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1 Laboratory of Molecular Epidemiology for Infectious Agents, Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan
2 Laboratory of Infectious Disease, Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan
Correspondence
Kimiko Ubukata
ubukatak{at}lisci.kitasato-u.ac.jp
Received April 14, 2008
Accepted July 28, 2008
To determine the prevalence of macrolide antibiotic and levofloxacin resistance in infections with Streptococcus pyogenes (group A streptococcus or GAS), strains were collected from 45 medical institutions in various parts of Japan between October 2003 and September 2006. Four hundred and eighty-two strains from patients with GAS infections were characterized genetically. Strains were classified into four groups according to the type of infection: invasive infections (n=74) including sepsis, cellulitis and toxic-shock-like syndrome; acute otitis media (AOM; n=23); abscess (n=53); and pharyngotonsillitis (n=332). Among all strains, 32 emm types were identified; emm1 was significantly more common in invasive infections (39.2 %) and AOM (43.5 %) than in abscesses (3.8 %) or pharyngotonsillitis (10.2 %). emm12 and emm4 each accounted for 23.5 % of pharyngotonsillitis cases. Susceptibility of GAS strains to eight β-lactam agents was excellent, with MICs of 0.0005–0.063 µg ml–1. Macrolide-resistant strains accounted for 16.2 % of all strains, while the percentages of strains possessing the resistance genes erm(A), erm(B) and mef(A) were 2.5 %, 6.2 % and 7.5 %, respectively. Although no strains with high resistance to levofloxacin were found, strains with an MIC of 2–4 µg ml–1 (17.4 %) had amino acid substitutions at either Ser-79 or Asp-83 in ParC. These levofloxacin-intermediately resistant strains included 16 emm types, but macrolide-resistant strains were more likely than others to represent certain emm types.
Abbreviations: AOM, acute otitis media; CDC, Centers for Disease Control and Prevention; FQ, fluoroquinolone; GAS, group A streptococcus.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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M protein encoded by the emm gene (Fischetti, 1989) has been exploited for M typing in epidemiological studies (Tewodros & Kronvall, 2005). M typing has progressed from identification methods using antisera to analysis of emm gene sequences of the N-terminal hypervariable region using the Centers for Disease Control and Prevention (CDC) database (Beall et al., 1996). Results for GAS surveillance by Rogers et al. (2007) demonstrated that emm1 predominated in invasive infections compared with other noninvasive infections.
Macrolide and levofloxacin resistance have gradually increased in GAS isolates, although the isolates remain uniformly susceptible to β-lactam antibiotics. Clonal spread of levofloxacin-resistant GAS has been reported (Malhotra-Kumar et al., 2005), and possible implications for public health have been pointed out.
Recently, penicillin-intermediately resistant Streptococcus agalactiae that has amino acid substitutions in penicillin-binding protein 2X has appeared in Japan (Kimura et al., 2008). Therefore, attention to annual trends of β-lactam susceptibility of GAS is also necessary.
In this study, we aimed to clarify (i) emm-type distributions in invasive strains compared with noninvasive isolates; (ii) susceptibility of these strains to 11 β-lactam antibiotics, four macrolides, clindamycin and levofloxacin; and (iii) resistance genes for macrolides, lincosamides and streptogramin B (MLSB), and levofloxacin.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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For the purposes of this study, isolates were divided into four groups according to the modified classification of Rogers et al. (2007). The invasive infection group (total n=74) included septicaemia (n=34), cellulitis (n=13), septic arthritis (n=8), meningitis (n=4), necrotizing fasciitis (n=5), toxic-shock-like syndrome (n=4), pneumonia (n=3), mastitis (n=2) and pleurisy (n=1) isolates. In this group, isolates defined tentatively as GAS were obtained from blood, joint fluid or pleural fluid. The other GAS infections were classified in abscess (n=53), pharyngotonsillitis (n=332) and acute otitis media (AOM) (n=23) groups.
Characterization of the resistance mechanism. PCRs for detection of the erm(A), erm(B) and mef(A) genes mediating macrolide resistance were performed as described previously (Sunaoshi et al., 2004).
Strains possessing the erm(A) gene express an inducible macrolide, lincosamide and streptogramin B resistance (iMLSB) phenotype (Seppälä et al., 1998) and strains possessing the erm(B) gene express a constitutive MLSB (cMLSB) phenotype. The iMLSB phenotype strains by the target site modifications due to methylase activity show high resistance to 14- and 15-membered ring macrolides but are susceptible or intermediately resistant to clindamycin without induction (Giovanetti et al., 1999). The cMLSB phenotype strains by methylase activity show high resistance to all macrolides and clindamycin without induction. By contrast, strains possessing the mef(A) gene express a 14- and 15-membered ring macrolide resistance (M) phenotype by an active drug efflux pump (Giovanetti et al., 1999; Roberts et al., 1999).
