Chromosomal mechanisms of aminoglycoside resistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients

In total, 40 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients were included in this study. Twenty of these were collected in 1994 and 1997, from six CF patients, and the rest were collected from different CF patients in 2000 and 2001. The relative expression of mRNA for the efflux pump protein MexY was determined by real-time PCR and correlated with susceptibilities to amikacin and tobramycin. The chromosomal genes mexZ , rplY , galU , PA5471 and nuoG , which were found to have a role in the gradual increase in MICs of aminoglycoside antibiotics in laboratory mutants of P. aeruginosa , were analysed. MexY mRNA overproduction was found in 17/20 isolates collected in 1994 and 1997, and was correlated with decreased susceptibility to aminoglycosides. Alteration of the MexXY–OprM efflux system has been the main mechanism of resistance to aminoglycoside antibiotics in CF P. aeruginosa isolates over the 3-year period. In several isolates, expression of the PA5471 gene product might have some effect on elevated MICs of aminoglycosides. Inactivation of rplY , galU and/or nuoG may explain the gradual increase in MICs of aminoglycosides in laboratory mutants but probably not in the CF environment, as rplY and galU were unaltered in all isolates, and nuoG was not expressed in only one isolate. No 16S rRNA A-site mutations were found in any of the four copies of the gene in 13 investigated isolates.     

Mechanisms of resistance in clinical isolates of Pseudomonas aeruginosa less susceptible to cefepime than to ceftazidime

The MIC of cefepime determined with the MicroScan WalkAway system was ≥2 times higher than that of ceftazidime for 105 clinical isolates of Pseudomonas aeruginosa. This phenotype was confirmed by reference microdilution in 68 (64.8%) isolates, corresponding to 48 different rep-PCR patterns. The PSE-1 blactamase was identified in only 13.2% isolates, while oxacillinases were not identified in any of the 68 isolates. The level of expression of mexB, mexD and mexY was determined by real-time RT-PCR in eight clinical isolates representative of the different clones and patterns of susceptibility to cefepime and ceftazidime and in strain PAO1. All clinical strains overexpressed the mexY gene (18.3- to 152.7-fold in comparison with PAO1), although there was not a linear relationship between MIC of cefepime and level of mexY expression. Five of these strains contained mutations in the regulatory gene mexZ. mexD and mexB were also overexpressed in three and two isolates, respectively. Different mutations were observed in the regulatory genes nalD, mexR, nfxB and nalC. In conclusion, we have documented in our institution a polyclonal spread of P. aeruginosa with higher MICs of cefepime than of ceftazidime, related to overexpression of Mex-XY-OprM, coincident in some isolates with the production of PSE-1, Mex-CD-OprJ or MexAB-OprM.     

多重耐药铜绿假单胞菌MexAB-OprM、MexXY-OprM主动外排系统的耐药作用

目的 探讨MexAB-OprM、MexXY-OprM主动外排系统（外排泵）在多重耐药铜绿假单胞菌耐药机制中的作用.方法 选取25株外科监护室分离的多重耐药铜绿假单胞菌,用实时定量RTPCR测定结构基因mexA、mexX的mRNA表达水平来判断MexAB-OprM、MexXY-OprM外排泵表达情况;用PCR分别扩增其调控基因mexR、mexZ,并对其产物测序,用Blast软件在GenBank与已知序列比较,研究其过度表达的机理.结果 25株多重耐药铜绿假单胞菌中,14株高表达MexAB-OprM系统（56%）,3株高表达MexXY-OprM系统（12%）,这3株菌同时高表达2种外排泵.在8株mexA高表达的菌株中,5株菌发生mexR基因突变,出现氨基酸替代,1株mexR提前出现终止密码子,2株菌未发现mexR基因突变.2株mexX高表达的菌株均发生基因mexZ突变,出现氨基酸替代.结论 主动外排系统MexAB-OprM、MexXY-OprM在多重耐药铜绿假单胞菌中过度表达,是此菌多重耐药机制之一;外排泵MexAB-OprM、MexXY-OprM高表达分别与调控基因mexR、mexZ发生变异有关,同时存在其他调控机制.     

Mechanism of adaptive resistance to aminoglycosides of Pseudomonas aeruginosa

Exposure of Pseudomonas aeruginosa to aminoglycosides frequently selects for recalcitrant subpopulations exhibiting an unstable, < adaptive > resistance to these antibiotics. In this study, we investigated the implication in the phenomenon of MexXY-OprM, an active efflux system known to export aminoglycosides in P. aeruginosa. Immunoblotting experiments demonstrated that the transporter MexY, but not the outer membrane pore OprM, was overproduced during the post-drug exposure adaptation period in wild-type strain PAO1. Furthermore, MexY production was dependent upon the degree of bacterial exposure to gentamicin (drug concentration). In contrast to parental strain PAO1, mutants defective in MexXY or in OprM were unable to develop adaptive resistance. Altogether, these results indicate that the resistance process requires the rapid production of MexXY and the interaction of these proteins with the constitutively produced component OprM.     

