Molecular Epidemiology and Prevalence of Macrolide Efflux Genes mef(a) and mef(e) in Streptococcus pneumoniae Obtained in Canada from 1997 to 2002

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1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Mar. 2005, p Vol. 49, No /05/$ doi: /aac Copyright 2005, Amerian Soiety for Mirobiology. All Rights Reserved. Moleular Epidemiology and Prevalene of Marolide Efflux Genes mef(a) and mef(e) in Streptoous pneumoniae Obtained in Canada from 1997 to 2002 Aleksandra K. Wierzbowski, 1,2 Dean Swedlo, 3 Dave Boyd, 4 Mihael Mulvey, 4 Kim A. Nihol, 1,2 Daryl J. Hoban, 1,2 and George G. Zhanel 1,2,3 * Department of Medial Mirobiology, Faulty of Mediine, University of Manitoba, 3 Departments of Mediine 2 and Clinial Mirobiology, 1 Health Sienes Centre, and Health Canada, 4 Winnipeg, Manitoba, Canada Reeived 30 Marh 2004/Returned for modifiation 26 June 2004/Aepted 28 Otober 2004 One hundred forty M phenotype Streptoous pneumoniae isolates were evaluated by PCR-restrition fragment length polymorphism, serotyping, and pulsed-field gel eletrophoresis. Moleular genotyping revealed that the predominant marolide resistane mehanism in S. pneumoniae in Canada is mef(e) and resistane dissemination is due to both spread of the geneti element MEGA as well as lonal dissemination of peniillinand/or marolide-resistant strains. Low-level resistane to 14- and 15-member marolides and suseptibility to linosamides and streptogramin B (M phenotype) in Streptoous pneumoniae (16) are onferred by the presene of a membrane-bound efflux protein, enoded by the mef gene mef(a) or mef(e) (1, 4, 5, 10, 13). The geneti elements arrying the mef genes, MEGA and Tn1207.1, have reently been desribed and are well desribed in the literature (5, 14). Clinial isolates of S. pneumoniae with redued suseptibility to marolides may arise through the horizontal aquisition of the geneti element arrying the mef gene or through lonal expansion of resistant strains. Unlike peniillin-resistant S. pneumoniae, the moleular epidemiology of marolide-resistant S. pneumoniae in Canada has not been examined extensively (9, 12). The aim of this study was to identify the prevalene of mef genes in a large olletion of S. pneumoniae strains isolated in Canada from 1997 to 2002 and to determine the geneti relatedness between mef(e)- and mef(a)-arrying isolates. One hundred forty marolide-resistant (erythromyin MIC, 1 g/ml) and lindamyin-suseptible (MIC, 0.25 g/ml) (M phenotype) S. pneumoniae linial isolates were seleted from among 6,991 isolates olleted between 1997 and 2002 as part of an ongoing annual national surveillane study, the Canadian Respiratory Organism Suseptibility Study (7). Study isolates were olleted from medial enters in 9 out of 10 Canadian provines. Isolates for this study were olleted from respiratory trat speimens only and were limited to one isolate per patient. Erythromyin, lindamyin, and peniillin suseptibilities were determined by the NCCLS M7-A4 broth mirodilution method (11). MIC interpretive standards for erythromyin, lindamyin, and peniillin were defined aording to the NC- CLS breakpoints for 2000 (11). The presene of the mef gene was determined by a previously desribed PCR assay that did * Corresponding author. Mailing address: Clinial Mirobiology, Health Sienes Centre, MS Sherbrook St., Winnipeg, Manitoba R3A 1R9, Canada. Phone: (204) Fax: (204) ggzhanel@ps.mb.a. not distinguish between the two variants (15). Disrimination between mef(a) and mef(e) was performed by PCR-restrition fragment length polymorphism analysis aording to a previously desribed protool (5). The relatedness among mef(a)- and mef(e)-arrying isolates was examined by pulsed-field gel eletrophoresis (PFGE) by published methods (9, 12). Genomi DNAs were digested with SmaI prior to eletrophoresis with a ontour-lamped homogenous eletri field apparatus (CHEF DRIII; Bio-Rad Laboratories, Herules, Calif.). Isolates that differed by one to three bands were onsidered lonally related (12). DNA patterns were digitized for analysis with Moleular Analyst (Fingerprinting Plus, version 1.12) software. A dendrogram was alulated by the unweighted pair group method with arithmeti averages. Isolates were serotyped by the apsular swelling in antisera (Quellung reation) from the Statens Serum Institut (Copenhagen, Denmark) aording to the manufaturer s instrutions. The distribution of the mef(a) and mef(e) variants of the mef gene among pneumooal isolates is summarized in Table 1. Among a sample of 140 M phenotype S. pneumoniae isolates, 133 (95%) isolates arried the mef(e) gene and 7 (5%) isolates arried the mef(a) gene. Both mef(e)- and mef(a)- arrying isolates were resistant to erythromyin (MIC, 1 g/ ml); however, all mef(a)-arrying isolates were suseptible to other antibiotis, inluding peniillin (peniillin-suseptible MIC, 0.06 g/ml), while 66% (92 of 140) of mef(e)-arrying TABLE 1. Prevalene of mef(e) and mef(a) S. pneumoniae genotypes in Canada between 1997 and 2002 Yr of isolation No. (%) of isolates Total With mef(e) With mef(a) Total (95) 7 (5) 1257

