Characterization of the molecular mechanisms of quinolone resistance in Yersinia enterocolitica O:3 clinical isolates

OBJECTIVES: The aim of this study was to determine the roles of mutations in the gyrA and parC genes and the overexpression of efflux pump(s) as mechanisms of resistance to quinolones. Forty-five Yersinia enterocolitica O:3 clinical isolates (41 nalidixic acid-resistant, three nalidixic acid-susceptible and one nalidixic acid-resistant strain obtained in vitro) were analysed. RESULTS: All the nalidixic acid-resistant strains showed mutations in the gyrA gene and none in the parC gene. The presence of the inhibitor produced decreases in the MIC values of nalidixic acid by two to six serial dilution steps in 37 of the 41 nalidixic acid-resistant strains. Meanwhile, the MIC value of ciprofloxacin was affected in two strains whose values diminished three serial dilution steps. The nalidixic acid-resistant mutant obtained in vitro was also affected by the inhibitor decreasing the MIC value of nalidixic acid three serial dilutions steps whereas the MICs for the nalidixic acid-susceptible strains were not affected. CONCLUSIONS: Our results show that the high level of resistance to nalidixic acid is likely due to an overexpression of an efflux pump plus a mutation in the gyrA gene, whereas decreased susceptibility to ciprofloxacin is only associated with the presence of a mutation in the gyrA gene.     

Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations

OBJECTIVES: Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones. METHODS: The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-beta-naphthylamide (PA beta N). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPC(CIP)) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. RESULTS: In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 --> Tyr, Gly, Asn or Ser-83 --> Phe, Tyr) or parC (Thr-57 --> Ser). While CCCP had no influence on the MICs, PA beta N decreased the MIC(CIP) values by 1-3 dilution steps and MIC(NAL) values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPC(CIP) values were 4-64-fold higher than the MICs and ranged between 1-16 and 0.12-1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB. CONCLUSIONS: In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes.     

High prevalence of nalidixic acid resistant,ciprofloxacin susceptible phenotype among clinical isolates of Escherichia coli and other Enterobacteriaceae

Therapeutic failure of infections during their treatment with quinolones has been often described. This may be due to the development of resistance during treatment of an infecting strain which already had diminished susceptibility to quinolones, even though the initial MIC did not exceed the breakpoint. In this study the prevalence of the nalidixic acid resistant, ciprofloxacin susceptible phenotype among Enterobacteriaceae was analyzed. The results showed that 113 out of 151 (74.83%) strains of the Enterobacteriaceae with diminished susceptibility to ciprofloxacin (MICs from 0.06 to 1 microg/ml) were resistant to nalidixic acid (MICs > 32 microg/ml). The Escherichia coli strains presenting this phenotype already have a mutation in the amino acid codon Ser-83 of the gyrA gene, so that the possibility of developing a second mechanism of resistance during treatment is very high.     

Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii

The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii. No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2-4 mg/L, and a ciprofloxacin MIC of 0.006-0.06 mg/L). The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr-->Ile), but no alteration in either parC or gyrB genes. In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8-32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr-->Ile and Asp-->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser-->Ile). The fourth isolate did not possess the mutation at codon 87 of gyrA. Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin. This difference was related to an efflux pump mechanism. Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.     

Changes in antimicrobial susceptibility in a population of Salmonella enterica serovar Dublin isolated from cattle in Japan from 1976 to 2005

