a Department of Oral Surgery, School of Medicine, Tokai University, Bouseidai, Isehara, Kanagawa, 259-1193 Japan; b Department of Genetics, and c Chemotherapy Division, Mitsubishi Kagaku Bio-Clinical Laboratories Inc., 3-30-1 Shimura, Itabashi-ku, Tokyo, 174-8555 Japan
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Abstract |
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Introduction |
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In recent years, however, strains of oral streptococci less susceptible to some fluoroquinolones have been isolated from patients,6 and a trend towards increasing fluoroquinolone resistance has been found in oral streptococci. A major mechanism of resistance to fluoroquinolones in Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae is target modification; mutations in the quinolone resistance determining region (QRDR) of the gyrA, parC and parE genes, result in decreased affinity for the quinolones.79 However, the mechanism of fluoroquinolone resistance in oral streptococci has not been documented.
The aim of the present study was to analyse the contribution to fluoroquinolone resistance of mutations in the QRDRs of the gyrA and parC genes of Streptococcus sanguis and Streptococcus anginosus isolated from dental infection. To this end, mutants were obtained by serial exposure of wild-type strains to ofloxacin and DU-6859a.
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Materials and methods |
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S. sanguis QS-951 (ofloxacin susceptible), S. sanguis QR-95101 (ofloxacin resistant) and S. anginosus QS-701 (ofloxacin susceptible) were isolated from patients with dental infections at Tokai University Hospital in 1995 and kept in skimmed milk at 80°C. The isolates were identified by the API 20 Strep test (bioMérieux, Marcy I'Etoile, France) as described in the manufacturer's manual. S. sanguis ATCC 10556 and S. anginosus ATCC 33397 were used as reference strains.
Antimicrobials
Antimicrobial agents used were ofloxacin (Daiichi Pharmaceutical Co., Tokyo, Japan), ciprofloxacin (Bayer Yakuhin, Osaka, Japan), norfloxacin (Kyorin Pharmaceutical Co., Tokyo, Japan), DU-6859a (Daiichi Pharmaceutical) and ampicillin (Sigma Chemical Co., St Louis, MO, USA).
Susceptibility testing
Drug susceptibility of test strains was determined by an agar dilution method according to the guidelines established by the NCCLS.10 Each strain (1 µL of 107 cfu/mL) was inoculated on to MuellerHinton agar (Difco Laboratories, Detroit, MI, USA), supplemented with 5% defibrinated sheep blood and containing serial dilutions of test drug. After incubation at 35°C for 20 h, the MIC was defined as the lowest concentration of the drug that completely inhibited bacterial growth.
Selection of fluoroquinolone resistance
The fluoroquinolone-susceptible strains S. sanguis QS-951 and S. anginosus QS-701 were used to generate fluoroquinolone-resistant mutants by a serial passage method. A 48 h growth of each strain on 5% defibrinated horse blood-containing Blood Agar base No. 2 (Oxoid, Basingstoke, UK; hereafter called blood agar) was inoculated with a swab on to the blood agar containing 0.5 x MIC of either ofloxacin or DU-6859a. The surface growth after 48 h incubation was subcultured on to the medium containing twice the previous concentration of antibiotic. After 20 h incubation, subculture was repeated twice more using medium containing the same concentration of antibiotic. This procedure was repeated serially until bacterial growth occurred on medium containing the drug at a final concentration of 256 times the original MIC.
Analysis of the gyrA gene
Chromosomal DNA was isolated from each strain by phenol/chloroform extraction and ethanol precipitation. PCR amplification of the DNA regions contributing to the expression of fluoroquinolone resistance (corresponding to nucleotide sequence 164380; codons 55127 of Escherichia coli KL-16) was performed with two primers; forward, 5'-TGGGTGTGACACC(AGCT)GA(GT)AA(AG)-3', and reverse, 5'-ATACGTGCTTC(AG)GTATA(AC)CG-3', which were designed on the basis of previously published sequence data (GenBank accession number X06744). Amplification was performed in a total volume of 50 µL containing 1.0 µL template DNA, 1.0 µL of each deoxynucleoside triphosphate (10 mM), 5.0 µL 10 x Taq DNA polymerase buffer (100 mM TrisHCl pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.1% gelatin) (Boehringer-Mannheim GmbH, Mannheim, Germany), 2.0 µL of each primer (25 pmol/µL) and 0.25 µL Taq DNA polymerase (5 U/µL, BoehringerMannheim). The temperature profile for the amplification was as follows: 40 cycles of denaturation at 93°C for 30 s, annealing at 52°C for 1 min and extension at 72°C for 1 min. DNA sequencing of PCR products was carried out with a model 373A DNA autosequencer (Perkin-Elmer, Applied Biosystems Division, Foster City, CA, USA).
