Abstract
We determined whether gyrA mutations were present in fluoroquinolone-resistant laboratory mutants derived from the Bacteroides fragilis reference strain ATCC 25285 and in clinical isolates of B. fragilis. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of gyrA mutations, whereas two of the three CIP-selected second-step mutants studied presented the same gyrA mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 μg/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of B. fragilis were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO.
Bacteroides fragilis is the anaerobic organism most frequently isolated from patients with bacteremia (with an attributable mortality of 19.3% [11]) and intra-abdominal infections (8). Older fluoroquinolones, including ciprofloxacin and ofloxacin, show little activity against anaerobes and B. fragilis in particular, but some of the newer compounds, notably clinafloxacin, moxifloxacin, and trovafloxacin, are much more active in vitro (1, 5) as well as in vivo (13, 14).
Fluoroquinolones block DNA replication by forming a stable ternary complex with DNA and type II DNA topoisomerases: DNA gyrase and DNA topoisomerase IV (4). Gyrase is a tetrameric enzyme consisting of two A and two B subunits encoded by the gyrA and gyrB genes, respectively. It catalyzes ATP-dependent negative supercoiling of double-stranded DNA. Topoisomerase IV, also a heterodimer encoded by the parC and parE genes, is involved in chromosomal partitioning. Mutational alterations of the subunits of DNA gyrase or topoisomerase IV, as well as altered permeation mechanisms, have been shown to be related to quinolone resistance (4).
The prevalence of acquired resistance of B. fragilis to the new fluoroquinolones, in particular trovafloxacin, appears to be low worldwide, less than 5% (12; L. Dubreuil, E. Singer, S. Bland, and A. Sedallian, Prog. Abstr. 9th Eur. Cong. Clin. Microbiol. Infect. Dis., abstr. 793, p. 297, 1999). However, two recent reports from the United States found surprisingly high frequencies of resistance to trovafloxacin (MIC, ≥4 μg/ml): 16% (D. R. Snydman, L. A. MacDermott, and N. V. Jacobus, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 906, p. 218, 1999) and 48% (D. R. Gustafson, L. M. Sloan, and J. E. Rosenblatt, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 905, p. 218, 1999). The gyrA and gyrB genes from B. fragilis have recently been cloned and sequenced (9), and levofloxacin-selected second-step but not first-step in vitro mutants were found to carry a gyrA mutation leading to the replacement of Ser82 (equivalent to resistance hot spot Ser83 of GyrA of Escherichia coli) with Phe (9). In addition, active efflux of norfloxacin, resembling that produced by the NorA/Bmr-type transporter in gram-positive bacteria (7), may also exist in B. fragilis (6).
Our aims were to determine the prevalence of resistance to trovafloxacin in clinical strains of B. fragilis isolated from our hospital and to look for the role of gyrA mutations in the resistance of clinical isolates as well as of laboratory mutants selected with either ciprofloxacin or trovafloxacin.
MATERIALS AND METHODS
Bacterial strains.
One hundred fifty strains of B. fragilis isolated from clinical samples between 1995 and 1999 and one reference strain, B. fragilis ATCC 25285, were studied. The origins of the strains were as follows: cutaneous suppurations, 34; intra-abdominal suppurations, 30; suppurations of unspecified origin, 20; blood, 29; stools, 1; unknown, 36. To evaluate the role of gyrA in fluoroquinolone resistance, we randomly selected 9 fluoroquinolone-resistant isolates, with various resistance levels, among the 150 clinical isolates, and we also studied 3 resistant clinical strains isolated in 1996 in eastern France: L1, L2 (Nancy), and L3 (Annecy). Identification to the species level was performed with the API 20A system (bioMérieux, Marcy l'Etoile, France).
Susceptibility testing.
MICs were determined by the agar dilution technique using Wilkins-Chalgren medium (Oxoid, Lyon, France) supplemented with 5% sheep blood and an inoculum of 105 CFU per spot. MICs were read after 48 h of incubation at 37°C in an anaerobic atmosphere. Breakpoints used for ciprofloxacin and trovafloxacin were those recommended by the Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) (2). The quinolone agents tested were provided by the manufacturers as powders suitable for susceptibility testing. We also tested the effects of reserpine (Sigma, Saint-Quentin Fallavier, France) at a concentration of 20 μg/ml on the MICs of norfloxacin and trovafloxacin.
Frequency of mutation.
The reference strain B. fragilis ATCC 25285 was used as a parental strain to select quinolone-resistant in vitro mutants in two successive steps by plating an inoculum of approximately 1010 bacteria onto Wilkins-Chalgren agar (Oxoid) supplemented with 5% sheep blood and containing either ciprofloxacin or trovafloxacin at two times the MIC. After 48 h of anaerobic incubation at 37°C, the colonies were counted and the frequencies of mutation were determined relative to the total viable count of organisms plated.