For the gyrA, gyrB, parC and parE genes involved in fluoroquinolone (FQ) resistance, PCR was carried out for 40 cycles under the conditions of 94 °C for 30 s, 52 °C for 30 s and 72 °C for 60 s. Analytical primer sets for FQ resistance, shown in Table 1
, were designed to detect these genes in GAS strains (GenBank accession nos NC_002737, NC_008023, NC_008024). The PCR product was then purified using the QIAquick PCR Purification kit (Qiagen). Sequencing was performed using the BigDye Terminator Cycle Sequencing kit version 3.1 (Applied Biosystems), with assessment of results using the Applied Biosystems 3130 Genetic Analyzer.
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Extraction of template DNA was done using the established procedure (Ubukata et al., 1996). In brief, one colony of GAS growing on a sheep blood agar culture plate was picked and suspended in lysis solution, which comprised 0.1 M Tris/HCl (pH 8.0), 4 µg proteinase K, 0.225 % Tween 20 and 0.225 % Nonidet 40. This was incubated at 60 °C for 20 min and then at 90 °C for 10 min.
The PCR for emm genotyping was carried out according to minor modifications of the method described previously by Beall et al. (1996). The resulting PCR fragments were purified and sequenced in the same way as described above.
The first 240 bases of the 5' end of the emm gene sequences were compared with those in the CDC S. pyogenes emm sequence database (http://www.cdc.gov/ncidod/biotech/strep/strepblast.htm). An emm type showing over 98 % homology with a CDC reference strain was identified as that particular emm type.
Antimicrobial susceptibility tests. Susceptibility testing of GAS strains was carried out by the microdilution method using cation-adjusted Mueller–Hinton broth (Becton Dickinson) supplemented with 5 % lysed horse blood according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2007) and with a final inoculum of 5x105 c.f.u. ml–1. Oral antimicrobial agents employed in this study were ampicillin, amoxicillin, cefdinir, cefpodoxime, cefditoren, cefcapen, faropenem, tebipenem, clarithromycin, azithromycin, josamycin, clindamycin, telithromycin and levofloxacin. Parenteral agents were cefotaxime, panipenem and meropenem. These antimicrobial agents were obtained from the respective pharmaceutical manufacturers.
Statistical analysis. Statistical analysis was performed using Microsoft Excel Statistics 2006 for Windows (Social Survey Research Information). The chi-square test was used to assess significance of differences involving categorical variables.
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RESULTS AND DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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shows the patient age distribution in the four groups of GAS infection cases described in Methods.
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Both AOM cases (n=23) and pharyngotonsillitis cases (n=332) mainly involved children between 3 and 12 years old, which accounted for 43.5 % and 67.4 %, respectively, although 20.1 % of the pharyngotonsillitis patients were 20–30 years old.
Abscess cases (n=53), in which GAS was isolated from either draining or nondraining localized abscesses, were distributed across all age brackets.
Statistically significant differences in age distribution were recognized between the invasive group and each of the other three groups (invasive vs AOM, P <0.0001; invasive vs abscess, P=0.0003; invasive vs pharyngotonsillitis, P <0.0001).
Typing for emm
Results of emm typing of GAS isolates in four groups, that is, invasive (A), AOM (B), abscess (C) and pharyngotonsillitis (D) groups, are listed in Table 2. Although GAS isolates included a variety of emm types, the predominant emm types in each group differed. In the invasive group, emm1 was prominent, accounting for 39.2 % of cases, while other emm types generally accounted for 10 % of cases or fewer. Similarly, emm1 was most frequent in the AOM group (43.5 %), followed by emm12 (30.4 %).
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accounted for 22.6 % and 26.4 % of cases each, while emm1 was responsible for only 3.8 %. In the pharyngotonsillitis group, emm4 and emm12 accounted for 23.5 % of cases each, in contrast to 10.2 % for emm1. A significant difference in prevalence of emm types was noted between the invasive group and each of the other three groups (invasive vs AOM, P=0.0895; invasive vs abscess, P=0.0013; invasive vs pharyngotonsillitis, P <0.0001).
Susceptibilities to β-lactam agents
All β-lactam agents showed sharp distributions for all GAS, indicating that none of these agents showed decreased efficacy against GAS.