siHybrids技术沉默铜绿假单胞菌外排泵 mexB基因体外效应的初步研究

目的 初步研究siHybrids技术对铜绿假单胞菌野生菌PAO1外排泵mexB基因体外沉默效应。方法 针对铜绿假单胞菌野生株PAO1外排泵mexB基因设计并合成3条特异性siHybrids分子和1条阴性对照siHybrids分子。在分子浓度为50 nmol/L下,分别以合成的siHybrids分子干扰铜绿假单胞菌野生菌PAO1,并设实验组为铜绿假单胞菌野生菌PAO1空白对照组,阴性对照组scamble(sc)-001,干预组siHybrids( si) -001、siHybrids( si) -002及siHybrids( si) -003,分别在干预12h及24h后采用real-time PCR法检测各实验组中靶基因mexB基因mRNA的表达水平。进一步采用Mueller-Hinton倍比稀释法检测50 nmol/L浓度下,siHy brids分子干预铜绿假单胞菌野生菌PAO1前后氯霉素(CP)、红霉素(EM)、左氧氟沙星(L-OrLX)、头孢他啶(CAZ)、美洛培南(MER)的最小抑菌浓度(MIC)值。结果 不同siHybrids分子干预PAO1 12 h后,mexB基因mRNA表达量无明显差异性;但干预24 h后,mexB基因mRNA表达量：干预组（si-001,si-002,si-003）比空白对照组、阴性对照组（sc-001）有明显下降。对比干预12h、24h后mexB基因mRNA表达量,可以发现空白对照组、阴性对照组（sc-001）mRNA的表达量成上升趋势,而干预组（si-001,si-002,si-003）mexB基因mRNA表达量均呈下降趋势。siHybrids分子在干预铜绿假单胞菌野生菌24h前后的氯霉素(CP)、红霉素(EM)、左氧氟沙星( L-OFLX)、头孢他啶(CAZ)、美洛培南(MER) MIC无明显差异性。结论 在mRNA表达水平上,siHybrids分子能体外干预铜绿假单胞菌PAO1 mexB基因mRNA表达,此种沉默作用呈现时间依赖性,且在24h能有效地发挥干预作用。     

Mutations in the cueA gene encoding a copper homeostasis P-type ATPase reduce the pathogenicity of Pseudomonas aeruginosa in mice

A sequencing project identified a putative copper homeostasis gene, cueA, in Pseudomonas aeruginosa strain PAO1. Strains with mutations of the cueA gene, encoding a P-type ATPase linked to copper homeostasis in P. putida, displayed greater sensitivity to copper compared to wild-type bacteria using MIC determinations and in vitro passage in growth media with different concentrations of copper added. An LD50 assay showed a cueA deletion mutant was 50-fold more attenuated than wild-type strain PAO1 bacteria. Complementation of the cueA mutation restored in vitro tolerance to copper and virulence in a systemic model of infection to near wild-type levels. Competition assays between cueA mutants and wild-type P. aeruginosa strains demonstrated 20-fold attenuation by the cueA mutants within spleens of mice. This data suggests the P. aeruginosa CueA protein may be important in maintaining copper homeostasis both in vitro and in vivo.     

Molecular characterization of an epidemic clone of panantibiotic-resistant Pseudomonas aeruginosa

We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired beta-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-beta-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.     

siRNA干扰铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达的研究

目的 探讨RNA干扰技术对铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达及有关功能的影响.方法 设计并合成4条小分子干扰RNA(small interfering RNA,siRNA)(siRNA1、siRNA2、siRNA3和siRNA4)序列,构建干扰RNA( short hair pin RNA,shRNA)表达载体,将带有siRNA质粒电转入PAO1和临床耐药株PAO3.应用E-test方法检测PAO1和PAO3抗生素MIC的变化.用半定量RT-PCR和定量PCR方法检测mexB的表达变化.结果 经酶切鉴定siRNA表达载体构建成功,并明显提高PAO和临床耐药株PAO3对抗生素的敏感性.RT-PCR结果显示干扰后的PAO1和临床耐药株PAO3 mexB基因表达明显下降(P＜0.05).结论 siRNA成功干扰MexA-MexB-OprM外排泵mexB基因的表达,提高铜绿假单胞菌对抗生素的敏感性,为临床上治疗铜绿假单胞菌感染,降低其耐药性提供了新的思路.     

Low-level resistance to fluoroquinolones conferred by efflux overproduction in Pseudomonas aeruginosa: clinical significance and routine detection

The aim of this study was (i) to assess the impact of stable overproduction of efflux systems MexAB-OprM and MexXY-OprM on the bacteriostatic activities of fluoroquinolones in clinical Pseudomonas aeruginosa strains and (ii) to find a convenient test for screening isolates with a low level resistance to fluoroquinolones. The minimal inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were determined for clinical isolates of P. aeruginosa overexpressing MexAB-OprM or MexXY-OprM. Efflux pumps derepression was associated with a modest two- to fourfold increase in resistance to the tested fluoroquinolones. Clinical significance of low level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC > or =0.25 mg/L) or levofloxacin (MIC > or =1 mg/L) such as those due to overproduced MexAB-OprM or MexXY-OprM were predicted to result in poor clinical outcomes. Altogether these data strongly suggest that when derepressed MexAB-OprM or MexXY-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones such as ciprofloxacin and levofloxacin. Routine detection of clinical strains that displayed low-level resistance to fluoroquinolones with a Mueller Hinton agar containing 0.20 mg/L of ciprofloxacin will help clinician in his therapeutical choice.     

