution of MRSA isolates (unique strains) recovered from subjects enrolled in a phase IV pneumonia clinical trial for linezolid

Clonal complex

No. (%) of strains

United States Europea Latin Americab Asiac Total

CC5 170 (64.4) 9 (16.4) 30 (66.7) 34 (50.7) 243 (56.0)CC5-MRSA-I 0 (0.0) 3 (5.5) 13 (28.9) 0 (0.0) 16 (3.7)CC5-MRSA-II 159 (60.2) 4 (7.3) 17 (37.8) 34 (50.7) 214 (49.3)CC5-MRSA-IV 11 (4.2) 2 (3.6) 0 (0.0) 0 (0.0) 13 (3.0)

CC8-MRSA-IV 76 (28.8) 19 (34.5) 1 (2.2) 5 (7.5) 101 (23.3)CC239-MRSA-III 4 (1.5) 7 (12.7) 13 (28.9) 22 (32.8) 49 (11.3)d

CC45-MRSA-II/III/IV 7 (2.7)e 6 (10.9)f 0 (0.0) 1 (1.5)g 14 (3.2)CC22-MRSA-IV 0 (0.0) 10 (18.2) 0 (0.0) 0 (0.0) 10 (2.3)CC30-MRSA-II 4 (1.5) 1 (1.8) 0 (0.0) 0 (0.0) 5 (1.2)CC59-MRSA-IV 1 (0.4) 0 (0.0) 0 (0.0) 3 (4.5) 4 (0.9)CC398-MRSA-III/IV 0 (0.0) 1 (1.8)f 0 (0.0) 1 (1.5)g 2 (0.5)CC1-MRSA-IV 1 (0.4) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.2)CC9-MRSA-II 0 (0.0) 0 (0.0) 0 (0.0) 1 (1.5) 1 (0.2)CC72-MRSA-IV 1 (0.4) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.2)CC80-MRSA-IV 0 (0.0) 1 (1.8) 0 (0.0) 0 (0.0) 1 (0.2)CC96-MRSA-III 0 (0.0) 1 (1.8) 0 (0.0) 0 (0.0) 1 (0.2)CC97-MRSA-IV 0 (0.0) 0 (0.0) 1 (2.2) 0 (0.0) 1 (0.2)

Total 264 (60.8) 55 (12.7) 45 (10.4) 67 (15.4) 434 (100.0)a Includes isolates from Belgium, France, Germany, Greece, Poland, Portugal, Russia, Spain, Turkey, and the United Kingdom.b Includes isolates from Argentina, Brazil, Chile, Colombia, Mexico, and Puerto Rico.c Includes isolates from Hong Kong, South Korea, Malaysia, Singapore, and Taiwan.d Includes three CC239-MRSA-III strains from South Africa.e MRSA strains carrying SCCmec type II.f MRSA strain carrying a SCCmec type IV.g MRSA strains carrying SCCmec type III.

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TABLE 2 Epidemiologic data of baseline MRSA isolates (unique strains) recovered during a phase IV pneumonia clinical trial for linezolid

Region or country (no. tested) No. (%) SCCmec type PVLa agr type PFGE spa type(s) CC no.

APAC (67)Hong Kong (1) 1 (100.0) III � 1 ASI-J 433 45South Korea (44) 22 (50.0) II � 2 ASI-Ab 232, 1095, 1478, 1479 5

3 (6.8) II � 2 ASI-B 2 52 (4.5) II � 2 ASI-C 2, 1095 55 (11.4) IV � 1 ASI-D 451, 554 81 (2.3) II � 2 ASI-E 2 51 (2.3) III � 1 ASI-G 3 2398 (18.2) III � 1 ASI-I 3 2391 (2.3) II � 2 ASI-M 2 51 (2.3) II � 2 ASI-N 1095 5

Malaysia (6) 1 (16.7) II � 1 ASI-F 1152 85 (83.3) III � 1 ASI-I 3 239

Singapore (1) 1 (100.0) III � 1 ASI-I 3 239Taiwan (15) 4 (26.7) II � 2 ASI-A 2, 14 5