2 1258 NOTES ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Dendrogram depiting the geneti relatedness of 140 linial S. pneumoniae isolates on the basis of PFGE results Continued on following page isolates demonstrated redued suseptibility to peniillin (MIC, 0.12 g/ml). Fourteen unique apsular serotypes were identified among the 140 isolates. Predominant serotypes inluded 12F (14%), 19F (13%), 23F (12%), and 14 (12%). Seventy-four of 140 (53%) mef-positive S. pneumoniae isolates belonged to serotypes 6B, 9V, 18C, 19F, 14, and 23F. One hundred seven of the 140 (76%) mef-positive S. pneumoniae isolates studied belonged to serotypes 8, 14, 11A, 12F, 18C, 19F, 23F, 6B, and 9V. Nontypeable strains aounted for 18% (25 of 140). Based on

3 VOL. 49, 2005 NOTES 1259 TABLE 2 Continued Continued on following page the inidene of the partiular serotypes among our S. pneumoniae population, we found that the urrently available heptavalent and 23-valent pneumooal vainations would provide potential overage for 53 and 76% of the isolates, respetively. This vaine overage was greater for peniillinresistant isolates, inreasing to 77 and 86%, respetively. All S. pneumoniae strains were typeable by PFGE; the results are summarized in Table 2. Moleular analysis by PFGE with SmaI-restrited hromosomal DNA revealed 127 distint DNA profiles among 140 marolide-resistant S. pneumoniae isolates. One hundred twenty-two unique genotypes were found among the 133 mef(e)-arrying isolates. Dendrogram analysis of the mef(e)-arrying isolates identified 19 lusters ( 85% geneti relatedness), eah ontaining between 2 and 11 isolates, whih aounted for 47% (63 of 133) of the mef(e) S. pneumoniae isolates. The majority of the mef(e) isolates within lusters were oresistant to peniillin. Among the 19 lusters, 13 (68%) demonstrated luster-speifi serotypes. Isolates

