Distribution of capsular serotypes and macrolide resistance mechanisms among macrolide-resistant Streptococcus pneumoniae isolates in Korea

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Diagnostic Microbiology and Infectious Disease 63 (2009) 213 – 216 www.elsevier.com/locate/diagmicrobio

Antimicrobial Susceptibility Studies

Distribution of capsular serotypes and macrolide resistance mechanisms among macrolide-resistant Streptococcus pneumoniae isolates in Korea Songmee Bae⁎, Kwangjun Lee Division of Bacterial Respiratory Infections, Center for Infectious Diseases, National Institute of Health, Korea Centers of Disease Control and Prevention, Seoul 122-701, South Korea Received 11 April 2008; accepted 3 October 2008

Abstract The mechanism of macrolide resistance was investigated in 251 Streptococcus pneumoniae isolates with reduced susceptibility to erythromycin (erythromycin-resistant S. pneumoniae [ERSP]) collected during the period from 2000 to 2004 in Korea. Among these strains, erm(B) was the most prevalent pneumococcal macrolide resistance genotype. In particular, dual mechanisms of both the erm(B) and mef (A) genes were detected in 77 (30.7%) of 251 ERSP isolates. All of the 77 ERSP isolates, with dual erm(B) and mef (A), showed resistance to 2 or more antimicrobial agents, including penicillin, cefotaxime, clindamycin, tetracycline, and levofloxacin. Serotypes 19F, 23F, 19A, 14, 11A, 6B, 6A, and 9V accounted for 73.3% of ERSP isolates. Most of the strains with serotypes 19F (77.2%) or 19A (87.5%) had the dual erm (B) and mef (A) genes. The prevalence and spread of serotype 19F or 19A isolates may have contributed to the high rate of macrolideresistant pneumococci in Korea. In addition, we identified the emergence of a macrolide-nonsusceptible nonvaccine serotype 35B, which carries mef(A)-mediated resistance to macrolides. These findings emphasize the need for a continuous monitoring of macrolide-resistant S. pneumoniae in Korea. © 2009 Published by Elsevier Inc. Keywords: Macrolide resistance; serotype; S. pneumoniae

1. Introduction Streptococcus pneumoniae is an important bacterial pathogen causing a variety of community-acquired respiratory tract infections (Alonso et al., 1995). High frequency of antimicrobial resistance among S. pneumoniae has become a clinical problem in Korea (Song et al., 1999). Particularly, penicillin-resistant (N80%) and macrolide-resistant (N60%) pneumococci have become a common finding in patients. Macrolide resistance in S. pneumoniae is mediated by 2 major mechanisms: ribosomal modification and active efflux (Farrel et al., 2002; Farrel et al., 2005). The methylation of ribosomal target sites is mediated by methylases, encoded by the erm gene. This modification leads to broad crossresistance to macrolides, lincosamides, and streptogramin B antibiotics (MLSB phenotype). The active drug efflux pump, encoded by the mef gene, has been associated with a ⁎ Corresponding author. Tel.: +82-2-380-2135; fax: +82-2-385-8043. E-mail addresses: [email protected] (S. Bae), [email protected] (K. Lee). 0732-8893/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.diagmicrobio.2008.10.002

resistance pattern, the M phenotype, to only 14- and 15member ring macrolides. Recent reports have shown the emergence of erythromycin-resistant isolates with the dual erm(B) and mef (A) genotypes in several countries (Farrel et al., 2002; Farrel et al., 2005; McGee et al., 2001; Waites et al., 2003). These strains have been shown to exhibit high rates of resistance to multiple antimicrobial agents, and their spread might represent a serious public health problem. In this study, erythromycin-resistant S. pneumoniae (ERSP) isolates were investigated for their resistance phenotypes and genotypes. In addition, all ERSP strains were further studied for a correlation between their serotypes and the presence of genes for macrolide resistance. 2. Materials and methods 2.1. Bacterial isolates A total of 251 S. pneumoniae with reduced susceptibility to erythromycin (MIC, ≥1 μg/mL) were selected among clinical isolates collected from 2 diagnostic laboratories located at Seoul between 2000 and 2004: 115 isolates