4 (26.7) III � 1 ASI-G 3 2391 (6.7) III � 1 ASI-H 3 2391 (6.7) III � 1 ASI-I 3 2392 (13.3) IV � 1 ASI-K 143, 778 591 (6.7) IV � 1 ASI-L 776 591 (13.3) III � 1 NTc 539 3981 (13.3) III � 1 NT 3 239

Europe (55)Belgium (18) 5 (27.8) IV � 1 EUR-Ad 756, 1484 45

1 (5.5) IV � 1 EUR-Be 382 221 (5.5) IV � 1 EUR-Df 1 85 (27.8) IV � 1 EUR-Eg 1, 4, 366 84 (22.2) II � 2 EUR-Gb 437 51 (5.5) IV � 2 EUR-H 2 51 (5.5) IV � 1 NT 1485 398

France (8) 6 (75.0) IV � 1 EUR-D 1 81 (12.5) III � 1 EUR-K 351 2391 (12.5) IV � 2 EUR-M 1480 5

Germany (2) 1 (50.0) IV � 1 EUR-B 382 221 (50.0) IV � 1 EUR-L 15 45

Greece (1) 1 (100.0) IV � 3 EUR-J 70 80Poland (1) 1 (100.0) I � 2 EUR-I 388 5Portugal (8) 7 (87.5) IV � 1 EUR-B 382, 816, 1473 22

1 (12.5) II � 3 EUR-C 16 30Russia (13) 3 (23.1) IV � 1 EUR-D 1 8

4 (30.8) IV � 1 EUR-F 1 85 (38.5) III � 1 EUR-K 351 2391 (7.7) III � 3 EUR-N 999 96

Spain (2) 2 (100.0) I � 2 EUR-I 1481, 1488 5Turkey (1) 1 (100.0) III � 1 EUR-K 351 239United Kingdom (1) 1 (100.0) IV � 1 EUR-B 382 22

South Africa (3) 3 (100.0) III � 1 SA-A 3 239

North America (United States; 264) 56 (21.2) IV � 1 USA-Ah 1, 59, 245, 363, 1487 8110 (41.7) II � 2 USA-Bb See footnote i 517 (6.4) II � 2 USA-C 2, 11, 24, 50, 230 51 (0.4) II � 2 USA-D 2 55 (1.9) II � 2 USA-E 2, 24, 29, 302 510 (3.8) II � 2 USA-F 2, 24, 58 54 (1.5) II � 2 USA-G 2, 11, 302 51 (0.4) IV � 1 USA-H 1 82 (0.8) II � 2 USA-I 2, 1475 51 (0.4) IV � 1 USA-J 1 81 (0.4) IV � 1 USA-K 363 8

(Continued on following page)

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clone) (Table 2). Among ASI-A strains, the vast majority (22/26[84.6%]) originated from four hospitals in South Korea, while theremaining four strains were from two hospitals in Taiwan (4/26[15.4%]). In contrast, strains clustering within ASI-I (10 sub-types) were from South Korea (8/15 [53.3%]), Malaysia (5/15[33.3%]), Singapore (1/15 [6.7%]), and Taiwan (1/15 [6.7%])(Table 2).

Antimicrobial susceptibility profiles. Table 3 displays the ac-tivities and susceptibility profiles of selected agents tested againstMRSA strains according to lineage. Overall, linezolid (MIC50, 2�g/ml), vancomycin (MIC50, 1 �g/ml), teicoplanin (MIC50, 0.5�g/ml), and daptomycin (MIC50, 0.5 �g/ml) showed stable MIC50

results when evaluated against each major CC. Exceptions wereobserved when teicoplanin was tested against the CC239 and

TABLE 2 (Continued)

Region or country (no. tested) No. (%) SCCmec type PVLa agr type PFGE spa type(s) CC no.