OBJECTIVES: We investigated the antimicrobial susceptibilities and resistance mechanisms of cattle-adapted Salmonella enterica serovar Dublin isolated in Japan in the past 30 years. This study is an example of evaluation of the impact of introduction of antimicrobials in veterinary medical practice on the selection of resistance in S. enterica. METHODS: The antimicrobial susceptibilities and prevalence of R-plasmids in Salmonella Dublin isolated in Japan from 1976 to 2005 were investigated. To evaluate the importance of gyrA mutation and active efflux, we derived the gyrA revertants and acrAB deletion mutants, and then compared with their parental strains the MICs of quinolone antimicrobials such as nalidixic acid, enrofloxacin, ofloxacin and ciprofloxacin. RESULTS: Salmonella Dublin isolates with R-plasmids and resistance to more than three antimicrobials were predominant between 1981 and 1995. From the latter half of the 1990s to the present, Salmonella Dublin isolates without R-plasmids became dominant. The introduction of nalidixic acid into the veterinary field in the mid-1980s was followed by the emergence of nalidixic acid-resistant isolates, which are now predominant. We found only a single gyrA mutation (Asp-87-->Tyr) among the nalidixic acid-resistant isolates. Although the reduced susceptibilities to the fluoroquinolones were observed among the nalidixic acid-resistant isolates, none of the isolates was resistant to the fluoroquinolones used in this study. The MIC data for the fluoroquinolones differed up to 4-fold. Results of the susceptibility test using gyrA revertants and acrAB mutants suggest that the isolates with the gyrA mutation were selected by the use of nalidixic acid, and the AcrAB-TolC system accounts for the decreased fluoroquinolone susceptibilities. CONCLUSIONS: These data suggest that the introduction of nalidixic acid in veterinary medicine seemed to affect the susceptibilities of Salmonella Dublin among the cattle population in Japan, whereas the introduction of enrofloxacin has not caused any additional effect. The prudent use of antimicrobials in the veterinary field should be continuously stressed.     

Role of efflux pumps and topoisomerase mutations in fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli

Point mutations in the topoisomerase (DNA gyrase A) gene are known to be associated with fluoroquinolone resistance in Campylobacter. Recent studies have shown that an efflux pump encoded by cmeABC is also involved in decreased susceptibilities to fluoroquinolones, as well as other antimicrobials. Genome analysis suggests that Campylobacter jejuni contains at least nine other putative efflux pumps. Using insertional inactivation and site-directed mutagenesis, we investigated the potential contributions of these pumps to susceptibilities to chloramphenicol, ciprofloxacin, erythromycin, and tetracycline in C. jejuni and Campylobacter coli. Insertional inactivation of cmeB resulted in 4- to 256-fold decreases in the MICs of chloramphenicol, ciprofloxacin, erythromycin, and tetracycline, with erythromycin being the most significantly affected. In contrast, inactivation of all other putative efflux pumps had no effect on susceptibility to any of the four antimicrobials tested. Mutation of gyrA at codon 86 (Thr-Ile) caused 128- and 64-fold increases in the MICs of ciprofloxacin and nalidixic acid, respectively. The replacement of the mutated gyrA with a wild-type gyrA allele resulted in a 32-fold decrease in the ciprofloxacin MIC and no change in the nalidixic acid MIC. Our findings indicate that CmeABC is the only efflux pump among those tested that influences antimicrobial resistance in Campylobacter and that a point mutation (Thr-86-Ile) in gyrA directly causes fluoroquinolone resistance in Campylobacter. These two mechanisms work synergistically in acquiring and maintaining fluoroquinolone resistance in Campylobacter species.     

Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli.

Fifteen strains of Escherichia coli with MICs of ciprofloxacin (CIP) between 0.015 and 256 micrograms/ml were examined for the presence of mutations in the quinolone resistance-determining region of the gyrA gene and in an analogous region of the parC gene. No mutation was found in a susceptible isolate (MIC of CIP, 0.015 microgram/ml). Four moderately resistant strains (MIC of CIP 0.06 to 4 micrograms/ml) carried one gyrA mutation affecting serine 83, but in only one strain was an additional parC mutation (Gly-78 to Asp) detected. All ten highly resistant strains examined (MIC of CIP, > 4 micrograms/ml) carried two gyrA mutations affecting residues serine 83 and aspartate 87, and at least one parC mutation. These parC mutations included alterations of serine 80 to arginine or isoleucine and glutamate 84 to glycine or lysine. The parC+ and two mutant alleles (parCI-80 and parCI-80,G-84) were inserted into the mobilizable vector pBP507. Transfer of a plasmid-coded parC+ allele into parC+ strains did not alter the susceptibilities towards ciprofloxacin or nalidixic acid, while a significant increase in susceptibility was detectable for parC mutants. This increase, however, did not restore wild-type susceptibility, whereas transfer of a plasmid-coded gyrA+ allele alone or in combination with parC+ did. These data are in agreement with the view that topoisomerase IV is a secondary, less sensitive target for quinolone action in Escherichia coli and that the development of high-level fluoroquinolone resistance in E. coli requires at least one parC mutation in addition to the gyrA mutation(s).     