Amplification by PCR of the QRDR of the gyrA gene.
The quinolone resistance-determining region (QRDR) of gyrA was PCR amplified with oligonucleotide primers 5′-TGGAACTGGGAAATACGTCAG and 5′-GCATCACTTTGGGTTCCATC, generating a DNA fragment of 320 bp corresponding to nucleotide positions 152 to 471 of the gyrA gene of B. fragilis (9). Chromosomal DNA was prepared using the QiaAmp Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's recommendations. PCR was carried out in a 100-μl volume containing 250 μM deoxynucleoside triphosphates (Pharmacia Biotech, Uppsala, Sweden), 10 μl of 10-fold diluted DNA extract, 100 pmol of each primer, and 2.5 U of Taq DNA polymerase (Boehringer Mannheim, Meylan, France). Amplification was performed over 40 cycles of 1 min at 95°C, 1 min at 50°C, and 1 min at 72°C.
Restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis of PCR-amplified DNA.
Amplification products were digested with HinfI restriction endonuclease (Pharmacia) and subjected to electrophoresis in a 2% agarose gel in order to detect a mutation at codon 81 or 82 of gyrA of B. fragilis. For nucleotide sequencing, amplification products were purified with the microspin columns S-400 HR (Pharmacia) and analyzed using the Taq-Dye-Deoxy-Terminator sequencing kit (Perkin-Elmer Biosystems, Courtaboeuf, France) and the automatic DNA sequencer ABI Prism 310 (Perkin-Elmer Biosystems). The oligonucleotide primers used were identical to those for DNA amplification.
RESULTS AND DISCUSSION
Prevalence of resistance to trovafloxacin and of high-level resistance to ciprofloxacin among isolates of B. fragilis from our hospital (1995 through 1999).
The MICs of trovafloxacin and ciprofloxacin at which 50% of the isolates were inhibited (MIC50s) were 0.25 and 4 μg/ml, respectively (Fig. 1). By use of the breakpoint concentrations recommended by the CA-SFM (2), 87% (131 of 150), 3% (4 of 150), and 10% (15 of 150) of the strains investigated were classified as susceptible (MIC ≤ 1 μg/ml), intermediate (MIC = 2 μg/ml), and resistant (MIC ≥ 4 μg/ml) to trovafloxacin, respectively. Furthermore, 25% (38 of 150) of the strains studied required a trovafloxacin MIC greater than or equal to 1 μg per ml and had high-level resistance to ciprofloxacin (MIC ≥ 32 μg/ml). Although the MICs of trovafloxacin for half of these isolates were not sufficiently increased for the strains to be considered intermediate or resistant by standard criteria, they most probably had already acquired at least one mechanism of resistance to fluoroquinolones. These high-level ciprofloxacin-resistant strains were more frequent in 1995 (5 of 16; 31%) and 1996 (5 of 15; 33%) than in 1997 (14 of 57; 25%), 1998 (11 of 47; 23%), and 1999 (3 of 15; 20%). This decrease probably parallels a decreased usage of fluroquinolones in our hospital, from ≈12 kg/year in 1995 to ≈7 kg/year in 1998.
FIG. 1.
Distribution of trovafloxacin and ciprofloxacin MICs against 150 clinical strains of B. fragilis.
The prevalence of resistance to trovafloxacin in our hospital was unexpectedly high compared to findings of less than 5% intermediate or resistant strains reported from previous studies in Europe and the United States (12; Dubreuil et al., 9th Eur. Cong. Clin. Microbiol. Infect. Dis.). However, two recent reports from the United States found even higher frequencies of trovafloxacin resistance in clinical isolates of B. fragilis (MIC ≥ 4 μg/ml) than that reported here: 16% (87 of 530 isolates from 1996 through 1997) in a multicenter study (Snydman et al., 39th ICAAC) and 48% (43 of 90 isolates from 1998 through 1999) in a report from the Mayo Clinic (Gustafson et al., 39th ICAAC). Taken together, these data suggest that the prevalence of fluoroquinolone resistance among B. fragilis clinical isolates may vary substantially from one hospital to another. These variations may correlate with the level of fluoroquinolone usage, but this remains to be determined.
Presence of gyrA mutations in fluoroquinolone-resistant clinical isolates.
We studied 12 strains for which the MICs of ciprofloxacin ranged from 16 to 128 μg/ml. Compared to trovafloxacin, sparfloxacin was two- to eightfold less active, moxifloxacin was approximately as active, and clinafloxacin was either as active or two- to fourfold more active. Thus, acquired resistance to fluoroquinolones in clinical isolates of B. fragilis does not modify the relative activities of the new fluoroquinolones.