3 (1.1) II � 2 USA-L 2 51 (0.4) II � 2 USA-M 303 511 (4.2) IV � 2 USA-Nj 2, 23, 24, 29, 203, 693 51 (0.4) II � 2 USA-O 24 51 (0.4) IV � 3 USA-P 175 110 (3.8) IV � 1 USA-Qg 1, 7 81 (0.4) IV � 1 USA-R 139 84 (1.5) III � 1 USA-S 3 2391 (0.4) IV � 1 USA-T 1 81 (0.4) II � 2 USA-U 12 52 (0.8) IV � 1 USA-V 1 81 (0.4) II � 2 USA-W 1070 57 (2.7) II � 1/NT USA-Xd 10, 15, 62, 1472 454 (1.5) II � 3 USA-Yk 16, 33 301 (0.4) IV � 1 USA-Z 1 81 (0.4) II � 2 USA-AA 337 51 (0.4) II � 2 USA-AB 11 51 (0.4) II � 2 USA-AC 11 51 (0.4) IV � 1 USA-AEl 206 591 (0.4) IV � 1 USA-AFm 1482 722 (0.8) IV � 1 USA-AG 68, 1486 8

Latin America (45)Argentina (1) 1 (100.0) I � 2 LAT-D 58 5Brazil (18) 2 (11.0) II � 2 LAT-B 2 5

1 (5.5) I � 2 LAT-E 2 55 (27.8) III � 1 LAT-G 3 2393 (16.7) III � 1 LAT-H 3 2392 (11.1) III � 1 LAT-I 3 2392 (11.1) III � 1 LAT-J 3 2391 (5.5) III � 1 LAT-K 3 2391 (5.5) IV � 1 LAT-M 92 971 (5.5) II � 2 LAT-N 2 5

Chile (8) 8 (100.0) I � 2 LAT-Cn 442 5Colombia (3) 3 (100.0) I � 2 LAT-C 442 5Mexico (10) 8 (80.0) II � 2 LAT-A 58, 1474 5

1 (10.0) II � 2 LAT-B 58 51 (10.0) II � 2 LAT-N 29 5

Puerto Rico (5) 3 (60.0) II � 2 LAT-B 2 51 (20.0) IV � 1 LAT-Fh 1 81 (20.0) II � 2 LAT-L 2 5

a PVL, Panton-Valentine leukocidin. �, present; �, absent.b PFGE type indistinguishable from or similar to that of USA100.c NT, nontypeable.d PFGE type indistinguishable from or similar to that of USA600.e PFGE type indistinguishable from or similar to that of EMRSA-15.f PFGE type indistinguishable from or similar to that of EMRSA-6.g PFGE type indistinguishable from or similar to that of USA500.h PFGE type indistinguishable from or similar to that of USA300.i Strains represented by spa types 2, 23, 24, 25, 29, 47, 50, 58, 65, 203, 230, 268, 300, 336, 442, 487, 841, 1206, 1476, 1477, and 1483.j PFGE type indistinguishable from or similar to that of USA800.k PFGE type indistinguishable from or similar to that of USA200.l PFGE type indistinguishable from or similar to that of USA1000.m PFGE type indistinguishable from or similar to that of USA700.n PFGE type indistinguishable from or similar to that of Cordobes/Chilean.

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CC22 strains, which showed MIC50 values 2-fold higher (MIC50, 1�g/ml) and 2-fold lower (MIC50, 0.25 �g/ml) than those of theother CC isolates, respectively. In addition, CC22 strains exhibiteddaptomycin MIC values (MIC50/90, 0.25/0.5 �g/ml) 2-fold lowerthan those of other tested CC strains (MIC50/90, 0.5/1 �g/ml). One

MRSA strain from the United States exhibited a nonsusceptiblephenotype to vancomycin (MIC, 4 �g/ml; intermediate) and clus-tered within USA-N (USA800). Tetracycline (MIC50, 0.25 �g/ml[�92.9% susceptible]) was active when tested against CC8, CC45,and CC22 strains, while 98.0% of CC239 MRSA isolates were

FIG 1 Proportion of unique CC8-MRSA-IV (PVL-positive; USA300) and CC5-MRSA-II strains recovered from subjects enrolled in the United States during thestudy period. CC8-MRSA-IV strains were agr type 1 and PVL positive and clustered within the PFGE USA-A group (USA300). CC5-MRSA-II strains were agrtype 2 and PVL negative.

