ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2002, p. 3311–3315 0066-4804/02/$04.00⫹0 DOI: 10.1128/AAC.46.10.3311–3315.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 10
Fluoroquinolone-Resistant Streptococcus pneumoniae Strains Occur Frequently in Elderly Patients in Japan Shin-ichi Yokota,1 Kiyoshi Sato,1,2 Osamu Kuwahara,3 Satoshi Habadera,3 Naoyuki Tsukamoto,4 Hironori Ohuchi,4 Hirotsugu Akizawa,5 Tetsuo Himi,6 and Nobuhiro Fujii1* Departments of Microbiology1 and Otolaryngology,6 Sapporo Medical University School of Medicine, Chuo-ku, Sapporo 060-8556, Hokkaido Wako Junyaku Co., Ltd., Kita-ku, Sapporo 001-0015,2 Sapporo Clinical Laboratory, Inc., Chuo-ku, Sapporo 060-0005,3 SRL Hokkaido, Inc., Chuo-ku, Sapporo 064-0919,4 and Laboratory Medicine, Hokkaido University Medical Hospital, Kita-ku, Sapporo 060-8648,5 Japan Received 29 April 2002/Returned for modification 3 June 2002/Accepted 6 July 2002
We identified and genetically characterized seven fluoroquinolone-resistant Streptococcus pneumoniae strains among 293 clinical strains isolated from 1999 to 2001 in Japan. The resistant strains were isolated only from adults, and 7 of 31 isolates (22.6%) were from patients more than 20 years old. Resistant strains were not found in 262 isolates from children under age 10. It is now feared that multidrug resistant Streptococcus pneumoniae strains are becoming more prevalent, and fluoroquinolone resistance has also become more common over the last few years (7, 10, 12, 17). In Japan, the prevalence of fluoroquinolone-resistant S. pneumoniae is thought to be very low (27). Here we report emergence of fluoroquinolone-resistant strains among 293 S. pneumoniae strains isolated in 1999 to 2001 in the Hokkaido prefecture, Japan. The isolates were obtained from rhinorrhea (235 samples), sputum (23), otorrhea (15), throat swab (14), nasal mucous membrane (5), and articular fluids (1). Two hundred fourteen strains were from outpatients. Seventy-nine strains were isolated from hospitalized patients. Identification of the isolates was routinely carried out by MicroScan WalkAway40 (Dade Behring, Tokyo, Japan). We also confirmed isolates as S. pneumoniae by detection of the pneumolysin gene by PCR and optochin sensitivity. Isolates were grown at 37°C with 5% CO2 on Trypticase soy agar (Nippon Beckton-Dickinson, Tokyo, Japan) supplemented with 5% defibrinized sheep blood. MICs of various fluoroquinolones were determined by the microdilution method using Mu ¨ller-Hinton broth (Nippon Beckton-Dickinson) supplemented with 5% defibrinized sheep blood according to the standard method described by the Japan Society of Chemotherapy (14). Reagent powders of antimicrobial agents were provided from manufacturers as follows: ciprofloxacin (Bayer, Osaka, Japan), levofloxacin (Daiichi Pharmaceuticals, Tokyo, Japan), tosufloxacin (Dinabot, Osaka, Japan), sparfloxacin (Dainippon Pharmaceuticals, Osaka, Japan), and gatifloxacin (Kyorin Pharmaceuticals, Tokyo, Japan). We found that seven strains (2.4% of total strains tested) were resistant to ciprofloxacin and levofloxacin. Six of the seven strains were also resistant to tosufloxacin, sparfloxacin, and gatifloxacin (Table 1). Table 2 indicates the age distribution of patients harboring resistant strains. Interestingly, none of the children harbored fluoroquinolone-resistant strains. All the re-