Novel gyrA point mutation in a strain of Escherichia coli resistant to fluoroquinolones but not to nalidixic acid.

We have previously described a clinical isolate of Escherichia coli (Q2) that is highly resistant to fluoroquinolones (MIC of ciprofloxacin, 16 micrograms/ml) but susceptible to nalidixic acid (MIC of nalidixic acid, 4 micrograms/ml) (N. Moniot-Ville, J. Guibert, N. Moreau, J.F. Acar, E. Collatz, and L. Gutmann, Antimicrob. Agents Chemother. 35:519-523, 1991). Transformation of strain Q2 with a plasmid carrying the wild-type gyrA gene from E. coli K-12(pAFF801) resulted in a 32-fold decrease in the MIC of ciprofloxacin, suggesting that at least one mutation in gyrA was involved in the resistance of Q2. Intragenic gyrA fragments of 668 and 2,500 bp from strain Q2 were amplified by the polymerase chain reaction. We sequenced the 668-bp fragment and identified a single novel point mutation (transition from G to A at position 242), leading to an amino acid substitution (Gly-81 to Asp) in the gyrase A subunit. We constructed hybrid plasmids by substituting either the 668-bp fragment or the 2,500-bp fragment from Q2 DNA, both of which contained the gyrA point mutation, for the corresponding fragments in wild-type gyrA (2,625 bp) of E. coli K-12. When introduced into E. coli KNK453 (gyrA temperature sensitive), both plasmids conferred an eightfold increase in the MIC of ciprofloxacin, but only a twofold increase in the MIC of nalidixic acid. When introduced into E. coli Q2, neither plasmid conferred any change in the MICs of ciprofloxacin or nalidixic acid, suggesting that only the point mutation found in gyrA was involved in the resistance that we observed.     

Characterization of a gyrB mutation responsible for low-level nalidixic acid resistance in Neisseria gonorrhoeae

Nalidixic acid-resistant derivatives of Neisseria gonorrhoeae WR302 were identified and categorized into two classes on the basis of their susceptibilities to this antimicrobial agent. The MIC of nalidixic acid for the derivative strain MUG116 was fourfold greater than that for its isogenic parental strain WR302 (2 versus 0.5 micrograms/ml, respectively). MUG324 was significantly more resistant to nalidixic acid (greater than 64 micrograms/ml). The MICs of other antimicrobial agents known to interact with either the gyrA or gyrB gene products were determined. Although the nalidixic acid MIC for MUG116 increased, no significant increases in the MICs of other agents that interact with the gyrA gene product were seen. The MICs of all agents that interact with the gyrA gene product were significantly increased for MUG324. The gene that imparts low-level nalidixic acid resistance was cloned from strain MUG116. The DNA sequence of this gene was determined, and by comparing the deduced amino acid sequence with sequences of proteins in data bases, this protein was found to be approximately 70% homologous with the gyrB gene product of Escherichia coli.     