4 1260 NOTES ANTIMICROB. AGENTS CHEMOTHER. TABLE 2 Continued Downloaded from a Provine: AB, Alberta; BC, British Columbia; MB, Manitoba; NB, New Brunswik; NS, Nova Sotia; ON, Ontario; QC, Quebe; SK, Saskathewan. b ERY, erythromyin; PEN, peniillin. NT, nontypeable. on June 15, 2018 by guest within these 13 lusters belonged to serotype 6B (3 lusters), 6A (2 lusters), 12F (2 lusters), 23F (2 lusters), 11A (1 luster), 9V (1 luster), 18C (1 luster), or 14 (1 luster). Among the seven mef(a)-arrying isolates, five unique genotypes were found. Dendrogram analysis identified one luster ( 80% geneti relatedness) whih aounted for 86% (six of seven) of the mef(a) S. pneumoniae isolates. All seven mef(a)-arrying isolates belonged to serotype 14, and all were suseptible to peniillin. The higher prevalene of the mef(e) variant found among the marolide-resistant S. pneumoniae population in Canada adds to the onlusion that the mef(e) gene is more prevalent in North Ameria than in Europe (1, 4 6, 10, 13). It has been proposed that the inidene of the two variants ours as a result of the arriage rates of Streptoous pyogenes and viridans group streptooi, whih arry mef(a) and mef(e), respetively (6). The low inidene of mef(a)-positive S. pyogenes isolates might explain the low inidene of mef(a) among mef-positive S. pneumoniae isolates in Canada (8); however, as the inidene of marolide-resistant mef(a)-arrying S. pyogenes isolates appears to be inreasing, it might affet the inidene of the mef(a) gene in the S. pneumoniae population (8). A low inidene of the mef(a) gene in the S. pneumoniae population might also be due to greater ability of the MEGA mef(e)-ontaining element to spread horizontally in S. pneumoniae ompared to the Tn mef(a)-ontaining element, whih has been referred to as defetive (5, 14). Genotyping of 133 mef(e)-arrying S. pneumoniae isolates showed that approximately half of the isolates were genetially related and the other half remained genetially unrelated (Ta-

5 VOL. 49, 2005 NOTES 1261 ble 2). This indiates that marolide resistane assoiated with the geneti element MEGA is a result of both lonal dissemination (vertial) as well as spread of the geneti element (horizontal). Further analysis showed that the majority of the isolates that are genetially related (luster) were also resistant to peniillin, while the majority of the genetially unrelated mef(e)-arrying isolates remained suseptible to peniillin, suggesting that peniillin resistane is driving the lonal spread of the MEGA element. Genotyping of seven mef(a)-arrying S. pneumoniae isolates demonstrated geneti relatedness among these isolates. As the isolates are not related in terms of date and loation of isolation, the presene of a single luster ontaining six of the seven mef(a) strains indiates that resistane due to the geneti element Tn is ourring through the expansion of a single peniillin-suseptible serotype 14 lone that has aquired the mef(a) gene. These PFGE patterns are similar to those of other investigators who found that mef(e) strains did not appear to be related by PFGE, while mef(a) strains were genetially indistinguishable (5). In onlusion, although both mef(e) and mef(a) genes were present in Canadian isolates of S. pneumoniae, the majority of isolates sreened were mef(e). This is in ontrast to the European studies that reported mef(a) as the major efflux gene among their S. pneumoniae isolates (1, 4, 5, 10, 13). Similar to the findings of others (5), all mef(a) isolates found in our study belonged to serotype 14, and unlike some studies (1, 10) that identified mef(a) isolates sattered over seven different serotypes (23F,19A,3,6B,15B,33A, and 9), no other serotypes were found in our mef(a) S. pneumoniae isolates. The mef(e)-arrying isolates, in onordane with other studies (1, 5, 10), were more sattered (over 14 serotypes). Beause mef(a) and mef(e) in pneumooi appear to originate from different essentially invariant elements, aquired from group A and viridans group streptooi, respetively, and beause aquisition of either gene may have impliations regarding streptooal physiology and antibioti resistane, partiularly peniillin, it remains important for mef(e) and mef(a) to be onsidered independently and to ontinue to doument their horizontal and vertial spread within S. pneumoniae as this may lead to a better understanding of the spread of marolide-resistant S. pneumoniae. In addition, sine both mef(e) and mef(a) have been found in S. pyogenes and Streptoous agalatiae in at least one study (2, 3), it would be interesting to see whether a similar mixed ourrene of mef(a) and mef(e) is also present in Canadian M phenotype strains of S. pyogenes and viridans group streptooi and this may lead to a better understanding of the dissemination of marolide resistane in Canada. REFERENCES 1. Amezaga, M. R., P. E. 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