sistant strains were found in adults (22.6%, 7 of 31 strains). In particular, more than a quarter of patients older than 65, who are a group at high risk for pulmonary diseases, such as pneumonia, harbored such fluoroquinolone-resistant strains. Similar observations about the age distribution of patients carrying fluoroquinolone-resistant S. pneumoniae have been reported by Canadian (7) and Hong Kong (8) groups. Quinolone resistance is imparted by mutations in a particular domain designated the quinolone resistance-determining region (QRDR) of the principal target enzymes, DNA gyrase (an A2B2 complex encoded by the gyrA and gyrB genes) and/or topoisomerase IV (a C2E2 complex encoded by the parC and parE genes) (19). We determined the DNA sequences of the QRDRs in these four genes. Genomic DNA was isolated from a colony grown on an agar plate according to the method of Ubukata et al. (25) and used as a template for PCR analysis. PCR fragments containing QRDRs were amplified as described elsewhere (20) and directly sequenced by the dye termination method. The QRDR DNA fragment of parC could not be amplified in two (SR27 and SR179) of the seven resistant strains, and thus the full-length parC genes of these were amplified by another primer set (11) and sequenced by DNA walking with a combination of primers. The QRDR DNA sequences of the resistant strains were compared with sensitive strains, including strain R6 (GenBank accession no. NC 003098) as a standard strain and five strains obtained in our study (Table 1). In the resistant strains, mutations at the amino acid level were found in one or more of the QRDRs in the parC, gyrA, and parE genes, but not in gyrB. The mutation patterns varied between the strains except for SR27 and SR179. Among these mutations, two site ParC mutations (Ser79 [TCT] to Phe [TTT] or Arg [AGA], and Asp83 [GAT] to Tyr [TAT]), one site GyrA mutation (Ser81 [TCC] to Tyr [TAC] or Phe [TTC]), and one ParE mutation (Asp453 [GAC] to Asn [AAC]) have been previously reported to be frequent mutations contributing to fluoroquinolone resistance (3, 6, 15, 16, 21, 23, 26). It is unclear whether other mutations relate to fluoroquinolone resistance. The Ile460 (ATC) to Val (GTC) mutation on ParE observed in six of the resistant strains was also found in two fluoroquinolone-sensitive strains (SR4 and
* Corresponding author. Mailing address: South-1, West-17, Chuoku, Sapporo 060-8556, Japan. Phone: 81-11-611-2111. Fax: 81-11-6125861. E-mail:
[email protected]. 3311
None None None None None None None None
Ser81 3 Phe, Ser114 3 Gly Ser81 3 Tyr None None None None None None None
Agea
No. of resistant strains/no. isolated
Frequency of resistant strains (%)
0 to 8 20 to 64 ⬎65
0/262 2/12 5/19
0 16.7 26.3
Total
7/293
2.4
Abbreviations of antibiotics: CIP, ciprofloxacin; LVX, levofloxacin; TSX, tosufloxacin; SPX, sparfloxacin; GAT, gatifloxacin.
a
a
73 88 71 48 1 1 1 61 HU86 SR69 SR166 SR4 SR11 SR12 HU1 HU2
F F M M F F F M
Sputum Sputum Sputum Sputum Rhinorrhea Rhinorrhea Otorrhea Rhinorrhea
16 (4) 16 (4) 8 (2) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1)
16 (8) 8 (4) 2 (1) 1 (1) 0.5 (1) 1 (1) 0.5 (1) 0.5 (1)
2 (1) 2 (1) 0.5 (0.25) 0.12 (0.12) 0.12 (0.12) 0.12 (0.12) 0.12 (0.12) 0.5 (0.25)
8 (4) 1 (2) 0.25 (0.5) 0.5 (0.5) 0.25 (0.25) 0.25 (0.5) 0.25 (0.25) 0.5 (0.5)
8 (8) 4 (4) 1 (0.5) 0.25 (0.25) 0.25 (0.5) 0.25 (0.25) 0.25 (0.25) 0.5 (0.5)
Ser52 3 Gly, Ser79 3 Arg, Asn91 3 Asp, Glu135 3 Asp None Ser79 3 Phe Asp83 3 Tyr None None None None Lys137 3 Asn 4 (2) 8 (4) 4 (4) 8 (4) 16 (16) Sputum 78 SR179
M
8 (4) 8 (4) 4 (2) 16 (8) 16 (4) Articular fluid 22 HU85
M
TABLE 2. Relationship between patient age and prevalence of fluoroquinolone-resistant S. pneumoniae strains
Asp435 3 Asn Ile460 3 Val Ile460 3 Val Ile460 3 Val None None None Ile460 3 Val
None
None
Asp435 3 Asn, Ile460 3 Val Ile460 3 Val
Ile460 3 Val Ile460 3 Val
