Mutations in topoisomerase genes of fluoroquinolone-resistant salmonellae in Hong Kong

A total of 88 salmonella isolates (72 clinical isolates for which the ciprofloxacin MIC was >0.06 microg/ml, 15 isolates for which the ciprofloxacin MIC was < or =0.06 microg/ml, and Salmonella enterica serotype Typhimurium ATCC 13311) were studied for the presence of genetic alterations in four quinolone resistance genes, gyrA, gyrB, parC, and parE, by multiplex PCR amplimer conformation analysis. The genetic alterations were confirmed by direct nucleotide sequencing. A considerable number of strains had a mutation in parC, the first to be reported in salmonellae. Seven of the isolates sensitive to 0.06 micro g of ciprofloxacin per ml had a novel mutation at codon 57 of parC (Tyr57-->Ser) which was also found in 29 isolates for which ciprofloxacin MICs were >0.06 micro g/ml. Thirty-two isolates had a single gyrA mutation (Ser83-->Phe, Ser83-->Tyr, Asp87-->Asn, Asp87-->Tyr, or Asp87-->Gly), 34 had both a gyrA mutation and a parC mutation (29 isolates with a parC mutation of Tyr57-->Ser and 5 isolates with a parC mutation of Ser80-->Arg). Six isolates which were isolated recently (from 1998 to 2001) were resistant to 4 micro g of ciprofloxacin per ml. Two of these isolates had double gyrA mutations (Ser83-->Phe and Asp87-->Asn) and a parC mutation (Ser80-->Arg) (MICs, 8 to 32 microg/ml), and four of these isolates had double gyrA mutations (Ser83-->Phe and Asp87-->Gly), one parC mutation (Ser80-->Arg), and one parE mutation (Ser458-->Pro) (MICs, 16 to 64 micro g/ml). All six of these isolates and those with a Ser80-->Arg parC mutation were S. enterica serotype Typhimurium. One S. enterica serotype Typhi isolate harbored a single gyrA mutation (Ser83-->Phe), and an S. enterica serotype Paratyphi A isolate harbored a gyrA mutation (Ser83-->Tyr) and a parC mutation (Tyr57-->Ser); both of these isolates had decreased susceptibilities to the fluoroquinolones. The MICs of ciprofloxacin, levofloxacin, and sparfloxacin were in general the lowest of those of the six fluoroquinolones tested. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation (Tyr57-->Ser or Ser80-->Arg), while those with double gyrA mutations were more resistant.     

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.     

Alterations in DNA gyrase and topoisomerase IV in resistant mutants of Clostridium perfringens found after in vitro treatment with fluoroquinolones

To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants.     

DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolone resistance-determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A

The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.     

DNA gyrase and topoisomerase IV mutations associated with fluoroquinolone resistance in Proteus mirabilis

Mutations associated with fluoroquinolone resistance in clinical isolates of Proteus mirabilis were determined by genetic analysis of the quinolone resistance-determining region (QRDR) of gyrA, gyrB, parC, and parE. This study included the P. mirabilis type strain ATCC 29906 and 29 clinical isolates with reduced susceptibility (MIC, 0.5 to 2 microg/ml) or resistance (MIC, > or =4 microg/ml) to ciprofloxacin. Susceptibility profiles for ciprofloxacin, clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were correlated with amino acid changes in the QRDRs. Decreased susceptibility and resistance were associated with double mutations involving both gyrA (S83R or -I) and parC (S80R or -I). Among these double mutants, MICs of ciprofloxacin varied from 1 to 16 microg/ml, indicating that additional factors, such as drug efflux or porin changes, also contribute to the level of resistance. For ParE, a single conservative change of V364I was detected in seven strains. An unexpected result was the association of gyrB mutations with high-level resistance to fluoroquinolones in 12 of 20 ciprofloxacin-resistant isolates. Changes in GyrB included S464Y (six isolates), S464F (three isolates), and E466D (two isolates). A three-nucleotide insertion, resulting in an additional lysine residue between K455 and A456, was detected in gyrB of one strain. Unlike any other bacterial species analyzed to date, mutation of gyrB appears to be a frequent event in the acquisition of fluoroquinolone resistance among clinical isolates of P. mirabilis.     

Chromosomal mutations responsible for fluoroquinolone resistance in Ureaplasma species in the United States

We sequenced the full lengths of the gyrA, gyrB, parC, and parE genes in 13 fluoroquinolone-resistant Ureaplasma isolates (levofloxacin MICs, 4 to 32 μg/ml) and 10 susceptible isolates (MICs ≤ 2 μg/ml). Mutations were detected in all resistant isolates but in none of the susceptible isolates. The most prevalent mutation was the S83L substitution in the ParC protein. No plasmid-mediated fluoroquinolone resistance genes were detected.     