Increasing incidence of quinolone resistance in human non-typhoid Salmonella enterica isolates in Korea and mechanisms involved in quinolone resistance

OBJECTIVES: We investigated the trends of nalidixic acid resistance in human non-typhoid Salmonella enterica in a Korean population, and examined some possible mechanisms involved in this resistance. METHODS: A total of 261 clinical strains were tested. For all strains, the MICs of nalidixic acid were determined. Nalidixic acid-resistant strains underwent further analysis, including determination of MICs of other antibiotics, mutation analysis within the topoisomerase genes, organic solvent tolerance test, western blotting for AcrA, marOR mutation analysis, ciprofloxacin accumulation test, and PCR for the qnr gene. The clonal relationships of Salmonella strains were examined by random amplified polymorphic DNA analysis. RESULTS: The incidence of nalidixic acid resistance increased from 1.8% in 1995-96 to 21.8% in 2000-02. The resistance rate was higher in S. enterica serotype Enteritidis (21.6%) than in serotype Typhimurium (12.1%). The nalidixic acid resistance rates in Salmonella Enteritidis varied according to the phage type (PT) and Salmonella Enteritidis PT 1 was most commonly associated with resistance to nalidixic acid. Several cases of clonal spread, especially by Salmonella Enteritidis PT 1, were identified. Of the 46 nalidixic acid-resistant strains, 43 had single mutations in the gyrA gene. Four strains were organic solvent-tolerant and were associated with decreased ciprofloxacin accumulation; three of these showed increased expression of AcrA and had novel mutations in marOR (84L). The qnr gene was not identified. CONCLUSIONS: Recently, the rate of nalidixic acid resistance in Korean clinical Salmonella strains markedly increased and it was partly due to the clonal spread of Salmonella Enteritidis, especially PT 1. The main mechanism of nalidixic acid resistance was a mutation in the gyrA region.     

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.     

Association between double mutation in gyrA gene of ciprofloxacin-resistant clinical isolates of Escherichia coli and MICs.

The mutations in the quinolone resistance-determining region of the gyrA and gyrB genes from 27 clinical isolates of Escherichia coli with a range of MICs of ciprofloxacin from 0.007 to 128 micrograms/ml and of nalidixic acid from 2 to > 2,000 micrograms/ml were determined by DNA sequencing. All 15 isolates with ciprofloxacin MICs of > or = 1 micrograms/ml showed a change in Ser-83 to Leu of GyrA protein, whereas in clinical isolates with a MIC of > or = 8 micrograms/ml (11 strains), a double change in Ser-83 and Asp-87 was found. All isolates with a MIC of nalidixic acid of > or = 128 micrograms/ml showed a mutation at amino acid codon Ser-83. Only 1 of the 27 clinical isolates of E. coli analyzed showed a change in Lys-447 of the B subunit of DNA gyrase. A change in Ser-83 is sufficient to generate a high level of resistance to nalidixic acid, whereas a second mutation at Asp-87 in the A subunit of DNA gyrase may play a complementary role in developing the strain's high levels of ciprofloxacin resistance.     

Activity of clinafloxacin, compared with six other quinolones, against Acinetobacter baumannii clinical isolates

The in vitro activity of clinafloxacin was studied in comparison with ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, sparfloxacin and trovafloxacin against Acinetobacter baumannii clinical isolates. Clinafloxacin showed a MIC(90) of 4 mg/L, whereas the remaining quinolones showed a MIC(90) equal to or higher than 16 mg/L. MIC(50) determination in the presence of reserpine resulted in a two-fold decrease, except for trovafloxacin, which decreased four-fold, and for moxifloxacin and nalidixic acid, which did not change. The effect of reserpine was most pronounced among strains with a low level of resistance to quinolones. The MIC of clinafloxacin for strains with no mutation in either gyrA or parC genes ranged from 0.008 to 0.25 mg/L. In strains with a single mutation at amino acid codon Ser83 of the gyrA gene, the MIC of clinafloxacin ranged from 0.12 to 1 mg/L, whereas strains with a double mutation, one in the gyrA gene and another in the parC gene, showed a range of MIC of clinafloxacin from 1 to 8 mg/L. Therefore, clinafloxacin shows good activity against strains carrying a single mutation in the gyrA gene, and hence a second mutation is required for the microorganism to express resistance.     