Analysis of the parC gene
Chromosomal DNA was isolated as described above. PCR amplification of the DNA regions contributing to the expression of resistance to fluoroquinolones (corresponding to nucleotides 148458; codons 50152 of S. pneumoniae) was performed with two primers: forward, 5'-AAGGATAGCAATACTTTT-3', and reverse, 5'-GTTGGTTCTTTCTCCGTATCG-3', which were published previously.11 Amplification was performed in a total volume of 50 µL containing 1.0 µL template DNA, 0.5 µL each deoxynucleoside triphosphate (10 mM), 5.0 µL 10 x Taq DNA polymerase buffer, 1.0 µL of each primer (25 pmol/µL) and 0.25 µL Taq DNA polymerase (5 U/µL). The temperature profile for the amplification was as follows: 40 cycles of denaturation at 93°C for 30 s, annealing at 48°C for 1 min and extension at 72°C for 1 min. DNA sequencing of PCR products was carried out as described for analysis of the gyrA gene.
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Results |
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As shown in Table I, the clinical isolate S. sanguis QR-95101 was resistant to ofloxacin, ciprofloxacin and norfloxacin with MICs of 32, 64 and 128 mg/L, respectively, but was susceptible to DU-6859a and ampicillin, with an MIC of 0.5 mg/L each. Another S. sanguis isolate, QS-951, was susceptible to ofloxacin and ciprofloxacin with an MIC of 2 mg/L each, but was resistant to norfloxacin (MIC 16 mg/L). The MIC of DU-6859a and ampicillin against S. sanguis QS-951 was 0.12 and 0.25 mg/L, respectively. S. sanguis QS-951 was exposed to ofloxacin to generate fluoroquinolone-resistant mutants. The resulting mutant, QS-951OFm, was resistant to ofloxacin, ciprofloxacin and norfloxacin (MICs 128 mg/L) and also to DU-6859a (MIC 16 mg/L). Furthermore, the resistant mutant QS-951DUm, obtained by exposure to DU-6859a, exhibited high-level resistance to fluoroquinolones (MIC of DU-6859a 64 mg/L; MICs of other fluoroquinolones >128 mg/L), but was as susceptible to ampicillin (MIC 0.25 mg/L) as the parental strain. When S. anginosus QS-701, a fluoroquinolonesusceptible isolate, was similarly exposed to ofloxacin, the resulting mutant QS-701OFm, appeared to be resistant to ofloxacin, ciprofloxacin and norfloxacin (MICs 32128 mg/L), but less so to DU-6859a (MIC 4 mg/L). QS-701DUm had high-level resistance to all the fluoroquinolones including DU-6859a (MIC 32 mg/L), but was still highly susceptible to ampicillin without any change in the initial MIC.
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The QRDRs of the gyrA and parC genes were sequenced in both the parental strains and fluoroquinolone-resistant mutants. As shown in Table II, the fluoroquinolonesusceptible isolates QS-951, QS-701 and the two reference strains of S. sanguis had the same amino acids at Ser83 and Glu87 in the gyrA gene and also at Ser79 and Glu135 in the parC gene. S. sanguis QS-951 and S. anginosus QS-701 were repeatedly exposed to either ofloxacin or DU-6859a to select mutants resistant to fluoroquinolones. The ofloxacin-selected resistant mutant QS-951OFm had a Ser83
Tyr substitution in the gyrA gene, but no change at Glu87. The resistant mutant also had a Ser79
Phe substitution in the parC gene but no change at Glu135. The DU-6859a-selected resistant mutant QS-951DUm had a Ser83
Phe and a Glu87
Lys substitution in the gyrA gene. The resistant mutant had also a Ser79
Ile substitution in the parC gene but no change at Glu135. S. sanguis QR-95101, a fluoroquinolone-resistant clinical isolate, had a Ser83
Phe substitution in the gyrA gene but no change at Glu87, and also had a Ser79
Phe substitution in the parC gene but no change at Glu135.
