High Prevalence of Fluoroquinolone-Resistant Group B Streptococci among Clinical Isolates in China and Predominance of Sequence Type 19 with Serotype III

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Group B streptococcus (GBS) is the leading cause of septicemia and meningitis in neonates (1). It is also associated with bacteremia, endocarditis, arthritis, and other infections in nonpregnant women with underlying diseases and in elderly adults (2). Clinical GBS isolates usually remain penicillin susceptible. Considering the 0.7% to 10% incidence of penicillin allergies (3), as well as resistance to erythromycin and clindamycin, fluoroquinolones (FQs) are important alternatives. However, FQ resistance has been reported (4–7).

Ten GBS serotypes, specified by the polysaccharide capsule, are known, and five of these (Ia, Ib, II, III, and V) are included in a vaccine that is in development. A knowledge of serotype distribution will be important for vaccine development. Few studies from mainland China have reported on antimicrobial resistance, serotypes, and molecular epidemiology in GBS. Therefore, investigating the current resistance status in GBS in China is necessary. From September to December 2011, 146 clinical consecutive nonduplicate GBS isolates were collected from 13 teaching hospitals in eight cities in China. All isolates were confirmed for identification at the central laboratory. Confirmation testing was done using a commercial latex agglutination technique (Slidex Strepto B; bioMérieux, Marcy L'Étoile, France) and PCR for the dltS gene.

A multiplex PCR assay containing a mixture of 19 primers was performed as described by Imperi et al. (8). Serotype III was the most prevalent, accounting for 63.7% of the isolates, followed by serotypes Ia (21.9%), II (9.6%), and V (4.8%). These findings are similar to previous reports from North America and Europe (9, 10). Serotype Ia was significantly higher in children than in adults (42.9% versus 23%, respectively; P = 0.0414) (Table 1).

The MICs of the antimicrobials were determined using the agar dilution method according to guidelines from the Clinical and Laboratory Standards Institute (CLSI) (11). The antimicrobials used were penicillin, cefaclor, cefuroxime, ceftriaxone, erythromycin, clindamycin, tetracycline (Sigma, St. Louis, MO), levofloxacin (Daiichi Sankyo Pharmaceutical, Japan), and moxifloxacin (Bayer AG, Leverkusen, Germany). The results were analyzed by WHONET 5.6 software and interpreted according to CLSI M100-S22 (12). For statistical analysis, χ² test or Fisher's exact test was used to analyze the qualitative variables, as appropriate. A P value of <0.05 was considered statistically significant.

All 146 isolates were susceptible to penicillin, cefuroxime, cefaclor, and ceftriaxone. Resistance to erythromycin, clindamycin, and tetracycline was found in 104 (71.2%), 78 (53.4%), and 119 (81.5%) isolates, respectively. Fifty-five isolates (37.7%) were levofloxacin resistant. The levofloxacin resistance rate was significantly higher in urinary tract infection (48.4%, 30/62) than in the other cases and among colonized pregnant women (29.8%, 25/84; P < 0.001). Significant differences were found in levofloxacin and clindamycin resistance among the 4 serotypes (P = 0.0015, P = 0.028, respectively). Levofloxacin resistance was significantly higher in serotype III than in serotypes Ia (P = 0.021) and II (P = 0.0006). Clindamycin resistance was lowest in serotype II (P < 0.05, Table 1).

The resistance determinants for FQ, macrolides and tetracycline were determined. PCR amplification and sequencing of the gyrA, gyrB, parC, and parE genes were performed as previously described (13). The macrolide resistance phenotype was determined using a double-disk test with erythromycin and clindamycin (12). Macrolides and tetracycline resistance genes, including erm(B), erm(A), mef(A/E), tet(M), and tet(O), were detected using PCR as previously described (14–16). The mef(A) and mef(E) were determined by sequencing the 1,431 bp mef(A/E) PCR product. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed for the FQ-resistant isolates as previously described (17–19).