We determined the nucleotide sequence of the QRDR of gyrA from codon 55 to 138. The same mutational alteration, Ser82Phe, was found for isolates C5, C14, and L1, which showed the highest level of resistance to trovafloxacin (MIC = 4 μg/ml [Table 1]). The Ser82 residue in the GyrA of B. fragilis is equivalent to resistance hot spot Ser83 in the GyrA of E. coli. Analysis of the restriction map of the PCR-amplified 320-bp fragment of gyrA of B. fragilis showed that this gyrA mutation was able to abolish one of the two existing HinfI sites (the restriction site GANTC was changed to AANTC in gyrA mutants). This was confirmed by RFLP analysis (data not shown).
TABLE 1.
Susceptibilities of clinical isolates of B. fragilis to fluoroquinolones and amino acid substitutions in the A subunit of DNA gyrase
| Strain | MICa (μg/ml)
|
Amino acid substitution in GyrA | ||||
|---|---|---|---|---|---|---|
| CIP | SPA | CLI | MOX | TRO | ||
| C3 | 16 | 2 | 0.25 | 0.5 | 1 | |
| C10 | 16 | 2 | 0.25 | 0.5 | 0.5 | |
| C13 | 16 | 2 | 0.25 | 0.5 | 1 | |
| C16 | 16 | 1 | 0.25 | 0.5 | 0.5 | |
| C9 | 32 | 4 | 0.25 | 0.5 | 1 | |
| L2 | 32 | 8 | 1 | 2 | 1 | |
| C5 | 64 | 16 | 1 | 4 | 4 | Ser82→Phe |
| C12 | 64 | 4 | 0.5 | 1 | 1 | |
| C14 | 64 | 32 | 2 | 8 | 4 | Ser82→Phe |
| C23 | 128 | 16 | 2 | 4 | 2 | |
| L1 | 128 | 32 | 2 | 8 | 4 | Ser82→Phe |
| L3 | 128 | 16 | 2 | 4 | 2 | |
CIP, ciprofloxacin; SPA, sparfloxacin; CLI, clinafloxacin; MOX, moxifloxacin; TRO, trovafloxacin.
Presence of gyrA mutations in fluoroquinolone-resistant in vitro mutants.
The reference strain B. fragilis ATCC 25285 (ciprofloxacin and trovafloxacin MICs, 4 and 0.25 μg/ml, respectively) was plated onto agar containing either ciprofloxacin or trovafloxacin at two times the MIC. With ciprofloxacin, first-step mutants were obtained at a frequency of 3 · 10−8. Two of these, Mc1 and Mc2, were selected at random for further study. The activity of trovafloxacin against these mutants was unchanged, whereas that of ciprofloxacin was reduced twofold. Mc1 and Mc2 were plated again at two times the MIC of ciprofloxacin. Second-step mutants were obtained at a mean frequency of 2 · 10−9. Three second-step mutants were further studied: Mc3, deriving from Mc1, and Mc4 and Mc5, deriving from Mc2. The relative activities of clinafloxacin, moxifloxacin, sparfloxacin, and trovafloxacin against these mutants (Table 2) were similar to those against the clinical isolates described above (Table 1).
TABLE 2.
Susceptibilities of in vitro mutants of B. fragilis to fluoroquinolones and amino acid substitutions in the A subunit of DNA gyrase
| Straina | MICb (μg/ml)
|
Amino acid substitution in GyrA | ||||
|---|---|---|---|---|---|---|
| CIP | SPA | CLI | MOX | TRO | ||
| ATCC 25285 | 4 | 1 | 0.12 | 0.25 | 0.25 | |
| Mc1 and Mc2 | 8 | 1 | 0.12 | 0.5 | 0.25 | |
| Mc3 | 32 | 8 | 1 | 2 | 2 | |
| Mc4 and Mc5 | 64 | 16 | 2 | 8 | 4 | Ser82→Phe |
| Mt1 | 4 | 1 | 0.12 | 0.25 | 2 | Ala118→Val |
| Mt2 | 8 | 8 | 0.5 | 2 | 2 | |
| Mt3 | 16 | 4 | 0.5 | 2 | 4 | Asp81→Asn |
Mutants Mc1 and Mc2 were selected from the parental strain ATCC 25285 on ciprofloxacin at two times the MIC. Mutant Mc3 and mutants Mc4 and Mc5 were selected from first-step mutants Mc1 and Mc2, respectively, on ciprofloxacin at two times the MIC. Mutants Mt1 through Mt3 were selected from the parental strain ATCC 25285 on trovafloxacin at two times the MIC.
CIP, ciprofloxacin; SPA, sparfloxacin; CLI, clinafloxacin; MOX, moxifloxacin; TRO, trovafloxacin.