In vitro selection and characterization of Bacillus anthracis mutants with high-level resistance to ciprofloxacin

Mutants of attenuated Bacillus anthracis with high-level ciprofloxacin resistance were isolated using a three-step in vitro selection. Ciprofloxacin MICs were 0.5 micro g/ml for first-step mutants, which had one of two gyrA quinolone resistance-determining region (QRDR) mutations. Ciprofloxacin MICs were 8 and 16 microg/ml for second-step mutants, which had one of three parC QRDR mutations. Ciprofloxacin MICs for third-step mutants were 32 and 64 microg/ml. Mutants for which MICs were 64 microg/ml had one of two additional mutations within the gyrA QRDR or one of two mutations within the gyrB QRDR. Mutants for which MICs were 32 microg/ml had no additional target modifications but showed evidence of enhanced ciprofloxacin efflux.     

Prevalence of mutations within the quinolone resistance-determining region of gyrA, gyrB, parC, and parE and association with antibiotic resistance in quinolone-resistant Salmonella enterica

Salmonella enterica isolates (n = 182) were examined for mutations in the quinolone resistance-determining region of gyrA, gyrB, parC, and parE. The frequency, location, and type of GyrA substitution varied with the serovar. Mutations were found in parC that encoded Thr57-Ser, Thr66-Ile, and Ser80-Arg substitutions. Mutations in the gyrB quinolone resistance-determining region were located at codon Tyr420-Cys or Arg437-Leu. Novel mutations were also found in parE encoding Glu453-Gly, His461-Tyr, Ala498-Thr, Val512-Gly, and Ser518-Cys. Although it is counterintuitive, isolates with a mutation in both gyrA and parC were more susceptible to ciprofloxacin than were isolates with a mutation in gyrA alone.     

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.     

Prevalence of Single Mutations in Topoisomerase Type II Genes among Levofloxacin-Susceptible Clinical Strains of Streptococcus pneumoniae Isolated in the United States in 1992 to 1996 and 1999 to 2000

Levofloxacin resistance in Streptococcus pneumoniae is rare, requiring at least two mutations in the quinolone resistance-determining region (QRDR) of topoisomerase IV and DNA gyrase. The prevalence of single QRDR mutations in these genes is unknown. Of 9,438 levofloxacin-susceptible pneumococci from the TRUST 4 surveillance study (1999-2000), 528 strains (MICs of 0.5 to 2.0 microg/ml) were selected for analysis. For comparison, 214 levofloxacin-susceptible strains (MICs of 0.5 to 1 microg/ml) isolated between 1992 and 1996 were analyzed. Oligonucleotide probe assay and DNA sequencing were used to detect QRDR mutations leading to changes at Ser79 and Asp83 in ParC, Ser81 in GyrA, and Asp435 in ParE, the most frequently found substitutions among levofloxacin-resistant strains. Among the 1992 to 1996 isolates only one strain (levofloxacin MIC, 1 microg/ml) had a mutation (Ser79 to Phe in ParC). No single mutations were found among 270 TRUST 4 strains with levofloxacin MICs of 0.5 microg/ml. Among 244 strains for which levofloxacin MICs were 1 microg/ml, 15 strains (6.1%) had a parC mutation and 3 strains (1.2%) had a parE mutation. Of 14 strains for which levofloxacin MICs were 2 microg/ml, 10 strains (71%) had a parC mutation; no parE mutations were found. No gyrA mutations were detected. It was estimated that 4.5% of the 9,438 levofloxacin-susceptible TRUST 4 isolates (MICs, < or =0.06 to 2 microg/ml) had a single parC or parE QRDR mutation. Although there has been an increase in the prevalence of single-step mutants, the increase may have been overestimated due in part to differences in geographical distribution for the two sets of isolates.     

In vitro induction and selection of fluoroquinolone-resistant mutants of Streptococcus pyogenes strains with multiple emm types

