Biological cost of single and multiple norfloxacin resistance mutations in Escherichia coli implicated in urinary tract infections

Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA, gyrB, parC, and parE, previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA, gyrB, and marOR. Subsequent selection steps yielded mutations in gyrA, parE, and marOR but not in gyrB, parC, or acrR. Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.     

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.     

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。     

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.     

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.     

Comparative Studies of Mutations in Animal Isolates and Experimental In Vitro- and In Vivo-Selected Mutants of Salmonella spp. Suggest a Counterselection of Highly Fluoroquinolone-Resistant Strains in the Field

The occurrence of mutations in the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) of Salmonella typhimurium experimental mutants selected in vitro and in vivo and of 138 nalidixic acid-resistant Salmonella field isolates was investigated. The sequencing of the quinolone resistance-determining region of these genes in highly fluoroquinolone-resistant mutants (MICs of 4 to 16 microg/ml) revealed the presence of gyrA mutations at codons corresponding to Gly-81 or Ser-83, some of which were associated with a mutation at Asp-87. No mutations were found in the gyrB, parC, and parE genes. An assay combining allele-specific PCR and restriction fragment length polymorphism was developed to rapidly screen mutations at codons 81, 83, and 87 of gyrA. The MICs of ciprofloxacin for the field isolates reached only 2 microg/ml, versus 16 microg/ml for some in vitro-selected mutants. The field isolates, like the mutants selected in vivo, had only a single gyrA mutation at codon 83 or 87. Single gyrA mutations were also found in highly resistant in vitro-selected mutants (MIC of ciprofloxacin, 8 microg/ml), which indicates that mechanisms other than the unique modification of the intracellular targets could participate in fluoroquinolone resistance in Salmonella spp. A comparison of experimental mutants selected in vitro, field strains, and mutants selected in vivo suggests that highly fluoroquinolone-resistant strains are counterselected in field conditions in the absence of selective pressure.     

铜绿假单胞菌的临床分布及对环丙沙星耐药机制的研究

目的 分析医院铜绿假单胞菌（PAE）的临床分布和耐药性,并了解其对环丙沙星的耐药机制.方法 我院2011年9月-2013年9月收集的287株铜绿假单胞菌.采用琼脂纸片扩散法（K-B法）进行药敏试验,微量肉汤稀释法测定环丙沙星对PAE的最低抑菌浓度（MIC）.应用PCR方法扩增PAE的gyrA、gyrB、parC及parE基因,并测序来确定基因的突变.结果 287株铜绿假单胞菌以痰液标本中分离最多（76.3％）;主要感染分布在重症监护室（29.3％）和呼吸内科（22.6％）;且对哌拉西林/他唑巴坦及亚胺培南敏感,敏感率均在70％以上;而对氨苄西林及环丙沙星耐药率高,分别达97.9％和44.9％.在45株（环丙沙星MICs≥32 μg/ml）铜绿假单胞菌中,有42株均存在gyrA基因突变,且28株存在着gyrA基因和parC基因双突变.结论 铜绿假单胞菌多重耐药现象严重,gyrA基因突变是PAE对环丙沙星耐药的主要机制之一.应加强对铜绿假单胞菌的临床分布及耐药性监测,更好地指导临床合理用药,控制医院感染.     

Genetic analyses of mutations contributing to fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.

Twenty-one clinical isolates of Streptococcus pneumoniae showing reduced susceptibility or resistance to fluoroquinolones were characterized by serotype, antimicrobial susceptibility, and genetic analyses of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. Five strains were resistant to three or more classes of antimicrobial agents. In susceptibility profiles for gatifloxacin, gemifloxacin, levofloxacin, moxifloxacin, ofloxacin, sparfloxacin, and trovafloxacin, 14 isolates had intermediate- or high-level resistance to all fluoroquinolones tested except gemifloxacin (no breakpoints assigned). Fluoroquinolone resistance was not associated with serotype or with resistance to other antimicrobial agents. Mutations in the QRDRs of these isolates were more heterogeneous than those previously reported for mutants selected in vitro. Eight isolates had amino acid changes at sites other than ParC/S79 and GyrA/S81; several strains contained mutations in gyrB, parE, or both loci. Contributions to fluoroquinolone resistance by individual amino acid changes, including GyrB/E474K, ParE/E474K, and ParC/A63T, were confirmed by genetic transformation of S. pneumoniae R6. Mutations in gyrB were important for resistance to gatifloxacin but not moxifloxacin, and mutation of gyrA was associated with resistance to moxifloxacin but not gatifloxacin, suggesting differences in the drug-target interactions of the two 8-methoxyquinolones. The positions of amino acid changes within the four genes affected resistance more than did the total number of QRDR mutations. However, the effect of a specific mutation varied significantly depending on the agent tested. These data suggest that the heterogeneity of mutations will likely increase as pneumococci are exposed to novel fluoroquinolone structures, complicating the prediction of cross-resistance within this class of antimicrobial agents.     