In data not shown here, MIC90 values for GAS were excellent in the following order: tebipenem (0.002 µg ml–1) > cefditoren=cefcapen (0.008 µg ml–1) > amoxicillin=cefdinir=cefpodoxime (0.016 µg ml–1) > ampicillin=faropenem (0.031 µg ml–1) for oral β-lactams; and panipenem=meropenem (0.008 µg ml–1) > cefotaxime (0.016 µg ml–1) for parenteral β-lactams. All isolates remained uniformly susceptible to β-lactam antibiotics.
Susceptibilities to macrolides and resistance genes
Table 3 shows the MIC ranges, MIC50 and MIC90 of clarithromycin, azithromycin, josamycin, telithromycin and clindamycin for GAS according to the macrolide-resistance genes identified. Of all 482 isolates, strains possessing erm(A), erm(B) and mef(A) represented 2.5 % (n=12), 6.2 % (n=30) and 7.5 % (n=36), respectively.
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64 µg ml–1). GAS strains with the mef(A) gene, which mediate an M phenotype, showed slight decreases in susceptibility, from 4- to 8-fold, to clarithromycin, azithromycin and telithromycin, but maintained undiminished susceptibility to josamycin and clindamycin. In Japan, the prevalence of macrolide resistance in GAS has remained at 5–7 % for a long time. In the survey in 2003, of the total of 533 GAS strains collected from all over the country, 0.5 % had the erm(A) gene, 3.2 % the erm(B) gene and 4.9 % the mef(A) gene (Sunaoshi et al., 2004). Macrolide resistance appears to increase gradually.
Table 4 shows the correlation between the emm type and macrolide-resistance genes. Of the GAS strains typed to emm1, 21.3 % had the mef(A) gene, which was prevalent in invasive infections, in contrast to the emm status of strains with erm(A) and erm(B) genes.
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Susceptibility to levofloxacin and mutations of the target gene
Table 4 also shows the emm type distribution of GAS strains (n=71) which were intermediately resistant to FQ. Although no strain was identified as showing high resistance, strains with an MIC of 2–4 µg ml–1 accounted for 17.4 % (n=84) of all isolates. Eighty-five per cent of strains with an MIC of at least 2 µg ml–1 (n=71) had an amino acid substitution at the Ser-79 or Asp-83 position in quinolone resistance-determining regions (QRDRs) encoded by parC. Ser-79 was changed to Phe-79, Ala-79 or Tyr-79 in 93.0 % (n=66), while Asp-83 was changed to Asn-83 in 7.0 % (n=5). There was no amino acid substitution affecting FQ resistance in gyrA, gyrB and parE genes.
Genotypic levofloxacin-intermediately resistant GAS strains belonged to 16 emm types, although emm6 and emm11 were prominent at 94.4 % and 76.9 %, respectively, as described previously (Orscheln et al., 2005). This finding suggests that genotypically FQ-intermediately resistant GAS is selected under the pressure of exposure to FQs including levofloxacin. In vitro experiments have indicated that all emm types seem equally prone to induction of FQ resistance (Billal et al., 2007).
Recently, we isolated a GAS strain, showing high resistance to FQ, from an adult patient (29 years old) with pharyngotonsillitis in September 2007. The strain possessed amino acid substitutions in QRDRs of both gyrA and parC, which had already been reported in several countries (Malhotra-Kumar et al., 2005; Reinert et al., 2004; Richter et al., 2003; Rivera et al., 2005; Yan et al., 2000).
According to the market research for oral antibiotics (Fujita et al., 2007) in Japan, oral FQs, including four respiratory FQs, have been prescribed for adult outpatients aged 15 years at the highest rate of 50 %, followed by oral cephalosporin antibiotics at the rate of 43 %, which is higher than that for penicillins. The status of the current usage of FQ causes concern that the incidence of GAS and Streptococcus pneumoniae strains possessing high FQ resistance might increase in the near future.
On the other hand, macrolides to which 14-membered ring macrolides and azalides belong have been widely prescribed to reduce inflammation in patients with diffuse panbronchiolitis, chronic bronchitis and chronic sinusitis at low doses for long periods, in addition to being used in the treatment of community-acquired respiratory tract infections. Such long-term non-chemotherapeutical usage of macrolides may result in a decrease in usefulness of macrolides for infectious disease and be related to selection and spread of macrolide resistance in S. pneumoniae and S. pyogenes.
In conclusion, continuous molecular epidemiological surveillance of GAS is necessary to ensure proper use of antimicrobial agents.
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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References |
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TOPINTRODUCTIONMETHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSReferences |
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