In vitro activities of garenoxacin (BMS-284756) against Haemophilus influenzae isolates with different fluoroquinolone susceptibilities

The in vitro activity of garenoxacin (BMS-284756) against 62 clinical Haemophilus influenzae isolates with different fluoroquinolone susceptibilities was determined by the microdilution susceptibility testing method and compared with the activities of other oral quinolones and nonquinolone oral antimicrobial agents. Cefixime presented the highest intrinsic activity (MIC at which 50% of the isolates tested were inhibited [MIC(50)], 0.01 microg/ml), followed by garenoxacin, moxifloxacin, and ciprofloxacin (MIC(50), 0.06 microg/ml), levofloxacin (MIC(50), 0.12 microg/ml), cefuroxime (MIC(50), 1.0 microg/ml), and amoxicillin-clavulanate (MIC(50), 1.0/0.5 microg/ml), amoxicillin (MIC(50), 2 microg/ml), azithromycin (MIC(50), 4 microg/ml), and erythromycin (MIC(50), 8 microg/ml). In strains with ciprofloxacin MICs of < or =0.06 microg/ml, ciprofloxacin and garenoxacin displayed similar MIC(50)s and MIC(90)s, one dilution lower than those of moxifloxacin and levofloxacin. For strains for which ciprofloxacin MICs were > or = 0.12 microg/ml, MIC(50)s were similar for the four quinolones tested, although garenoxacin presented the widest activity range (0.03 to 32 microg/ml) and the highest MIC at which 90% of the isolates tested were inhibited (16.0 microg/ml). For strains without amino acid changes in the quinolone resistance determining region (QRDR) of GyrA and ParC, garenoxacin MICs were < or =0.03 microg/ml; with a single amino acid change in GyrA, garenoxacin MICs were 0.06 to 0.12 microg/ml; with one amino acid change each in GyrA and ParC, garenoxacin MICs were 0.5 to 2.0 micro g/ml; one amino acid change in ParC combined with two amino acid changes in GyrA increased the MICs to > or = 4 microg/ml for all assayed quinolones. We conclude that garenoxacin has excellent activity against H. influenzae, although progressive acquired resistance was observed by step-by-step mutation in the QRDR of gyrA and parC.     

Increasing prevalence of quinolone resistance in human nontyphoid Salmonella enterica isolates obtained in Spain from 1981 to 2003

From January 1981 to December 2003, susceptibility to nalidixic acid was tested in 10,504 nontyphoid Salmonella enterica isolates from patients with acute enteric disease in Gipuzkoa, Spain. The prevalence of nalidixic acid resistance steadily increased from less than 0.5% before 1991 to 38.5% in 2003, mainly due to the increase in resistance among isolates of the most prevalent serovar, S. enterica serovar Enteritidis. For nalidixic acid-resistant isolates, the ciprofloxacin MIC was eightfold higher than that for susceptible isolates, and the nalidixic acid-resistant isolates contained a single point mutation in the gyrA gene (at codons for Ser83 or Asp87). The same mutations were found in a sample of nalidixic acid-resistant nontyphoid Salmonella strains isolated between 1999 and 2003 from retail food for human consumption. In 2003, we identified five S. enterica serovar Typhimurium clinical isolates with high-level fluoroquinolone resistance (ciprofloxacin MIC, 16 microg/ml) with two point mutations in the gyrA gene (coding for Ser83-->Phe and Asp87-->Asn) and one point mutation in the parC gene (coding for Ser80-->Arg). Strict sanitary controls are needed to avoid the spread of ciprofloxacin-resistant serovar Typhimurium isolates, and a more efficient veterinary policy must be adopted to decrease the large burden of Salmonella serovar Enteritidis infections in humans in our region.     