Detection of mutations in Salmonella enterica gyrA, gyrB, parC and parE genes by denaturing high performance liquid chromatography (DHPLC) using standard HPLC instrumentation

AIMS: Quinolone antibiotics are the agents of choice for treating systemic Salmonella infections. Resistance to quinolones is usually mediated by mutations in the DNA gyrase gene gyrA. Here we report the evaluation of standard HPLC equipment for the detection of mutations (single nucleotide polymorphisms; SNPs) in gyrA, gyrB, parC and parE by denaturing high performance liquid chromatography (DHPLC). METHODS: A panel of Salmonella strains was assembled which comprised those with known different mutations in gyrA (n = 8) and fluoroquinolone-susceptible and -resistant strains (n = 50) that had not been tested for mutations in gyrA. Additionally, antibiotic-susceptible strains of serotypes other than Salmonella enterica serovar Typhimurium strains were examined for serotype-specific mutations in gyrB (n = 4), parC (n = 6) and parE (n = 1). Wild-type (WT) control DNA was prepared from Salmonella Typhimurium NCTC 74. The DNA of respective strains was amplified by PCR using Optimase proofreading DNA polymerase. Duplex DNA samples were analysed using an Agilent A1100 HPLC system with a Varian Helix DNA column. Sequencing was used to validate mutations detected by DHPLC in the strains with unknown mutations. RESULTS: Using this HPLC system, mutations in gyrA, gyrB, parC and parE were readily detected by comparison with control chromatograms. Sequencing confirmed the gyrA predicted mutations as detected by DHPLC in the unknown strains and also confirmed serotype-associated sequence changes in non-Typhimurium serotypes. CONCLUSIONS: The results demonstrated that a non-specialist standard HPLC machine fitted with a generally available column can be used to detect SNPs in gyrA, gyrB, parC and parE genes by DHPLC. Wider applications should be possible.     

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.     

Role of novel gyrA mutations in the suppression of the fluoroquinolone resistance genotype of vaccine strain Salmonella Typhimurium vacT (gyrA D87G)

OBJECTIVES: This study was aimed at characterizing the gyrA locus and determining its impact on fluoroquinolone susceptibility, DNA supercoiling degree and growth rate of Salmonella Typhimurium live vaccine strain vacT in comparison with its parent M415. Furthermore, the role of multiple drug resistance efflux in the susceptibility of vacT to fluoroquinolones and macrolides was investigated. METHODS: DNA sequences were determined for genes gyrA, gyrB, parC and parE of M415 and three consecutive mutants Nal2ori, Nal2passage and vacT. The impact of gyrA mutations on the fluoroquinolone susceptibilities and relative DNA supercoiling degrees was investigated by a complementation assay using wild-type gyrA (gyrA+) and a reporter gene system, respectively. Doubling times of the strains and MICs of different antibiotics in the absence and presence of an efflux pump inhibitor (EPI) were determined. RESULTS: Besides the gyrA mutation D87G, two novel mutations (G75A and A866S) were identified in the three mutants and a third novel mutation W59R in vacT. Fluoroquinolone susceptibilities and DNA supercoiling degrees of all three mutants were reduced compared with those of M415. Introduction of the gyrA+ allele restored fluoroquinolone susceptibilities of the two intermediate strains to the wild-type level; however, for vacT, MICs of fluoroquinolones were reduced below those of M415. VacT had a higher susceptibility to macrolides and the EPI compared with M415. CONCLUSIONS: The data point to a combination of at least one non-gyrA mutation and novel gyrA mutation(s) as the basis for the unusual fluoroquinolone susceptibility of vacT.     

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.     

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.     

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。     

Frequency of 1st- and 2nd-step topoisomerase mutations in Streptococcus pneumoniae following levofloxacin and moxifloxacin exposure

Seven Streptococcus pneumoniae isolates were exposed to inhibitory concentrations of levofloxacin and moxifloxacin in antibiotic-containing agar dilution plates. Colony counts were used to calculate the frequency of mutation. DNA was sequenced to detect mutations in the quinolone resistance-determining regions of the gyrA, gyrB, parC, and parE genes. The wild-type S. pneumoniae isolate developed a parC mutation after exposure to levofloxacin more frequently than it developed a gyrA mutation after exposure to moxifloxacin. The 1st-step gyrA mutant developed a 2nd-step gyrA-parC mutation more frequently after exposure to levofloxacin. Conversely, the transformation from a 1st-step parC mutant to a 2nd-step parC-gyrA mutant occurred more frequently following exposure to moxifloxacin. Our data suggest that the occurrence of a 2nd mutation will be contingent on the location of the 1st mutation and the preferential binding site of the fluoroquinolone that drives the transformation from 1st- to 2nd-step mutant.     