The same gyrA mutation that was found in clinical isolates (derived substitution Ser82Phe) was found only for second-step mutants Mc4 and Mc5, for which the MICs of ciprofloxacin and trovafloxacin showed the highest increase (16-fold) and which were classified as trovafloxacin resistant (MICs, 4 μg/ml [Table 2]). The fluoroquinolone resistance phenotypes of these two laboratory mutants were similar to those of the above-described clinical isolates carrying gyrA mutations. Thus, with ciprofloxacin as a selector, two consecutive mutational events, of which the second is a typical gyrA mutation, can confer trovafloxacin resistance in B. fragilis. Similar results have been obtained with levofloxacin as a selector: levofloxacin-selected first-step mutants were devoid of gyrA or gyrB mutations, but a gyrA mutation identical to that described above occurred in second-step mutants (9). Taken together, these data suggest that DNA gyrase of B. fragilis could be only a secondary target of ciprofloxacin and levofloxacin, similar to what is observed in gram-positive bacteria (4), topoisomerase IV being the primary target. This remains to be verified by amplification and sequencing of the critical regions of the parC and parE genes from first-step in vitro mutants and low-level-resistant clinical isolates.
With trovafloxacin at two times the MIC, first-step mutants were obtained at a frequency of 1 · 10−9. Three of these, Mt1, Mt2, and Mt3, were selected at random for further study. Despite repeated attempts, we were not able to obtain second-step mutants with trovafloxacin at two times the MIC as a selector (frequency of mutation, <8 · 10−11). Sharp increases in the MICs of trovafloxacin, 8- to 16-fold, were observed for the three trovafloxacin-selected first-step mutants studied, whereas the activity of ciprofloxacin was only slightly reduced (2- to 4-fold) in two cases and was even unchanged for mutant Mt1. Our findings confirm those of a previous study that had shown that ciprofloxacin and trovafloxacin selected for mutants of B. fragilis with specific phenotypes (L. J. V. Piddock and V. Ricci, Abstr. 38th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C180, p. 121, 1998). Furthermore, these data suggest that DNA gyrase may be the primary target of trovafloxacin in B. fragilis. This may represent another example of the fact that different fluoroquinolones can have different primary targets in the same bacterial species, as has already been shown for Streptococcus pneumoniae (10).
Mutant Mt1 was as susceptible as the parental strain to ciprofloxacin and carried an unusual gyrA mutation leading to an Ala118Val substitution. Mutant Mt3 carried a novel, yet undescribed gyrA mutation leading to an Asp81Asn substitution; Asp81 is equivalent to Asp82 of the GyrA of E. coli. Thus, trovafloxacin appears to be able to select for unusual gyrA mutations that diminish the activity of trovafloxacin selectively. This is particularly striking for the Ala118Val substitution in mutant Mt1, which is associated with an eightfold increase in the MIC of trovafloxacin, whereas the MICs of ciprofloxacin, sparfloxacin, moxifloxacin, and clinafloxacin are unchanged (Table 2). The reason for that dissociated resistance could be the bulkiness of both the side chain of valine (three methyl groups instead of one for alanine) and the N-1 substituent of trovafloxacin (a difluorophenyl instead of a cyclopropyl for the other drugs tested). An identical substitution in GyrA has been found in an in vitro mutant of Salmonella enterica serovar Typhimurium selected on nalidixic acid (mutant L30 nalA), and the MIC of ciprofloxacin for that mutant remained low (0.03 μg/ml) (3).
Effects of reserpine on the MICs of norfloxacin and trovafloxacin.
It has been shown that norfloxacin is actively pumped out in wild-type B. fragilis and that norfloxacin efflux is decreased in the presence of reserpine (6). In addition, a one-step norfloxacin-selected mutant with threefold increases in the MICs of norfloxacin and ethidium bromide and no change in the MIC of sparfloxacin has been described (6). This suggests that enhanced active efflux with a specificity similar to that of the NorA/Bmr-type transporter of gram-positive bacteria could be involved in the fluoroquinolone resistance of B. fragilis.
To detect such a mechanism, we determined the MICs of norfloxacin and trovafloxacin with or without 20 μg of reserpine/ml for reference strain ATCC 25285, the 12 fluoroquinolone-resistant clinical isolates described above, and the resistant in vitro mutants. For all strains tested, the MICs of trovafloxacin were unaffected by the presence of reserpine whereas the activity of norfloxacin was reduced only twofold in the presence of reserpine (data not shown). Thus, it was unlikely that a NorA/Bmr-type efflux was implicated in the resistance of the strains studied.
In conclusion, the resistance phenotypes and genotypes observed in clinical isolates were similar to those obtained in vitro with ciprofloxacin. In contrast, trovafloxacin selected for peculiar mutants. This raises the possibility of similar phenomena occurring in vivo, either with trovafloxacin or with other new fluoroquinolones.
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