TABLE 3 Antimicrobial activity and susceptibility results of selected drugs tested against baseline MRSA clinical isolates (unique strains) by clonalcomplex recovered during a phase IV pneumonia clinical trial for linezolid

Antimicrobial agent

MIC50/MIC90, �g/ml (% susceptible) bya:

CC USA clone

CC5 (n � 243) CC8 (n � 101) CC239 (n � 49) CC45 (n � 14) CC22 (n � 10)USA300(n � 56)

USA100(n � 110)

Linezolid 2/4 (100.0) 2/2 (100.0) 2/2 (100.0) 2/4 (100.0) 2/2 (100.0) 2/2 (100.0) 2/4 (100.0)Vancomycin 1/1 (99.6) 1/1 (100.0) 1/1 (100.0) 1/2 (100.0) 1/1 (100.0) 1/1 (100.0) 1/1 (100.0)Teicoplanin 0.5/2 (100.0) 0.5/1 (100.0) 1/2 (100.0) 0.5/2 (100.0) 0.25/0.5 (100.0) 0.5/1 (100.0) 0.5/1 (100.0)Daptomycin 0.5/1 (100.0) 0.5/1 (100.0) 0.5/1 (100.0) 0.5/1 (100.0) 0.25/0.5 (100.0) 1/1 (100.0) 0.5/1 (100.0)Erythromycin �64/�64 (1.2) 64/�64 (8.9) �64/�64 (0.0) 0.5/�64 (57.1) �64/�64 (40.0) 64/�64 (1.8) �64/�64 (0.0)Clindamycin �64/�64 (4.5)b 0.25/�64 (61.4)c �64/�64 (0.0)d 0.12/�64 (57.1)e 0.12/�64 (40.0)f 0.25/�64 (85.7)g �64/�64 (0.0)h

Gatifloxacin 8/�16 (4.9) 2/16 (28.7) 4/16 (0.0) 4/8 (7.1) 8/16 (0.0) 2/8 (32.1) 8/�16 (2.7)Tetracycline 0.5/64 (87.3) 0.25/1 (93.1) 32/�64 (2.0) 0.25/1 (92.9) 0.25/0.25 (100.0) 0.25/0.5 (98.2) 0.5/1 (98.2)Q/Di 0.5/1 (99.6) 0.25/0.5 (99.0) 0.5/0.5 (100.0) 0.25/0.5 (100.0) 0.25/0.25 (100.0) 0.5/0.5 (100.0) 0.5/0.5 (100.0)TMP/SMXj 0.12/0.25 (98.0) 0.06/0.5 (90.1) 16/�32 (18.4) 0.12/0.5 (92.9) 0.06/0.06 (100.0) 0.06/0.12 (100.0) 0.12/0.25 (95.5)a MICs were interpreted according to the M100-S22 document (13).b Nonsusceptible strains are represented by 73.0% and 22.5% of constitutive and inducible resistance phenotypes, respectively.c Nonsusceptible strains are represented by 32.6% and 6.0% of constitutive and inducible resistance phenotypes, respectively.d Nonsusceptible strains are represented by 81.6% and 18.4% of constitutive and inducible resistance phenotypes, respectively.e Nonsusceptible strains are represented by 42.9% of constitutive resistance phenotypes.f Nonsusceptible strains are represented by 20.0% and 40.0% of constitutive and inducible resistance phenotypes, respectively.g Nonsusceptible strains are represented by 10.7% and 3.6% of constitutive and inducible resistance phenotypes, respectively.h Nonsusceptible strains are represented by 66.4% and 33.6% of constitutive and inducible resistance phenotypes, respectively.i Q/D, quinupristin-dalfopristin.j TMP/SMX, trimethoprim-sulfamethoxazole.

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tetracycline resistant. Trimethoprim-sulfamethoxazole (TMP/SMX) showed potent activity (MIC50/90, 0.06 to 0.12/0.06 to 0.5�g/ml [�90.1% susceptible]) against all major CC groups, exceptfor CC239 strains (MIC50/90, 16/�32 �g/ml [18.4% susceptible]).