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.     

上海地区肺炎链球菌对氟喹诺酮类的敏感性及其喹诺酮耐药决定区突变研究

目的了解上海地区临床分离肺炎链球菌对氟喹诺酮类等抗菌药物的敏感性，并对氟喹诺酮类敏感菌株的喹诺酮耐药决定区（QRDR）突变进行初步研究。方法收集上海地区部分医院2004-2005年共176株肺炎链球菌临床分离株，用琼脂稀释法测定环丙沙星、左氧氟沙星、加替沙星和莫西沙星等15种抗菌药物的抗菌活性，并选取部分左氧氟沙星敏感肺炎链球菌菌株进行QRDRPCR扩增、测序。结果176株受试菌株中．PSSP、PISP和PRSP各占48．9％、47．1％和4．0％，受试菌株对环丙沙星、左氧氟沙星、加替沙星和莫西沙星敏感率均为100％。QRDR的扩增测序结果发现，20株左氧氟沙星MIC1～2mg／L的敏感菌株中，19株的parC、parE基因上已存在不同程度的氨基酸突变．包括ParC：Phe 105→Leu／Asp136→Tyr，Pare：Asp435→AsnjIle460→ValjAsn477→Lys．而在gyrA、gyrB基因上仅发现点突变，未导致相关氨基酸突变。结论上海地区未发现氟喹诺酮类耐药肺炎链球菌临床分离株，但左氧氟沙星MIC1～2mg／L的敏感株中已发现QRDR一级突变现象，突变的基因位点均见于parC、pare基因。     

耐喹诺酮铜绿假单胞菌药物作用靶位改变的研究

目的：探讨铜绿假单胞菌对喹诺酮类药物的耐药机制。方法30株对环丙沙星耐药的铜绿假单胞菌,采用 PCR方法扩增DNA解旋酶和拓扑异构酶Ⅳ的基因 gyrA,gyrB,parC和 parE,再测序查找是否存在位点突变,同时用脉冲场电泳（pulsed-field gel electrophoresis,PFGE）对菌株进行同源性分析。结果30株菌中,28株菌出现 gyrA基因扩增片段的137位点均有C→T突变,导致T83I改变;17株菌gyrB基因扩增片段的351位出现G→C突变,导致G466A改变;21株菌的 parC基因扩增片段的277位点有 C→U 突变导致 S87L 改变;2株菌 parE 基因在不同位点出现 C→U 突变,导致A425V和 A473V改变。30株菌可分为6个克隆,其中 A克隆4株,仅有 gyrA突变;B克隆7株,有 gyrA和 parC两种基因发生突变;C克隆3株,有gyrA和gyrB两种基因发生突变;D克隆14株,同时有 gyrA,gyrB和 parC三种基因发生突变;其他克隆2株,仅在 parE位点发生突变。结论该组菌株的靶位突变型与流行克隆型密切相关,同一流行型的菌株药物作用靶位的改变相同,并与环丙沙星的 MICs值的高低呈正比,突变基因数越多,MICs 值越高。4种基因中 gyrA基因突变频率最高,且该突变比其他靶位的突变对药物与靶位结合的影响更大,是需要关注的重点。     

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.     

南京地区肺炎链球菌的耐药变迁及喹诺酮耐药机制研究

目的了解南京地区肺炎链球菌临床分离株的耐药性变迁及对喹诺酮类的耐药机制。方法收集2010-2012年南京7所教学医院共147株肺炎链球菌,琼脂稀释法测定9种抗菌药物的MIC,与南京2006-2007年分离的肺炎链球菌耐药情况进行比较。并对氟喹诺酮耐药株的gyrA、gyrB、parE和parC基因喹喏酮耐药决定区域（QRDR）进行PCR扩增及测序。结果147株肺炎链球菌中,对红霉素的耐药率为89．1％;青霉素不敏感肺炎链球菌（MIC≥4mg／L）占3．4％;对头孢呋辛、头孢曲松、美罗培南、左氧氟沙星、莫西沙星的耐药率分别为26．5％、1．4％、3．4%、1．4％、0．7％;所有分离株对万古霉素、利奈唑胺敏感。与2006-2007年分离株相比,2010-2012年临床分离株对青霉素、头孢呋辛耐药率下降,对红霉素、头孢曲松耐药率变化不大,出现了少数左氧氟沙星及莫西沙星耐药株。对喹诺酮耐药株进行基因测序发现1菌株有gyrA突变（Asn167-Ile）和ParE突变（Ile460～Val）;另1菌株则有ParC突变（Ser81-Gly;Asn94-Asp）。结论南京地区肺炎链球菌对红霉素耐药率居高不下,对青霉素仍较敏感,出现了少数莫西沙星、左氧氟沙星耐药株。喹诺酮耐药株有gyrA、parE和parC的QRDR突变。