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between 2000 and 2001, 56 from 2002, and 80 between 2003 and 2004. These laboratories were requested to provide all S. pneumoniae isolated from patients with respiratory tract infections (community-acquired pneumonia, acute otitis media, acute bacterial sinusitis, etc.). All strains were isolated from clinical specimens including sputum, throat, blood, cerebrospinal fluid, or other respiratory sites. Identification of S. pneumoniae was confirmed by colony morphology, α-hemolysis, Gram staining, optochin susceptibility, bile solubility, and agglutination in the Pneumo-kit slidex test (bioMérieux, France). 2.2. Serotyping Serotyping was performed using the capsular swelling procedure (quellung reaction); the serum pool and serotype/group-specific antisera were provided by the Staten Seruminstitut (Copenhagen. Denmark) (Lund and Henrichsen, 1978). 2.3. Antimicrobial susceptibility testing Minimum inhibitory concentrations of erythromycin and clindamycin were determined by the broth microdilution method in cation-adjusted Mueller–Hinton broth supplemented with 3% lysed horse blood according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (2005). S. pneumoniae ATCC49619 was used as a reference strain. 2.4. Macrolide resistance phenotypes The double-disk method with erythromycin (15 μg) and clindamycin disks (2 μg) (Oxoid, UK), as well as MIC data, was used for determination of macrolide resistance phenotypes. Blunting of the clindamycin inhibition zone near the erythromycin disk was characteristic of an iMLSB phenotype, and resistance to both erythromycin and clindamycin indicated a cMLSB phenotype. Susceptibility to clindamycin with no blunting was observed in the M resistance phenotype (M type) (Waites et al., 2000). 2.5. Detection of macrolide resistance determinants by polymerase chain reaction Chromosomal DNA was extracted with a QIAmp Tissue kit (QIAGEN, Germantown, MD, USA) according to the manufacturer's protocol. The presence of the resistance mechanisms for both MLSB (erm) and M resistance (mef) was analyzed using polymerase chain reaction. Erythromycin resistance genes erm(A) and erm(B) were screened using primer sets designed by Sepälä et al. (1998) and by Sutcliffe et al. (1996), respectively. The mef(A) gene was amplified using the primer pair described by Sutcliffe et al. (1996). 2.6. Statistical analysis Statistical analysis was performed by a χ2 test using SAS software version 9.2 (SAS Institute, Cary, NC), as appropriate.

3. Results All 251 ERSP isolates were isolated from sputum (60.2%), throat (14.3%), blood (10.8%), pus (3.2%), ear (2.4%), cerebrospinal fluid (2.0%), bronchoalveolar lavage (1.6%), wound (1.2%), and others (4.4%). These strains were recovered from patients with age ranging from 0 to 101 years (Interquartile Range [IQR] = 53, mean = 47.3 years, median = 59 years), except the 14 patients with no information; 55 (21.9%) were from children younger than 10 years, 9 (3.6%) from 10 to 19 years, 24 (9.5%) from 20 to 49 years, and 149 (59.4%) from adults 50 years and older. The phenotype of macrolide resistance of all 251 ERSP isolates were determined using the disk diffusion method with a 15-μg erythromycin disk and a 2-μg clindamycin disk. Two hundred fourteen (85.3 %) were assigned to the cMLSB phenotype and 37 (14.7%) to the M phenotype (Table 1). Of the 251 ERSP, erm(B) and mef(A) genes were detected in 214 (85.3%) and 114 (45.4%) isolates, respectively, and erm(B) gene was more prevalent than mef(A) gene in macrolide-resistant genotype by χ2 test (P = 0.000). There were 77 (30.7%) isolates that had both erm(B) and mef(A) genotypes. All isolates with the MLSB phenotype had the erm (B) gene. For the 214 pneumococci with cMLSB phenotype, 137 (64.0%) isolates carried the erm(B) gene alone and 77 (36.0%) had both the erm(B) and mef(A) genes. All 37 M phenotype strains carried the mef(A) gene only. Thirty-four different serotypes were identified among all ERSP isolates (Table 2). The predominant serotypes were 19F (22.7%), 23F (9.9%), 19A (9.5%), 14 (7.2%), 11A (6.8%), 6B (6.4%), 6A (5.6%), and 9V (5.2%), accounting for 184 (73.3%) of the 251 ERSP. Among the 137 erm(B) isolates, the serotype 23F, 14, 6B, 6A, 9V, and 11A was most frequently identified. The mef(A) gene was most prevalent in isolates with serotype 19F and 35B; most of the dual erm(B) + mef(A) isolates were of serotype 19F or 19A. Strikingly, 44 (77.2%) of 57 isolates with serotype 19F carried both erm(B) and mef(A), and 21 (87.5%) of 24 isolates with serotype 19A also had both the erm(B) and mef(A) genotypes. All of the 77 ERSP with dual erm(B) and mef(A) genotypes demonstrated to be not susceptible to 2 or more antimicrobial agents, including penicillin, cefotaxime, clindamycin, tetracycline, and levofloxacin. The predominant antibiotic resistance patterns are shown in Table 3: CLN, PEN, TET or CEF, CLN, PEN, and TET. Among the 77 dual erm(B) and mef(A) isolates, 4 (5.2%) were resistant to levofloxacin. Table 1 Macrolide resistance phenotypes and their distribution of erythromycin resistance genes among 251 ERSP isolates Phenotype of macrolide resistance

