Comparison of gyrA and parC mutations and resistance levels among fluoroquinolone-resistant isolates and laboratory-derived mutants of oral streptococci

Akihiro Kanekoa,*, Jiro Sasakia, Mitsunobu Shimadzub, Akiko Kanayamac, Takeshi Saikac and Intetsu Kobayashic

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Laboratory-derived fluoroquinolone-resistant mutants were obtained by serial passage of Streptococcus sanguis and Streptococcus anginosus isolates on agar containing increasing concentrations of old and new fluoroquinolones, ofloxacin and DU-6859a, respectively. Sequencing of an S. sanguis isolate exposed to DU-6859a showed that resistance was associated with two mutations in the quinolone resistance determining region (QRDR) of the gyrA gene (Ser83->Phe; Glu87->Lys), and with a mutation in the parC gene (Ser79->Ile). However, different mutations in the gyrA gene (Ser83->Tyr) and parC gene (Ser79->Phe) were found in a S. sanguis isolate exposed to ofloxacin. A fluoroquinolone-resistant isolate, QR-95101, from a dental infection, had a single mutation in the gyrA gene (Ser83->Phe) and in the parC gene (Ser79->Phe). Two fluoroquinolone-resistant mutants, QS-701OFm and QS-701DUm, were obtained from S. anginosus QS-701, by exposure to ofloxacin and DU-6859a, respectively. These mutants showed a common substitution at codon 83 (Ser->Phe) in the gyrA gene but had different substitutions at codon 87 (QS-701OFm, Glu->Gln; QS-701DUm, Glu->Lys). They also had different substitutions at codons 79 and 135 in the parC gene (QS-701OFm, Ser79->Leu but no change at Glu135; QS-701DUm, Ser79->Ile and Glu135->Gln). The resistance levels of the DU-6859a-selected resistant S. sanguis mutant QS-951DUm to DU-6859a, ofloxacin, ciprofloxacin and norfloxacin were higher than those of the ofloxacin-selected resistant mutant QS-951OFm. However, ampicillin susceptibilities of these mutants were not different from the parental strains. In S. anginosus, the DU-6859a-selected fluoroquinolone-resistant mutant QS-701DUm was resistant to all the fluoroquinolones tested, while the ofloxacin-selected mutant QS-701OFm was resistant to three fluoroquinolones, but not DU-6859a. The results indicate that different fluoroquinolones select distinct mutations in the QRDR of the gyrA and parC genes in oral streptococci. The gyrA or parC mutation in oral streptococci may determine the levels of fluoroquinolone resistance.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Ofloxacin was the first fluoroquinolone used to treat oral and dental infections in Japan. Following its introduction in 1988, six different fluoroquinolone derivatives are presently used for infections in this field. Fluoroquinolones formerly found only limited use in oral or dental infections, since the drugs were generally less active than penicillins against oral streptococci, the main pathogens in such infections.1 However, fluoroquinolones have become more widely used for treatment of oral infections owing to decreased susceptibility of the bacteria to penicillins2 and the development of new fluoroquinolones with enhanced activity against both Gram-positive and -negative bacteria.35

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.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test strains

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 Mueller–Hinton 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 164–380; codons 55–127 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 Tris–HCl 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, Boehringer–Mannheim). 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 148–458; codons 50–152 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.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Drug susceptibility

As shown in Table IGo, 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 32–128 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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Table I. Drug susceptibilities of ofloxacin- or DU-6859a-selected resistant mutants of S. sanguis and S. anginosus
 
gyrA and parC mutations

The QRDRs of the gyrA and parC genes were sequenced in both the parental strains and fluoroquinolone-resistant mutants. As shown in Table IIGo, 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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Table II. gyrA and parC mutations of ofloxacin- or DU-6859a-selected strains of S. sanguis and S. anginosus
 
In the case of S. anginosus QS-701, the ofloxacin-selected mutant QS-701OFm and the DU-6859a-selected resistant mutant QS-701DUm each exhibited the same Ser83->Phe substitution in the gyrA gene but had distinct substitutions at Glu87->Gln (ofloxacin-selected mutant) and Glu87->Lys (DU-6859a-selected mutant). Furthermore, the QS-701OFm and QS-701DUm mutants exhibited distinct substitutions in the parC gene; the QS-701OFm mutant had a Ser79->Leu substitution but no change at Glu135, and the QS-701DUm mutant had a Ser79->Ile and a Glu135->Gln substitution.