USA300 strains were susceptible to most antimicrobial agentstested (�98.2% susceptible), except for erythromycin (1.8% sus-ceptible) and gatifloxacin (32.1% susceptible), which were inac-tive. Clindamycin demonstrated a potent MIC50 result (0.25 �g/ml) when tested against MRSA belonging to the USA300 lineage.However, 10.7 and 3.6% of isolates exhibited, respectively, consti-tutive and inducible resistance phenotypes for clindamycin, re-sulting in a suboptimal overall susceptibility result (85.7% suscep-tible) and an elevated MIC90 result (�64 �g/ml). CC5 isolates andtheir regional U.S. subset (USA100) showed a similar susceptibil-ity profile, except for tetracycline. When tested against all CC5strains, tetracycline demonstrated MIC90 results (MIC50/90, 0.5/64�g/ml) higher than those of the USA100 subset (MIC50/90, 0.5/1�g/ml), which translated into slightly different susceptibility pro-files (87.3 versus 98.2%, respectively). Among tetracycline-resis-tant CC5 strains, 27 of 31 (87.1%) originated from four hospitalsin South Korea, and 19 of 27 (70.4%) belonged to a unique PFGEtype (ASI-A; data not shown). Other tetracycline-resistant CC5isolates were from the United States (three strains) or Belgium(one strain).

Overall, 14.5% (63/434) isolates were characterized as hVISA,according to the employed method. The countries with the high-est hVISA rates (countries with �10 strains) were as follows:South Korea (61.4% [27/44]), Brazil (40.4% [8/18]), Russia(23.1% [3/13]), Mexico (20.0% [2/10]), Taiwan (13.3% [2/15]),and the United States (5.3% [14/264]) (data not shown). The oc-currences of hVISA strains were higher within CC30-MRSA-IIstrains (40.0% [2/5]), followed by CC239-MRSA-III (22.4%; [11/49]), CC45-MRSA-II/III (21.4% [3/14]), CC5-MRSA-I/II/IV(16.5% [40/243]), and CC8-MRSA-IV (6.9% [7/101]) strains.Among CC5 hVISA strains, the majority were collected fromSouth Korea (65.0% [26/40]) and belonged mostly (76.9% [20/26]) to a unique PFGE type (ASI-A). Two CC30 hVISA isolateswere recovered from the United States and Portugal (one each),while CC45 hVISA strains originated from three U.S. hospitalsand clustered within USA600 (USA-X). hVISA strains associatedwith CC239-MRSA-III were collected from scattered countries,such as Brazil (six strains), Russia (three strains), Malaysia (onestrain), and Turkey (one strain). Differences in the susceptibilityprofiles between hVISA and non-hVISA strains were not ob-served.

DISCUSSION

The majority (56.0%) of the microbiologically evaluable baselineMRSA isolates responsible for NP and included in this study be-longed to CC5-MRSA-I/II/IV. However, when analyzing the prev-alence of MRSA lineages according to the geographic regions, twomajor clones predominated in each area. In the APAC and LatinAmerica regions, isolates associated with CC5-MRSA-I/II pre-vailed, followed by CC239-MRSA-III. Isolates responsible for NPamong U.S. hospitals were mostly associated with CC5-MRSA-II/IV and CC8-MRSA-IV, while in Europe, a greater clonal diver-sity was observed and CC8-MRSA-IV predominated, followed bysimilar occurrences of CC22-MRSA-IV and CC5-MRSA-I/II/IV.Overall, these lineages are comprised of the so-called pandemicclones, such as ST239-MRSA-III (Brazilian/Hungarian), ST5-

MRSA-II (New York/Japan), ST5-MRSA-IV (pediatric), ST22-MRSA-IV (UK-EMRSA-15) and ST8-MRSA-IV (USA300,USA500, and UK-EMRSA-2 and -6) (18, 33, 36, 41).