OBJECTIVES: To perform a systematic analysis of point mutations in the quinolone resistance determining regions (QRDRs) of the DNA gyrase and topoisomerase genes of emm type 6 and other emm types of Streptococcus pyogenes strains after in vitro exposure to stepwise increasing concentrations of levofloxacin. METHODS: Twelve parent strains of S. pyogenes, each with a different emm type, were chosen for stepwise exposure to increasing levels of levofloxacin followed by selection of resistant mutants. The QRDRs of gyrA, gyrB, parC and parE correlating to mutants with increased MICs were analysed for point mutations. RESULTS: Multiple mutants with significantly increased MICs were generated from each strain. The amino acid substitutions identified were consistent regardless of emm type and were similar to the mechanisms of resistance reported in clinical isolates of S. pyogenes. The number of induction/selection cycles required for the emergence of key point mutations in gyrA and parC was variable among strains. For each parent-mutant set, when MIC increased, serine-81 of gyrA and serine-79 of parC were the primary targets for amino acid substitutions. No point mutations were found in the QRDRs of gyrB and parE in any of the resistant mutants sequenced. CONCLUSIONS: Despite its intrinsic polymorphism in the QRDR of parC, emm type 6 is not more likely to develop high-level resistance to fluoroquinolones when compared with other emm types. All emm types seem equally inducible to high-level fluoroquinolone resistance.     

Activity of gemifloxacin against penicillin- and ciprofloxacin-resistant Streptococcus pneumoniae displaying topoisomerase- and efflux-mediated resistance mechanisms

Nine penicillin-resistant Streptococcus pneumoniae clinical isolates from Northern Ireland, resistant to ciprofloxacin (MICs, 2 to 64 microg/ml) through topoisomerase- and/or reserpine-sensitive efflux mechanisms, were highly susceptible to gemifloxacin (MICs, 0.03 to 0. 12 microg/ml). Two strains (requiring a ciprofloxacin MIC of 64 microg/ml) carried known quinolone resistance mutations in parC, parE, and gyrB, resulting in S79F, D435V, and E474K changes, respectively. Thus, gemifloxacin is active against clinical strains exhibiting altered topoisomerase and efflux phenotypes.     

耐左氧氟沙星大肠埃希菌gyrA/gyrB/parC/parE基因突变研究

目的了解耐氟喹诺酮大肠埃希菌gyrA／gyrB／parC／parE基因突变情况。方法用Kirby—Bauer琼脂扩散法筛选耐左氧氟沙星大肠埃希菌;采用聚合酶链反应（PCR）对gyrA／gyrB／parC／parE基因进行扩增,并进行PCR扩增产物直接双向测序,分析上述基因突变情况。结果PCR扩增耐菌株gyrA／gyrB／parC／parE基因,获得目的片段,测序结果显示,氟喹诺酮耐药基因突变集中在gyrA基因83、87位ser→Leu、Asp→Asn突变;parC基因55位Ser→Leu突变,部分菌株尚存在62位Gln→Glu第二突变点;未发现gyrB、parE突变。结论靶位点gyrA和parC的改变,是本地大肠埃希菌对氟喹诺酮类耐药的主要机制。     

Contribution of target gene mutations and efflux to decreased susceptibility of Salmonella enterica serovar typhimurium to fluoroquinolones and other antimicrobials

The mechanisms involved in fluoroquinolone resistance in Salmonella enterica include target alterations and overexpression of efflux pumps. The present study evaluated the role of known and putative multidrug resistance efflux pumps and mutations in topoisomerase genes among laboratory-selected and naturally occurring fluoroquinolone-resistant Salmonella enterica serovar Typhimurium strains. Strains with ciprofloxacin MICs of 0.25, 4, 32, and 256 microg/ml were derived in vitro using serovar Typhimurium S21. These mutants also showed decreased susceptibility or resistance to many nonfluoroquinolone antimicrobials, including tetracycline, chloramphenicol, and several beta-lactams. The expression of efflux pump genes acrA, acrB, acrE, acrF, emrB, emrD, and mdlB were substantially increased (>or=2-fold) among the fluoroquinolone-resistant mutants. Increased expression was also observed, but to a lesser extent, with three other putative efflux pumps: mdtB (yegN), mdtC (yegO), and emrA among mutants with ciprofloxacin MICs of >or=32 microg/ml. Deletion of acrAB or tolC in S21 and its fluoroquinolone-resistant mutants resulted in increased susceptibility to fluoroquinolones and other tested antimicrobials. In naturally occurring fluoroquinolone-resistant serovar Typhimurium strains, deletion of acrAB or tolC increased fluoroquinolone susceptibility 4-fold, whereas replacement of gyrA double mutations (S83F D87N) with wild-type gyrA increased susceptibility>500-fold. These results indicate that a combination of topoisomerase gene mutations, as well as enhanced antimicrobial efflux, plays a critical role in the development of fluoroquinolone resistance in both laboratory-derived and naturally occurring quinolone-resistant serovar Typhimurium strains.     

Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals.