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Discussion |
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Although few studies to date have examined the mechanism of fluoroquinolone resistance in oral streptococci, the mechanism of resistance in S. pneumoniae has been well investigated. Janoir et al.13 studied resistance to fluoroquinolones in in vitro-selected mutants and clinical isolates of S. pneumoniae, and found that mutations in both gyrA and parC were required for high-level resistance, whereas either a gyrA or a parC mutation alone was associated with expression of low-level resistance. Similar findings were reported by Tankovic et al.14 who noted that parC was the primary target for low-level fluoroquinolone resistance in S. pneumoniae. In the present study, we confirmed that highly resistant strains of oral streptococci had mutations in both gyrA and parC. In a recent study, Jorgensen et al.9 indicated that gyrA and parE, instead of parC, also participated in decreased susceptibility of some strains of highly ofloxacin-resistant S. pneumoniae. To evaluate the resistance of oral streptococci to fluoroquinolones, genetic analysis of the QRDR, including gyrA, parC and parE, is necessary. We isolated S. sanguis QR-95101, which was resistant to ofloxacin, ciprofloxacin and norfloxacin but susceptible to DU-6859a, from a patient with dental infection. To assess whether fluoroquinolone derivatives may generate different mutations in the gyrA and parC genes of oral streptococci, we generated fluoroquinolone-resistant mutants by serial passage of S. sanguis and S. anginosus on agar containing increasing concentrations of an old (ofloxacin) and a new (DU-6859a) fluoroquinolone.15 It was reported that DU-6859a had potent activity against Gram-positive and -negative bacteria and was active against fluoroquinolone-resistant strains of Pseudomonas aeruginosa with altered DNA gyrase and of Klebsiella pneumoniae and Enterobacter cloacae with altered DNA gyrase and parC.16 In the present study, considerable differences were found in levels of resistance to DU-6859a, ofloxacin and ciprofloxacin between a clinical isolate (QR-95101) and laboratory-derived resistant mutants (QS-951OFm and QS-951DUm) of S. sanguis. Both the laboratory-derived resistant strains were resistant to all the quinolones tested, whereas the clinical isolate QR-95101 was highly susceptible to DU-6859a. These results suggested the possible presence of different types of mutation in the gyrA or parC genes in these resistant strains. The effect of ciprofloxacin (an older fluoroquinolone) and clinafloxacin (a newer type) on the selection of drug resistance in S. pneumoniae strains was studied by the transfer method, as used in our study.17 With ciprofloxacin, high-level resistance was selected in the second step in the serial transfers, whereas with clinafloxacin, resistance was selected in a stepwise manner, involving both gyrA and parC mutations.17 These results were similar to ours with DU-6859a. DNA sequence analysis of S. sanguis QS-951DUm showed that the DU-6859a-selected resistance was associated with gyrA substitutions at codons 83 and 87, and the same changes were also found in the gyrA gene of S. anginosus QS-701DUm. However, the gyrA mutations of S. sanguis QS-951OFm and QR-95101 (a clinical isolate) were associated with a single amino acid substitution, Ser83Tyr or Phe, respectively. Furthermore, S. sanguis QS-951DUm had a mutation in the parC gene (Ser79
Ile) that differed from that seen in QS-951OFm and QR-95101 (Ser79
Phe). Although Streptococcus spp., including oral streptococci, were originally susceptible to fluoroquinolones, recent studies indicate that resistance to currently available fluoroquinolones is an emerging problem.6 The clinical isolate S. sanguis QR-95101 used in this study was resistant to fluoroquinolones such as ofloxacin, ciprofloxacin and norfloxacin, but remained fully susceptible to DU-6859a. This strain had one mutation each in gyrA at codon 83 and in parC at codon 79.
If new fluoroquinolones such as DU-6859a become widely used in place of the fluoroquinolones currently available, resistance may still be expected to arise, in part via gyrA or parC mutations. It was noteworthy that the same amino acid substitution (Glu87Lys) was also associated with fluoroquinolone resistance in S. sanguis after serial exposure to DU-6859a. Under the selective pressure of intense fluoroquinolone use, a variety of clinical isolates with different mechanisms and levels of resistance may be expected to develop from the previously susceptible population of Gram-positive bacteria, including oral streptococci from oral and dental infections.
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Notes |
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References |
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Pan, X. S. & Fisher, L. M. (1998). DNA gyrase and topoisomerase IV are dual targets of clinafloxacin action in Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 42, 281016.
Received 23 August 1999; returned 12 November 1999; revised 2 December 1999; accepted 12 January 2000