In the United States, ST5-MRSA-II (USA100) has been themost common nosocomial MRSA clone, which has been impli-cated for approximately 60 to 68% of hospital-onset or hospital-acquired infections (HA-MRSA) (10, 27). The rate (60.2% [159/264]) of CC5-MRSA-II (USA100) strains observed in this studycorroborates those previously reported and confirmed USA100 asan important cause of HA-MRSA infections. However, studiesreported that USA300 strains are now responsible for 15.7 to20.0% of the invasive HA-MRSA infections in the United States(10, 22, 27). An overall USA300 prevalence rate (21.2%) similar tothose reported previously was observed in this study. In addition,the proportion of USA300 strains appears to have increasedslightly throughout the study period (Fig. 1). Overall, rates ofUSA300 were 10.0 to 22.4% during 2005 to 2008, while CC5-MRSA-II represented 58.7 to 67.2% of strains included. In 2009,USA300 and CC5-MRSA-II strains comprised 33.3 and 45.1% ofall isolates, respectively. These results support previous data indi-cating that the proportion of infection cases caused by USA300has increased and that it may be slowly replacing the traditionalHA-MRSA strains (31, 39). However, this piece of data needs to beanalyzed carefully, since the inclusion of the isolates in this pneu-monia trial did not follow prevalence mode design criteria.

Grundmann et al. (19) recently reported the detection of ge-netic diversity among MRSA strains causing invasive infections inEurope, with CC5 (30.3%) being the most prevalent lineage, fol-lowed by CC22 (16.9%), CC8 (15.3%), and CC239 (5.0%).Changes within the MRSA population have been well docu-mented in European countries, such as Germany (2), Ireland andSpain (38, 47), and Portugal (1, 4). Additional reports have de-scribed alterations in the MRSA epidemiology among other Eu-ropean countries (6, 9, 14, 16). The results reported here corrob-orate those reported by Grundmann et al. (19), who showed agreater genetic diversity and similar distribution of MRSA CCs. Inaddition, the results described here and elsewhere indicate thatpreviously prevalent clones, such as ST247-MRSA-I (Iberian;CC8), ST228-MRSA-I (Southern Germany; CC5), ST239-MRSA-III (CC239; Brazilian/Hungarian), and ST45-MRSA-IV (CC45;Berlin, Germany), have decreased occurrences or have no longerbeen detected (15).

In contrast, Alp et al. (3) described the persistence of ST239-MRSA-III among eight university hospitals in Turkey over a 10-year period. Previous reports have also described a significantdominance (�80.0%) of ST239-MRSA-III within Russian hospi-tals (5, 52, 54). Interestingly, this study detected a higher preva-lence (53.8%) of CC8-MRSA-IV causing NP in Russia, whileST239-MRSA-III represented 38.5% of strains from this region.We are unaware of any previous report describing this clonal al-teration in the HA-MRSA strain population in Russia, except foran earlier publication reporting that 80.0% of the MRSA strainscausing skin and skin-structure infections during a phase IV clin-ical trial in this country were CC8-MRSA-IV (30), although ahigher CC8-MRSA-IV rate might be expected among strains re-sponsible for skin infections. Still in Europe, one strain belongingto a livestock-associated clone (spa type 1485/ST398) was col-lected from Belgium and one CC80-MRSA-IV strain (spa type 70;PVL positive) was noted in Greece. The latter has been implicated

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as an important CA-MRSA clone in Europe, but it has also beenresponsible for documented HA-MRSA infections (26).

CC5-MRSA-I, CC5-MRSA-II, and CC239-MRSA-III predom-inated among Latin American countries and represented 95.6% ofall strains. CC239-MRSA-III was the predominant clone found inthis region, but recent investigations demonstrated that CC239-MRSA-III has been partially replaced by the Cordobes/Chilean(CC5-MRSA-I) and New York/Japan (CC5-MRSA-II) clones (30,42). However, CC239-MRSA-III and CC5-MRSA-I were detectedequally in the South America, while CC5-MRSA-II was most fre-quently observed in Mexico. Moreover, the latter has been thepredominant clone in Mexico after 2002, when this lineage dis-placed ST30-MRSA-IV (30). More recently, the CC8-MRSA-IV(USA300) clone has also emerged in several South Americancountries (30, 40, 42). One USA300 strain (PVL-positive) col-lected from Puerto Rico was observed here.