All 119 tetracycline-resistant isolates carried tet(M), and 4 of these isolates also harbored tet(O). Previous studies also found more than one tet gene in the minority of tetracycline-resistant GBS isolates (20, 21). Seventy-eight of 104 erythromycin-resistant isolates showed cMLS_B resistance, and the remaining 26 isolates showed an M resistance phenotype. All 78 cMLS_B isolates harbored erm(B), and 45 of these isolates also harbored mef(E). The isolates with the M resistance phenotype carried mef(E).

Six STs and seven PFGE types (14 subtypes) were identified for 55 FQ-resistant isolates. ST19-CC19/serotype III, corresponding to PFGE types A to E, was the most predominant type, accounting for 80.0% of the FQ-resistant isolates (Table 2). ST10 and ST23 were associated with serotype Ia, ST17 with serotype III, and ST1 with serotype V, which are consistent with the previous studies (22).

Of the 51 isolates with 32-μg/ml levofloxacin MICs, 44 belonged to ST19/serotype III. All ST19/serotype III isolates had three amino acid substitutions, and five ST10/Ia isolates and one ST1/V had two substitutions (Table 2). Most of the isolates with 8-μg/ml levofloxacin MICs had only one substitution. The specific GyrA-ParC-ParE triple mutation (S81L in GyrA, S79Y in ParC, and H225Y in ParE) and the GyrA-ParC double mutation (S81L in GyrA and S79F in ParC) both conferred high levels of levofloxacin resistance.

Of the 55 FQ-resistant isolates, 80.0%, 70.9%, and 83.6% were resistant to erythromycin, clindamycin, and tetracycline, respectively. Table 2 shows that 45 tetracycline-resistant isolates possessed tet(M) and 1 possessed both tet(M) and tet(O). Among the 41 erythromycin- and tetracycline-resistant ST19/serotype III isolates, 25 harbored the erm(B), mef(E), and tet(M) genes. All five M-phenotype resistant isolates harbored mef(E) and tet(M) (Table 2).

This is the first study on serotype distribution, antimicrobial resistance, and molecular characterization of GBS isolates in mainland China. In the present study, we found high rates of multiple drug resistance in GBS, including resistance to FQ, macrolides, and tetracycline. The incidence of macrolide resistance of GBS is relatively low in western countries, ranging from 11.5% to 32% (9, 20, 22–24), and higher in Taiwan, at 58.3% (25). The first FQ-resistant GBS was reported in 2003 in Japan (26) at a relatively lower prevalence (27, 28). To our knowledge, the FQ resistance rate in the present study is the highest among the latest reports. With the wide use of empirical FQ in various community-acquired infections, FQ-resistant GBS will be encountered in other regions worldwide.

The present study found that the high prevalence of FQ-resistant GBS was associated with the emergence and spread of multidrug-resistant ST19/serotype III clone. The finding of predominance, multiple sources, and distribution of serotype III/ST19 in eight distant areas suggests that its emergence is affecting the whole country. At least two major lineages of GBS isolates of serotype III, ST17 and ST19, have been reported. ST19/serotype III was reported as the predominant clone in Canada (29) and the United States (30), whereas ST23/serotype Ia and ST17/III are the most prevalent in Portugal, Italy, and Spain (20, 23, 24). However, in the Mediterranean region, ST19 was associated with serotypes Ib, II, III, and IV, which may be ascribed to the horizontal transfer of capsular genes among different clones.

In the present study, the FQ-resistant ST19/serotype III isolates also showed resistance to macrolide and tetracycline. Most of these isolates carried the tet(M), erm(B), and mef(E) genes, which may represent multiple horizontal gene transfer events. Supporting the importance of the mechanism, a Polish study reported the identification of the tet(M) and erm(B) genes on the same conjugative Tn916-family transposons in GBS (21). Further study of genetic mobile elements with resistance determinants in GBS is needed in China.

In summary, clonal expansion of multidrug-resistant ST19/serotype III is the primary cause of the high prevalence of FQ resistance in China, which increases concerns regarding its future global spread. The study data emphasizes the need for careful epidemiologic monitoring of this FQ-resistant clone with erm(B), mef(E), and tet(M) genes in invasive diseases.