No. (%) of strains

No. (%) of strains with the following gene erm(B) only

erm(B) + mef(A)

cMLSB M

214 (85.3) 37 (14.7)

137 (64.0) –

–

77 (36.0)

mef(A) only – 37 (100)

S. Bae, K. Lee / Diagnostic Microbiology and Infectious Disease 63 (2009) 213–216 Table 2 Distribution of macrolide resistance determinants and their association with serotypes in 251 ERSP Serotype

19F 23F 19A 14 11A 6B 6A 9V 35B 9A 23A 3 7C 10A 20 Others Total

No. (%) of strains

No. of strains with the following gene

57 (22.7) 25 (9.9) 24 (9.5) 18 (7.2) 17 (6.8) 16 (6.4) 14 (5.6) 13 (5.2) 9 (3.6) 6 (2.4) 6 (2.4) 5 (2.0) 4 (1.6) 4 (1.6) 4 (1.6) 29 (11.5) 251 (100.0)

6 19 3 13 13 14 13 13 1 6 6 3 4 3 2 18 137

erm(B)

erm(B) + mef(A) 44 5 21 3 – – – – 1 – – – – – 1 2 77

mef(A) 7 1 – 2 4 2 1 – 7 – – 2 – 1 1 9 37

Others: 4(1), 7A(1), 9N(2), 13(3), 15A(2), 15B(1), 15C(2), 16F(1), 19B (1), 22F(2), 23B(1), 23C(1), 24F(3), 28F(1), 29(1), 31(1), 33F(1), 35A(2), 36(1), ND(1).

The susceptibility to penicillin of the pneumococcal isolates carrying erm(B) or mef(A) genes is summarized in Table 4. Among all 251 ERSP, 92.4% were penicillin intermediate or resistant. One hundred twenty-six (92.0%) of 137 erm(B)-positive pneumococci exhibited reduced sensitivity to penicillin, and 29 (78.4%) of 37 mef(A)-positive

Table 3 Antibiotic resistance patterns and serotype distribution of 77 ERSP isolates that carried both erm(B) and mef(A) genes Resistance genotype

Serotype Total No. of Antibiotic resistance strains strains

erm(B) + mef(A)

19F

44

19A

21

23F

5

14

3

15B

1

18 9 8 7 1 1 9 6 4 1 1 3 1 1 2 1 1

20 35A 35B

1 1 1

1 1 1

CLN, PEN, TET CEF, CLN, PEN, TET CLN, PEN CEF, CLN, PEN CEF, CLN, PEN, LEV CLN, PEN, TET, LEV CLN, PEN CEF, CLN, PEN, TET CLN, PEN, TET CEF, CLN, PEN CLN, PEN, LEV CLN, PEN, TET CLN, PEN CEF, CLN, PEN, TET CEF, CLN, PEN, TET CLN, PEN, TET CEF, CLN, PEN, TET, LEV CLN, PEN, TET CLN, PEN, TET CLN, PEN, TET

CEF = cefotaxime.; CLN = clindamycin; PEN = penicillin; TET = tetracycline; LEV = levofloxacin.