Ser81 3 Phe Ser81 3 Phe, Ser114 3 Gly Ser81 3 Phe None Ser52 3 Gly, Ser79 3 Arg, Asn91 3 Asp, Glu135 3 Asp None 32 (16) 8 (4) 32 (32) 8 (8) 8 (4) 8 (4) ⬎32 (⬎32) 32 (16) M F 33 85 SR68 SR27
Age
Sex
Sputum Sputum
32 (32) 16 (8)
ParE GyrA CIP
LVX
TSX
SPX
GAT
ParC
Mutation in QRDRs MIC (MIC in the presence of reserpine) (g/ml) Source Profile of patient Strain
TABLE 1. Characterization of fluoroquinolone susceptibilities and the mutations in quinolone target gene QRDRs of seven resistant and five sensitive strainsa
ANTIMICROB. AGENTS CHEMOTHER. None None
NOTES GyrB
3312
Isolates from 9- to 19-year-old individuals were not included in this study.
HU2), suggesting that it does not contribute to fluoroquinolone resistance. Pestova et al. also showed that this mutation is unrelated to resistance by a DNA recombination study (21). SR166, whose resistance to ciprofloxacin is imparted mainly by an efflux mechanism as shown below, bears an Asp83 (GAT) to Tyr (TAT) mutation in ParC. This mutation should be providing SR166 with the intermediate resistance to fluoroquinolones other than ciprofloxacin. For determination of resistance originating with an efflux mechanism, MICs were determined in the presence and absence of reserpine (10 g/ml; Daiichi Pharmaceuticals) (4, 5, 9, 18). SR166 showed complete susceptibility to ciprofloxacin in the presence of reserpine. MICs of ciprofloxacin for HU85, HU86, and SR69 were only partially affected. Reserpine did not markedly affect the resistance of any of the strains to the other agents, indicating the lack of efflux mechanisms, which are inhibited by reserpine, assisting resistance to these drugs. Many nucleotide mutations, whose expressed amino acid residues were altered or not, were found in parC and gyrA QRDRs of SR27 and SR179, so we characterized the full DNA sequences of the gyrA and parC genes in these strains. The PCR fragments containing the entire coding region of the gyrA gene were obtained as described elsewhere (1). The nucleotide sequences of both genes were identical between the two strains. The two strains should be derived from the same clone. Randomly applied polymorphic DNA-PCR analysis confirms this observation (data not shown). Notably, SR27 and SR179 were isolated from the same hospital, suggesting the possibility of hospital infection. The sequence homologies of the parC and gyrA genes of SR27 and the S. pneumoniae standard strain R6 were 91.42 and 97.40%, respectively. The homology plot analysis (performed with DNASIS-Mac 3.5; Hitachi Soft Engineering, Tokyo, Japan) indicated that sequence dissimilarities of parC genes of SR27 and R6 were scattered throughout the whole coding region (Fig. 1). In contrast, the dissimilarities of the gyrA genes were concentrated on the 5⬘-terminal side of the gene. The failure of PCR amplification of the fragment containing the parC QRDR should be due to the sequence dissimilarity of parC genes in these strains. We performed phylogenetic tree analysis on the QRDRs in the parC and gyrA genes of S. pneumoniae strains and viridans streptococcus strains, including Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis (Fig. 2). Horizontal transfer of various genes among S. pneumoniae and viridans streptococci has been previously reported (13, 22); the sequences used in the analysis were either the S. pneumoniae sequences we determined in this study or S. pneumoniae and viridans streptococcus sequences derived from the GenBank
FIG. 1. Homology plots comparing the DNA sequences of the entire parC and gyrA genes of the fluoroquinolone-resistant S. pneumoniae strain SR27 and the standard strain R6. The nucleotide sequence of strain R6 was taken from the GenBank database (accession no. NC 003098).