    Discussion
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 Materials and methods
 Results
 Discussion
 References
 
Owing to their excellent antibacterial activity, tissue distribution and oral bioavailability, fluoroquinolones have been widely used in place of penicillins for treatment of dental and oral infections. However, in a survey of oral streptococci isolated from dental and oral infections in 1995, we found a trend towards decreased susceptibility to fluoroquinolones.12

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, Ser83->Tyr 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 (Glu87->Lys) 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.


    Notes
 
* Corresponding author. Tel: +81-463-93-1121; Fax: +81-463-91-5902; E-mail: akihiro{at}is.icc.u-tokai.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Chin, N. X. & Neu, H. C. (1984). Ciprofloxacin, a quinolone carboxylic acid compound active against aerobic and anaerobic bacteria. Antimicrobial Agents and Chemotherapy 25, 319–26.[ISI][Medline]

2 . Bantar, C., Fernandez Canigia, L., Relloso, S., Lanza, A., Bianchini, H. & Smayevsky, J. (1996). Species belonging to the ‘Streptococcus milleri’ group: antimicrobial susceptibility and comparative prevalence in significant clinical specimens. Journal of Clinical Microbiology 34, 2020–2.[Abstract]

3 . Piddock, L. J. (1994). New quinolones and gram-positive bacteria. Antimicrobial Agents and Chemotherapy 38, 163–9.[ISI][Medline]

4 . Sato, K., Hoshino, K., Tanaka, M., Hayakawa, I. & Osada, Y. (1992). Antimicrobial activity of DU-6859, a new potent fluoroquinolone, against clinical isolates. Antimicrobial Agents and Chemotherapy 36, 1491–8.[Abstract]

5 . Cantón, E., Pemán, J., Jimenez, M. T., Ramón, M. S. & Gobernado, M. (1992). In vitro activity of sparfloxacin compared with those of five other quinolones. Antimicrobial Agents and Chemotherapy 36, 558–65.[Abstract]

6 . Kaneko, A., Tomita, F., Karakida, K. & Yamane, N. (1994). In-vitro antibacterial activity of SY5555 in dentistry and oral surgery. Chemotherapy (Tokyo) 42, 94–100.

7 . Margerrison, E. E., Hopewell, R. & Fisher, L. M. (1992). Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins. Journal of Bacteriology 174, 1596–603.[Abstract]

8 . Ferrero, L., Cameron, B. & Crouzet, J. (1995). Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 39, 1554–8.[Abstract]

9 . Jorgensen, J. H., Weigel, L. M., Ferraro, M. J., Swenson, J. M. & Tenover, F. C. (1999). Activities of newer fluoroquinolones against Streptococcus pneumoniae clinical isolates including those with mutations in the gyrA, parC and parE loci. Antimicrobial Agents and Chemotherapy 43, 329–34.[Abstract/Free Full Text]

10 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically—Fourth Edition: Approved Standard M7-A4. NCCLS, Villanova, PA.

11 . Muñoz, R. & De La Campa, A. G. (1996). ParC subunit of DNA topoisomerase IV of Streptococcus pneumoniae is a primary target of fluoroquinolones and cooperates with DNA gyrase A subunit in forming resistance phenotype. Antimicrobial Agents and Chemotherapy 40, 2252–7.[Abstract]

12 . Sasaki, J., Karakida, K., Yamane, N., Ohta, Y., Busujima, Y. & Takakura, A. (1995). Antibacterial activity of balofloxacin against oral bacteria and the penetration into saliva and dental extraction wounds. Japanese Journal of Chemotherapy 43, 490–4.

13 . Janoir, C., Zeller, V., Kitzis, M. D., Moreau, N. J. & Gutmann, L. (1996). High-level fluoroquinolone resistance in Streptococcus pneumoniae requires mutations in parC and gyrA. Antimicrobial Agents and Chemotherapy 40, 2760–4.[Abstract]

14 . Tankovic, J., Perichon, B., Duval, J. & Courvalin, P. (1996). Contribution of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of Streptococcus pneumoniae obtained in vivo and in vitro. Antimicrobial Agents and Chemotherapy 40, 2505–10.[Abstract]

15 . Kitamura, A., Hoshino, K., Kimura, Y., Hayakawa, I. & Sato, K. (1995). Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy 39, 1467–71.[Abstract]

16 . Deguchi, T., Yasuda, M., Kawamura, T., Nakano, M., Ozeki, S., Kanematsu, E. et al. (1997). Improved antimicrobial activity of DU-6859a, a new fluoroquinolone, against quinolone-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates with alterations in GyrA and ParC proteins. Antimicrobial Agents and Chemotherapy 41, 2544–6.[Abstract]

17 . 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, 2810–16.[Abstract/Free Full Text]

Received 23 August 1999; returned 12 November 1999; revised 2 December 1999; accepted 12 January 2000