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pneumococci were not susceptible to penicillin. Of note, all the 77 isolates carrying both erm(B) and mef(A) genes were not susceptible to penicillin (intermediate and resistant). 4. Discussion Recent studies have shown that the prevalence of macrolide resistance is alarmingly high in Asian countries including Vietnam (88.3%), Taiwan (87.2%), Korea (85.1%), Hong Kong (76.5%), and China (75.6%) (Song et al., 2004). Globally, the distribution of phenotypes and genotypes of macrolide-resistant pneumococci varies considerably depending on geographic location. In this study, the mechanism of macrolide resistance in 251 ERSP isolates collected during the period of 2000 to 2004 was observed to be predominantly mediated by the erm (B) gene. Of note was the finding that dual mechanisms of both the erm(B) and mef(A) genes were detected in 77 (30.7%) of 251 ERSP isolates. Pneumococci carrying both genes have been reported in South Africa, Vietnam, South Korea, the United States, New Zealand, Canada, Italy, and Scotland (Amezaga et al., 2002; Bean and Klean, 2002; Farrell et al., 2005; Song et al., 2004). These strains have shown high rates of resistance to multiple classes of antimicrobial agents; consequently, their potential spread is a serious public health problem. The distribution of serotypes among macrolide-resistant pneumococci was largely limited to serotypes 19F, 23F, and 19A. In our study, the distribution of serotypes among ERSP showed similar situation in the previous Korean studies (Song et al., 2004; Waites et al., 2003). The most prevalent serotype was 19F, which accounted for 22.7% of the 251 ERSP. Particularly, the significant finding was the very high rate of serotype 19A possessing both erm(B) and mef(A) genes among ERSP. In addition, most of strains with serotype 19F (77.2%) or 19A (87.5%) identified in this study carried the dual erm(B) and mef(A) genes. The prevalence and spread of serotype 19F or 19A isolates may have in part contributed to the high rate of macrolideresistant pneumococci observed in Korea. Even before the introduction of the 7-valent conjugate vaccine, it should be noted that the emerging increase of serotype 19A may perhaps occur independent of vaccination because of the overuse of antibiotics in Korea. Recent reports

Table 4 Distribution of macrolide resistance determinants and penicillin susceptibility among 251 ERSP Resistance genotype

erm(B) erm(B) + mef(A) mef(A)

No. (%) of strains with susceptibility to penicillin Susceptible

Intermediate

Resistant

11 (8.0) – 8 (21.6)

27 (19.7) 4 (5.2) 10 (27.0)

99 (72.3) 73 (94.8) 19 (51.4)

Total

137 77 37

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have shown that serotype 19A, not included in the 7-valent conjugate vaccine, is emerging globally and has become a replacement strain that causes significant problems in diverse populations (CDC, 2007). In a multihospital surveillance study of respiratory tract pathogens by the Korea Centers for Disease Control and Prevention, Seoul, Korea, the serotype 19A, not susceptible to penicillin and/or other antibiotics, has shown a dramatic increase since the early 2000s in Korea (manuscripts submitted for publication). Of interest was the observation of the emergence of the uncommon serotype 35B; this was the 9th most frequently identified serotype among ERSP isolates in this study. Most of the 35B serotype isolates displayed mef(A)-mediated resistance to macrolides. Serotype 35B has been regarded as a serotype associated with adult disease and is not included in the current pneumococcal vaccines. In the United States, the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance reported 68 invasive penicillinnonsusceptible serotype 35B (PN35B) pneumococci recovered from 1995 to 2001 (Beal et al., 2002). This surveillance program identified and emphasized the need for continued monitoring for penicillin and other antibiotic-resistant PN35B serotypes throughout the world. Judging from the high rate of serotype 19F or 19A with dual erm(B) and mef(A) genes and the finding of macrolidenonsusceptible pneumococci with nonvaccine serotype 35B, the need for further studies is clear to monitor for trends in the emergence and dissemination of novel macrolideresistant clones in Korea. Acknowledgment This study was supported by the intramural grant from the National Institute of Health, Korea Centers for Disease Control and Prevention. References Alonso DE, Verheul AF, Verhoef J, Snippe H (1995) Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccine. Microbiol Rev 59:591–603. Amezaga MR, Carter PE, Cash P, McJenzie H (2002) Molecular epidemiology of erythromycin resistance in Streptococcus pneumoniae isolates from blood and noninvasive sites. J Clin Microbiol 40: 3313–3318. Beal B, McEllistrem MC, Gertz RE, Boxrud DJ, Besser JM, Harrison LH, Jorgensen JH, Whitney GG, for the Active Bacterial Core Surveillance/ Emerging Infections Program Network (2002) Emergence for a novel

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