FIG. 2. Phylogenetic trees based on the nucleotide sequences of the parC and gyrA QRDRs of various Streptococcus spp. The 218-nucleotide parC sequence (positions 187 to 404) and the 170-nucleotide gyrA sequence (positions 190 to 359) were analyzed by the multiple-alignment program Clustal W 1.5 and expressed as a phylogenetic tree by Dendromaker 4.1. The DNA sequences of strains without an asterisk have been previously reported and were taken from the GenBank database. The sequences of strains indicated by asterisk(s) were determined in this study. The fluoroquinolonesensitive and -resistant strains are marked by single and double asterisks, respectively. Sp, S. pneumoniae; Sm, S. mitis; So, S. oralis; Ss, S. sanguinis. 3313
3314
NOTES
database. Multiple sequence alignment was performed with Clustal W version 1.5 (24). Phylogenetic analysis and tree construction were performed with DendroMaker ver.4.1 (developed by T. Horiguchi and T. Imanishi, Center for Information Biology, National Institute of Genetics, Mishima, Japan) available on the Internet at http://www.cib.nig.ac.jp/dda/timanish/ dendromaker/home.html). Of the 12 strains examined in this study, only the parC and gyrA QRDR sequences of SR27 and SR179 showed a close phylogenetic relationship with sequences from some strains of viridans streptococci (Fig. 2). On the other hand, parE and gyrB QRDRs of all strains examined did not possess any mutations compared with the standard strain R6, except one, which results in an Ile460Val substitution as described above, in ParE. That horizontal transfer of these genes occurs between streptococcal strains had been previously suggested for strains 3180 and 3870 for both genes (11) and SPN1506 for parC (2), although the QRDR sequences differed from SR27 and SR179. In contrast, the parC and gyrA QRDR sequences of five other S. pneumoniae strains examined in this study are highly similar to the typical S. pneumoniae QRDRs. These observations suggest that while SR27 and SR179, which are identical, may have acquired fluoroquinolone resistance by spontaneous mutations together with horizontal gene transfer from viridans streptococci, the remaining five resistant strains gained resistance only from point mutations. Ferra´ndiz et al. (11) have suggested that interspecies horizontal gene transfer between S. pneumoniae and viridans streptococci is a mechanism by which these bacteria gain fluoroquinolone resistance. Similar genetic exchange is well known for genes encoding penicillin binding proteins, and this process is thought to mainly contribute to the emergence and worldwide prevalence of -lactram resistance (22). However, that such recombination also largely contributes to fluoroquinolone resistance has been disputed by Bast et al. (2), who noted that strains sharing such chimeric quinolone target genes are rarely found. In support of this conjecture, we identified only two strains (SR27 and SR179), which are suggested to be derived from one clone, as showing a relationship between their parC and gyrA QRDRs and those of other viridans streptococci. Thus, interspecies horizontal gene transfer does not appear to be a major mechanism by which S. pneumoniae develops fluoroquinolone resistance. In conclusion, we have found that fluoroquinolone-resistant S. pneumoniae strains occur frequently in the elderly. These strains showed high resistance to ciprofloxacin and levofloxacin and intermediate or high resistance to fluoroquinolones targeted for gram-positive bacteria, namely tosufloxacin, sparfloxacin, and gatifloxacin. Genetic analysis of QRDRs of target genes indicated that six distinct mutation patterns were identified among the seven resistant strains. This suggests that fluoroquinolone resistance occurs sporadically through acquired point mutations rather than by the spreading of a specific resistant mutant strain. The reason fluoroquinolone-resistant strains were not found in the children we tested may be that fluoroquinolones other than norfloxacin are not applicable to children in Japan. The usage of fluoroquinolones, including new-generation ones, against S. pneumoniae infection should merit more attention. This is also the case for -lactams and macrolides.
ANTIMICROB. AGENTS CHEMOTHER.
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