Twenty-eight human isolates of Escherichia coli from Argentina and Spain and eight veterinary isolates received from the Ministry of Agriculture Fisheries and Foods in the United Kingdom required 2 to > 128 micrograms of ciprofloxacin per ml for inhibition. Fragments of gyrA and parC encompassing the quinolone resistance-determining region were amplified by PCR, and the DNA sequences of the fragments were determined. All isolates contained a mutation in gyrA of a serine at position 83 (Ser83) to an Leu, and 26 isolates also contained a mutation of Asp87 to one of four amino acids: Asn (n = 14), Tyr (n = 6), Gly (n = 5), or His (n = 1). Twenty-four isolates contained a single mutation in parC, either a Ser80 to Ile (n = 17) or Arg (n = 2) or a Glu84 to Lys (n = 3). The role of a mutation in gyrB was investigated by introducing wild-type gyrB (pBP548) into all isolates; for three transformants MICs of ciprofloxacin were reduced; however, sequencing of PCR-derived fragments containing the gyrB quinolone resistance-determining region revealed no changes. The analogous region of parE was analyzed in 34 of 36 isolates by single-strand conformational polymorphism analysis and sequencing; however, no amino acid substitutions were discovered. The outer membrane protein and lipopolysaccharide profiles of all isolates were compared with those of reference strains, and the concentration of ciprofloxacin accumulated (with or without 100 microM carbony cyanide m-chlorophenylhydrazone [CCCP] was determined. Twenty-two isolates accumulated significantly lower concentrations of ciprofloxacin than the wild-type E. coli isolate; nine isolates accumulated less then half the concentration. The addition of CCCP increased the concentration of ciprofloxacin accumulated, and in all but one isolate the percent increase was greater than that in the control strains. The data indicate that high-level fluoroquinolone resistance in E. coli involves the acquisition of mutations at multiple loci.     

Type II Topoisomerase Mutations in Fluoroquinolone-Resistant Clinical Strains of Pseudomonas aeruginosa Isolated in 1998 and 1999: Role of Target Enzyme in Mechanism of Fluoroquinolone Resistance

The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.     

Frequencies and mechanisms of resistance to moxifloxacin in nosocomial isolates of Acinetobacter baumannii

OBJECTIVES: To compare the in vitro activity of moxifloxacin and ciprofloxacin against 226 nosocomial isolates of Acinetobacter baumannii from 44 hospitals in the UK. METHODS: MICs of ciprofloxacin and moxifloxacin were determined by Etest. PCR analysis was used to detect chromosomal mutations in the gyrA and parC genes. Isolates resistant to ciprofloxacin and susceptible to moxifloxacin were examined for the ability to generate spontaneous moxifloxacin-resistant isolates. RESULTS: Of 226 isolates, 49.1% were resistant to ciprofloxacin and 39.4% were moxifloxacin-resistant according to BSAC criteria. Approximately 20% of isolates resistant to ciprofloxacin remained susceptible to moxifloxacin. A GyrA mutation at Ser-83 was found in all ciprofloxacin-resistant isolates. Single mutations in both the gyrA and parC genes at codons Ser-83 and Ser-80, respectively, were found in ciprofloxacin- and moxifloxacin-resistant isolates. Isolates that were ciprofloxacin-resistant but moxifloxacin-susceptible generated spontaneous moxifloxacin-resistant mutants when grown on medium containing up to 8x their initial MIC. However, these mutants were not stable and none displayed high-level moxifloxacin resistance. CONCLUSIONS: Moxifloxacin retained in vitro activity against some ciprofloxacin-resistant clinical A. baumannii isolates. Mutations in both gyrA and parC were necessary for resistance to moxifloxacin in most isolates of A. baumannii.     

gyrA、gyrB和外排系统共同介导铜绿假单胞菌对喹诺酮类耐药机制研究

目的探讨临床分离的对环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌耐药机制。方法收集临床分离经VITEK-2（C0mpact细菌鉴定仪检测环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌,琼脂稀释法测定环丙沙星和左氧氟沙星的MIC值,PCR扩增DNA促旋酶基因gyrA和gyrB以及DNA拓扑异构酶Ⅳ的parC和parE基因,实时-RT-PCR分析细菌外排系统表达情况。结果琼脂稀释法检测结果与VITEK-2 Compact细菌鉴定仪检测结果相符。PCR扩增测序发现以DNA促旋酶基因gyrA（在位点941处插入碱基C）和gyrB（3株在位点1588处缺失碱基A,其他菌株在位点1543处插入碱基T）基因缺失或者插入导致移码突变为主,parE基因有3株在位点1895处插入碱基C。实时定量PCR检测发现以mexA和mexC表达增加为主。结论检出的耐环丙沙星和左氧氟沙星铜绿假单胞菌是由于DNA促旋酶基因gyrA和gyrB基因的突变和mexAB-OprM和mexCD-OprJ表达增加共同作用的结果。     

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

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.     

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.