Among those MRSA strains originating from the APAC region,the CC5-MRSA-II and CC239-MRSA-III clones represented83.5% of all strains included. Previous investigations have dem-onstrated the predominance of these clones in China (28), Malay-sia (17), and other Asian countries (23). Surprisingly, a great pro-portion of hVISA strains detected by the method employedoriginated from South Korea (27/63 [42.9%]), and 61.4% of theMRSA strains from this country were considered hVISA. In addi-tion, the vast majority (26/27 [96.3%]) of these hVISA strainswere CC5-MRSA-II. An elevated hVISA rate (37.7%) has beenpreviously reported by Park et al. (37) among isolates responsiblefor bacteremia in a hospital in South Korea, and several studiesalso reported that a greater number of hVISA strains were associ-ated with CC5-MRSA-II (11, 37, 44, 48). The clinical significanceof hVISA remains controversial (37, 44), but the overall rate ofhVISA strains (14.5%) observed in this study appears elevated andmostly driven by a high rate noted in South Korea due to thedissemination of genetically related strains (PFGE, ASI-A). How-ever, the hVISA-positive results obtained here were not confirmedby a second Etest macromethod testing result or population anal-ysis profile-area under the curve (PAP-AUC). Some key pheno-typic characteristics were observed according to the MRSA clonallineage. Most notably, CC239-MRSA-III strains were resistant toerythromycin, clindamycin, gatifloxacin, tetracycline, and TMP/SMX, which has been a characteristic profile observed among iso-lates belonging to this clone (4). Although USA300 strains werepreviously described as susceptible to fluoroquinolones and clin-damycin (29), fluoroquinolone (gatifloxacin) resistance has be-come commonplace among these strains (29, 30, 50) and wasconfirmed here (67.9% resistance). In contrast, clindamycin resis-tance remains uncommon among USA300 strains (27, 30). How-ever, this study (14.3% resistance) and results from previous re-ports suggest that clindamycin resistance may be emerging amongUSA300 strains (27, 50).

It is important to mention that some countries, such as HongKong, Singapore, Germany, Greece, Poland, Spain, Turkey, theUnited Kingdom, South Africa, Argentina, and Colombia, con-tributed limited numbers of strains (�5 isolates) to this clinicaltrial study, which may compromise some epidemiological conclu-sions and/or comparison with other investigations. However, thisstudy provides some insights regarding the MRSA population re-sponsible for NP in most evaluated regions. In summary, the datadocumented a clear dominance of the CC5 lineage and confirmedan increased prevalence of USA300 strains causing NP in the

United States. In addition, higher fluoroquinolone (67.9%) andclindamycin (14.3%) resistance rates (inducible and constitutive)were observed among USA300 strains. Moreover, a shift in theMRSA population was observed in Russia, with CC8-MRSA-IVreplacing CC239-MRSA-III, and a high prevalence of hVISAstrains was observed in the APAC region, especially South Korea.

ACKNOWLEDGMENTS

We thank the following staff members at JMI Laboratories (North Liberty,IA) for technical support and manuscript assistance: S. Benning, M. Cas-tanheira, A. Costello, S. Farrell, and P. R. Rhomberg. We also thank Her-minia de Lencastre and Keiichi Hiramatsu for providing S. aureus strainsHDE288 and WIS, respectively, used in this study as positive controls(SCCmec types V and VI) during the SCCmec typing procedures.

This study was sponsored by the Pfizer, Inc., Specialty Care BusinessUnit, Collegeville, PA. JMI Laboratories, Inc. (R.E.M., L.M.D., D.J.F., andR.N.J.) received research and educational grants in 2009 to 2011 fromAchaogen, Aires, American Proficiency Institute (API), Anacor, Astellas,AstraZeneca, Bayer, bioMérieux, Cempra, Cerexa, Cosmo Technologies,Contrafect, Cubist, Daiichi, Dipexium, Enanta, Furiex, GlaxoSmithKline,Johnson & Johnson (Ortho McNeil), LegoChem Biosciences Inc., MeijiSeika Kaisha, Merck, Nabriva, Novartis, Paratek, Pfizer (Wyeth), PPDTherapeutics, Premier Research Group, Rempex, Rib-X Pharmaceuticals,Seachaid, Shionogi, Shionogi USA, the Medicines Co., Theravance, Ther-moFisher, TREK Diagnostics, Vertex Pharmaceuticals, and some othercorporations.

Some JMI Laboratories employees are advisors and/or consultants forAstellas, Cubist, Pfizer, Cempra, Cerexa-Forest, J&J, and Theravance.D.S.S. and B.S. have no potential conflicts of interest to declare.

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