Comparison of the Etest and agar dilution for in vitro antimicrobial susceptibility testing of Campylobacter

The performance of the Etest and agar dilution for in vitro antimicrobial susceptibility of Campylobacter spp. was evaluated using a quality control strain Campylobactor jejuni ATCC 33560, and 81 C. jejuni and 54 Campylobacter coli isolates recovered from retail raw meats. Seven antimicrobial agents: chloramphenicol, ciprofloxacin, doxycycline, erythromycin, gentamicin, nalidixic acid and tetracycline, were tested using the two methods, whereas azithromycin was tested using the Etest only. The correlation between the Etest and agar dilution MICs varied greatly depending on the antimicrobial agents tested. The overall agreement of MICs (+/-1 log(2) dilution) between the two methods was 61.9%, ranging from 21.4% for nalidixic acid to 92.6% for gentamicin. MICs obtained using the Etest were generally lower than those by agar dilution regardless of the species of organism tested. MIC(50) and/or MIC(90) values were at least one dilution lower for the Etest than for agar dilution when testing chloramphenicol, ciprofloxacin, doxycycline, erythromycin and nalidixic acid. Based on the agar dilution MICs, the resistant rate of the 135 Campylobacter isolates was highest for tetracycline (82.2%), followed by doxycycline (78.5%), nalidixic acid (21.5%), ciprofloxacin (20.7%) and erythromycin (17.0%). None of the isolates demonstrated resistance to chloramphenicol or gentamicin. The study indicated that the Etest results were not in complete agreement with the agar dilution test. Although the Etest has been proven to be a satisfactory testing method, its use for Campylobacter susceptibility testing requires further standardization. The study also showed that C. jejuni and C. coli isolates resistant to antimicrobials used for treating campylobacteriosis were common in retail raw meats.     

Phenotypic characterization of quinolone-resistant mutants of Enterobacteriaceae selected from wild type, gyrA type and multiply-resistant (marA) type strains.

The NCTC type strains of Escherichia coli, Enterobacter cloacae, Serratia marcescens and Klebsiella pneumoniae were exposed to 3, 5 and 10 x MIC of nalidixic acid, enoxacin, ciprofloxacin, PD 117596 and PD 127391. From each strain a mutant with a high MIC of quinolones alone (gyrA) and a mutant with intermediate resistance to quinolones, some beta-lactams, chloramphenicol and tetracycline (multiply resistant, m-r) were selected on agar containing antibiotics. The gyrA mutants required a higher concentration of quinolone to inhibit DNA synthesis by 50% but quinolone uptake kinetics and outer membrane profile were the same as the wild type. The m-r mutants had similar DNA synthesis IC50 as the wild type, decreased quinolone uptake kinetics and had decreased expression of an OMP of approximately 40 kD. The gyrA and m-r mutants were then exposed to 3, 5 and 10 x MIC of the same quinolones and new mutants (F2) selected. The F2 mutants from the gyrA mutants displayed a further increase in quinolone MIC; the F2 mutants from the m-r mutants had several phenotypes: high quinolone MICs with cross resistance to other agents, high quinolone resistance alone, or intermediate quinolone resistance alone. Most F2 mutants had MICs above the recommended breakpoint concentrations for quinolones. The F2 mutants often had altered biochemical profiles (API 20E), however, only in the case of E. cloacae did this affect speciation with the strains being identified as Rhanella aquatalis.     

In vivo selection during ofloxacin therapy of Escherichia coli with combined topoisomerase mutations that confer high resistance to ofloxacin but susceptibility to nalidixic acid

OBJECTIVES: To investigate quinolone resistance mechanisms in an Escherichia coli clinical isolate (Ar2) resistant to ofloxacin but susceptible to nalidixic acid selected after 10 days of ofloxacin therapy in a patient with prostatitis. METHODS: Molecular typing (ERIC-PCR and RAPD), antibiotic susceptibility and gyrA, gyrB, parC and parE QRDR sequences were compared for E. coli Ar2 and a wild-type E. coli (Ar1) isolated 2 months earlier in the same patient. Ofloxacin-resistant mutants were selected in vitro in order to reproduce the mutations observed and the original phenotype. RESULTS: The two strains were similar with regard to antibiotic susceptibility except quinolones and for ERIC-PCR and RAPD patterns, suggesting a clonal relationship and acquisition of quinolone resistance by chromosomal mutation. Quinolone MICs were 3, 0.12, 0.05 and 0.02 mg/L of nalidixic acid, ofloxacin, levofloxacin and ciprofloxacin, respectively, for E. coli Ar1 and 6, 32, 8 and 1 mg/L, respectively, for E. coli Ar2. The strain Ar2 harboured two substitutions, Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC. Introduction into E. coli Ar2 of the wild-type gyrA fully complemented fluoroquinolone resistance. Although the strain was not a hypermutator, ofloxacin first-step resistant mutants with gyrA mutations were easily obtained from E. coli Ar1 and 25% of them were at codon 81. In vitro stepwise combination of Gly-81-->Asp in GyrA and Ser-80-->Arg in ParC reproduced the original phenotype in E. coli KL16. CONCLUSIONS: A double topoisomerase mutant was selected in vivo by 10 days ofloxacin. The mutations were originally combined for a result of ofloxacin resistance but nalidixic acid susceptibility.     

