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Antimicrobial Agents and Chemotherapy, May 2008, p. 1888-1890, Vol. 52, No. 5
0066-4804/08/$08.00+0     doi:10.1128/AAC.00035-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Emergence of Fluoroquinolone Resistance in Group B Streptococcal Isolates in Taiwan

Hsiu-Mei Wu,¹ Rajendra Prasad Janapatla,¹ Yueh-Ren Ho,^2,4 Kuei-Hsiang Hung,⁴ Chi-Wen Wu,¹ Jing-Jou Yan,³ and Jiunn-Jong Wu^1,4*

Departments of Medical Laboratory Science and Biotechnology,¹ Biochemistry and Molecular Biology,² Pathology,³ Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan, Taiwan⁴

Received 9 January 2008/ Returned for modification 13 February 2008/ Accepted 18 February 2008

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Of 1,994 group B streptococcal isolates collected, 26 (1.3%) of the isolates were resistant to levofloxacin, and cross-resistance to other fluoroquinolones was observed. The emergence and prevalence of high-level fluoroquinolone resistance in genetically unrelated isolates were linked to the presence of gyrA, parC, and parE triple mutations in each isolate.

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Resistance to fluoroquinolones has emerged in different Streptococcus spp. (4, 5, 8, 15), mainly due to efflux or mutations in the quinolone resistance-determining regions (QRDRs) of the genes coding for type II topoisomerase enzymes, i.e., DNA gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) (7). Recently, Biedenbach et al. (1) showed that fluoroquinolone-resistant group B streptococci (FR-GBS) were identified in the United States in 1997; however, in the western Pacific region in Japan, they were detected in 2002 (8). Although ciprofloxacin has been available since 1990 and levofloxacin was introduced in 2000 in Taiwan, none of the studies reported the presence of fluoroquinolone resistance in GBS isolates from 1990 to 2007 (6). The objectives of this study were (i) to determine the prevalence of fluoroquinolone resistance, (ii) to characterize the QRDR mutations, and (iii) to determine the clonal relationship among resistant isolates by pulsed-field gel electrophoresis (PFGE).

A total of 1,994 GBS isolates were collected from 1993 to 2006 in the Department of Pathology, National Cheng Kung University Hospital, in southern Taiwan. Clinical isolates of gram-positive cocci with beta-hemolysis and a positive CAMP test were tested with Lancefield group B antiserum (Streptex; Murex Biotech, United Kingdom). Those cocci agglutinating with grouping-specific antiserum were identified as S. agalactiae. Clinical isolates were stored in Todd-Hewitt medium (Difco Laboratories, Detroit, MI) with 15% glycerol at –70°C until further testing. Susceptibilities to ampicillin, cefazolin, clindamycin, erythromycin, gentamicin, levofloxacin, penicillin, and vancomycin were determined by disk diffusion (2). MICs of levofloxacin (MIC breakpoint, 8 µg/ml) and other fluoroquinolones, including moxifloxacin, gatifloxacin, trovafloxacin, garenoxacin, and ciprofloxacin, were determined by the agar dilution method (3). PCR amplification and DNA sequencing of QRDRs responsible for the fluoroquinolone resistance phenotype were performed as described previously (1, 14). PFGE of SmaI-digested genomic DNA samples was carried out with a contour-clamped homogeneous electric field system (CHEF Mapper XA; Bio-Rad Laboratories, Hercules, CA) according to the instruction manual. PFGE banding patterns were interpreted as described previously (13).

Of the 1,994 GBS isolates, 26 isolates (1.3%) were resistant to levofloxacin. The MICs of levofloxacin-resistant GBS isolates were found to be 16 µg/ml. Since mutations in the QRDR lead to cross-resistance to fluoroquinolones, we determined the MICs by the agar dilution method and found that all the levofloxacin-resistant GBS isolates had elevations of MICs to ciprofloxacin (MIC, 32 to 64 µg/ml), garenoxacin (MIC, 2 to 4 µg/ml), gatifloxacin (MIC, 4 to 8 µg/ml), moxifloxacin (MIC, 2 to 4 µg/ml), and trovafloxacin (MIC, 8 to 16 µg/ml) (Table 1). The first FR-GBS clinical isolate was isolated in November 2004 from the urogenital tract of a 40-year-old female patient. Since 2004, the annual prevalence of FR-GBS isolates significantly increased from 0.33% (1 isolate) in 2004 to 3.83% (13 isolates) in 2005 and 5.04% in 2006 (12 isolates). The antimicrobial susceptibility pattern test showed that all the FR-GBS isolates were totally susceptible to penicillin, ampicillin, vancomycin, and cefazolin. Resistance to gentamicin was found in 100% of isolates, followed by resistance to erythromycin (65%) and clindamycin (62%).