Molecular basis of high-level ciprofloxacin resistance in Neisseria gonorrhoeae strains isolated in Denmark from 1995 to 1998

In Denmark surveillance of the in vitro susceptibility to ciprofloxacin of Neisseria gonorrhoeae was established in 1990. The proportion of N. gonorrhoeae strains with decreased susceptibility or resistance to ciprofloxacin (MIC >/= 0.06 microg/ml) was low (0.3 to 2.3%) up to 1995. Between 1995 and 1998 the rate of less-susceptible and resistant strains rose from 6.9 to 13.2%. Among ciprofloxacin-resistant strains (MIC >/= 1 microg/ml), 81% were highly resistant (MIC >/= 4 microg/ml). Thirty-five N. gonorrhoeae strains (40 isolates) for which ciprofloxacin MICs were 4 to 32 microg/ml were investigated for the frequency and patterns of mutations within the gyrA and parC genes. The quinolone resistance-determining regions of the gyrA and parC genes were amplified by PCR, and the amplicons were directly sequenced. Alterations at Ser-91 and Asp-95 in GyrA and a single or double alteration in ParC were identified in 32 strains (91%). Ser-91-to-Phe and Asp-95-to-Gly alterations in GyrA were detected in 28 strains (80%). The most common ParC alteration, Asp-86 to Asn, was found in 19 strains (54%). The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). The analysis showed that nine strains with the same mutation pattern in the gyrA and parC genes, originating from different geographical areas over 3 years, had the same PFGE patterns after SpeI as well as NheI digestion (only one strain with one band difference in the NheI pattern), suggesting that a resistant clone had spread worldwide. The results from this study strongly suggest that double gyrA mutations plus a parC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae.     

Mupirocin resistance in methicillin-resistant Staphylococcus aureus clinical isolates in a Spanish hospital. Co-application of multiplex PCR assay and conventional microbiology methods

A total of 33 Stenotrophomonas maltophilia clinical isolates were tested for their susceptibility to clinafloxacin in comparison with ciprofloxacin, levofloxacin, moxifloxacin, nalidixic acid, norfloxacin, sparfloxacin and trovafloxacin. The MIC(50) and MIC(90) were as follows: ciprofloxacin 4 and 64 microg/mL; clinafoxacin 0.5 and 4 microg/mL; levofloxacin 2 and 32 microg/mL; moxifloxacin 1 and 8 microg/mL; nalidixic acid 8 and 128 microg/mL; norfloxacin 64 and 256 microg/mL; sparfloxacin 1 and 16 microg/mL; and trovafloxacin 1 and 8 microg/mL. Clinafloxacin was the most active quinolone, with only a 15.1% of strains showing resistance. When the MICs were determined in the presence of 25 microg/ml of reserpine, the MIC(90) of trovafloxacin and moxifloxacin did not change, whereas decreased 2-fold for clinafloxacin, levofloxacin, sparfloxacin and nalidixic acid, and 4- and 8-fold for ciprofloxacin and norfloxacin respectively. No clinafloxacin-resistant strains were observed when the MIC was performed in the presence of reserpine. Therefore, clinafloxacin shows the better "in vitro"activity against these 33 strains of S.maltophilia.