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TABLE 1. MICs of fluoroquinolones and mutations in the QRDRs of gyrase (gyrA/gyrB) and topoisomerase IV (parC/parE) in GBS

gyrA, parC, and parE triple mutations were identified in all the FR-GBS isolates (Table 1). In gyrA, a Ser-81-to-Leu (Ser81Leu) mutation was predominant, while in gyrB, mutations were absent. However, in parC, a Ser79Tyr mutation was present in 16 isolates, and 10 isolates had a Ser79Phe mutation. In parE, all the isolates had a unique Ile495Leu mutation. Isolates with the parE mutation also had gyrA (Ser81Leu) and parC (Ser79Phe) mutations predominantly. To determine the clonal relationship of isolates resistant to fluoroquinolones, we performed PFGE. Among the 26 resistant isolates, 17 pulse types (A to Q) were identified (Fig. 1; Table 1).

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FIG. 1. PFGE patterns of 26 fluoroquinolone-resistant GBS isolates. The designations for isolates that were represented by each pulse type are shown below the gels. Lanes M contain a lambda ladder (Gibco) that served as a molecular marker.

In Taiwan, the presence of fluoroquinolone resistance in GBS isolates has not been reported before. After isolation of the first FR-GBS strain in November 2004, the annual incidence rate, which was 0.33% in 2004, significantly increased to 3.83% in 2005 and 5.04% in 2006. This indicates that FR-GBS isolates not only emerged but became significantly more prevalent since late 2004 in this region. Overall, the FR-GBS prevalence rate in our study was 1.3%, which is similar to prevalence rates reported earlier in Barcelona, Spain (1.16%, 2003 to 2004), and higher than those in the United States (0.7%, 1997 to 2004) (1, 12).

In GBS, QRDR mutations have been reported previously in gyrA and parC only. High-level resistance could be linked to the presence of a triple mutation (gyrA-parC-parE) in each isolate. Mutations in gyrA occurred predominantly at amino acid position 81 (Ser-81 to Leu) and those in parC were predominantly Ser79Tyr and Ser79Phe, which were similar to earlier reports (1, 9, 14). The QRDR of gyrA is the primary target which mediates fluoroquinolone resistance in several gram-negative bacteria, while in gram-positive bacteria parC is the primary target, which substantially increases the probability of a second mutation in gyrA, resulting in high-level fluoroquinolone resistance (4, 7, 10). Based on our data, we were unable to determine whether gyrase or topoisomerase IV is the primary target for fluoroquinolones or the other antibiotic(s) associated with the triple mutations. Further analysis of the minor targets gyrB and parE revealed that mutations were absent in gyrB in FR-GBS isolates, similar to the previous reports on fluoroquinolone resistance in GBS (1, 8, 14). A unique mutation (Ile495Leu) was present in parE, but since gyrB and parC mutations were also present in the isolates with the parE mutation, its significance is unknown. It can be either a silent mutation or a mutation similar to an S. pneumoniae parE (Ile460Val) variant that led to reduced susceptibility to fluoroquinolones (9). The presence of 17 pulse types and the absence of a predominant pulse type indicate that the fluoroquinolone-resistant isolates are genetically unrelated. Spread of fluoroquinolone resistance due to single and multiple clones was observed in other S. pneumoniae and S. pyogenes isolates (5, 11). For GBS, Wehbeh et al. suggested the nosocomial spread of levofloxacin-resistant GBS isolates based on two major clusters identified by PFGE (14).

Since all the FR-GBS isolates were totally susceptible to ampicillin, penicillin, cefazolin, and vancomycin, these antibiotics remain the preferred choice to treat GBS infections empirically. Since resistance to fluoroquinolones can develop during therapy and cross-resistance to other fluoroquinolones is likely to occur, when prescribing fluoroquinolones for GBS infections, susceptibility testing and monitoring during therapy should be done to avoid treatment failure (5, 10). Continuous molecular-level surveillance is needed to prevent further dissemination of FR-GBS clones in Taiwan.

In conclusion, our study confirmed the emergence of genetically unrelated FR-GBS isolates in Taiwan and showed a significant increase in prevalence from 2004 to 2006. High-level fluoroquinolone resistance in the isolates could be linked to the presence of gyrA-parC-parE triple mutations in each isolate.

 
This work was partially supported by grants DOH93-DC-1110, DOH94-DC-1008, and DOH95-DC-1038 from the Bureau of Center of Disease Control, Department of Health, Taiwan.

 
* Corresponding author. Mailing address: Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng-Kung University, No. 1 University Rd., Tainan, Taiwan 70101. Phone: 886-6-2353535, ext. 5775. Fax: 886-6-2363956. E-mail: jjwu{at}mail.ncku.edu.tw

Published ahead of print on 25 February 2008.

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Antimicrobial Agents and Chemotherapy, May 2008, p. 1888-1890, Vol. 52, No. 5
0066-4804/08/$08.00+0     doi:10.1128/AAC.00035-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wu, H.-M. Articles by Wu, J.-J. Search for Related Content PubMed PubMed Citation Articles by Wu, H.-M. Articles by Wu, J.-J.