铜绿假单胞菌oprD2基因突变及表达量改变与碳青霉烯类耐药的关系

目的 研究铜绿假单胞菌外膜通道蛋白OprD2的表达减弱或缺失,以及OprD2蛋白自身突变是否会影响铜绿假单胞菌对碳青霉烯类药物的耐药性.方法 收集分离自临床对亚胺培南(IPM)的最低抑菌浓度(MIC)值≥8μg/m1的铜绿假单胞菌共101株,采用肉汤稀释法检测菌株对比阿培南(BPM)、美罗培南(MEM)、帕尼培南(PEM)的MIC值;荧光定量RT-PCR检测铜绿假单胞菌膜通道蛋白oprD2基因的表达量情况;针对oprD2相对表达量正常并对亚胺培南耐药的铜绿假单胞菌,采用普通PCR的方法扩增oprD2全长基因并测序.结果 根据铜绿假单胞菌的OprD2蛋白相对表达量结果,将101株铜绿假单胞菌分成两组,组1为OprD2相对表达量降低组;组2为OprD2相对表达量正常组;组1与组2相比,对IPM、BPM、MEM、PEM的MIC值≥16μg/ml的菌株比例差异均有统计学意义(P＜0.01).组1中,28株同时对4种药物的MIC均≥16 μg/ml,其中有25株的OprD2的相对表达量明显减低(＜0.4);外膜孔蛋白OprD2转录水平与4种碳青霉烯类药物MICs之间呈负相关趋势.组2中,有16株9prD2基因发生突变,按照突变的类型主要分成4组;与PAO1相比,这些菌株对IPM、BPM、MEM、PEM的MIC值有不同程度的增加.结论 OprD2外膜蛋白的表达量减少或缺失是铜绿假单胞菌对亚胺培南耐药的主要机制,可能也与其他3种碳青霉烯类药物耐药有密切关系;铜绿假单胞菌的oprD2基因发生突变,可能会降低铜绿假单胞菌对这儿种碳青霉烯类药物的敏感性.     

Rapid identification of mutations in a multidrug efflux pump in Pseudomonas aeruginosa

The gene mexR regulates negatively the expression of the MexA-MexB-OprM efflux pump in Pseudomonas aeruginosa, and mutations in mexR cause a multiple antibiotic resistance phenotype. Five hundred and forty resistant clones of P. aeruginosa PAO503 were isolated after selection for resistance to chloramphenicol or tetracycline. All isolates showed similar phenotypes and were resistant to tetracycline, chloramphenicol and norfloxacin. Nineteen randomly selected isolates were analyzed. Since mutational analysis by direct sequencing of all regions of interest in several strains is time-consuming and expensive, a screening method, Non-Isotopic RNase Cleavage Assay (NIRCA), was applied to identify mutant genes so that they could be targeted for DNA sequencing. NIRCA is a simple but rapid method for mutational analysis and can be performed in 3-4 h. Results of NIRCA analysis were compared with DNA sequencing. Both NIRCA and DNA sequencing analysis showed mexR gene mutations in 11 of 19 isolates but no alterations in 8 strains. An immunoblot assay showed overexpression of OprN, a component of another multidrug efflux pump, MexE-MexF-OprN, in those eight isolates. Nucleotide sequencing of quinolone resistance-determining regions of DNA gyrase (gyrA) or topoisomerase IV (parC) showed no alterations in any of the 19 mutants. The data indicate that two efflux pump systems, MexA-MexB-OprM and MexE-MexF-OprN, were involved in multidrug resistance including quinolones and that NIRCA is a sensitive method for screening mutations.     

Role of efflux pumps and mutations in genes for topoisomerases II and IV in fluoroquinolone-resistant Pseudomonas aeruginosa strains

We have investigated the occurrence of mutations in topoisomerase II (DNA gyrase) subunit B(gyrB) and topoisomerase IV subunit E(parE) and the hyperexpression of genes for four efflux pump proteins in 20 previously described, fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa. Amino acid alterations were found in GyrB in five strains and in ParE in three strains with MIC of norfloxacin > or = 8 mg/L, and it is likely that some of the alterations contribute to the quinolone resistance exhibited by these strains. Seventeen of the 20 strains overproduced mRNA for one or more pump proteins (MexB, MexD, MexF, or MexY), which caused multidrug resistance phenotype in more than half of strains. Two strains were hypermutable and one of them was highly resistant, but the other strain was only moderately resistant.     

Multicentre study of the in vitro activity of cefepime, a broad-spectrum cephalosporin, compared to other broad-spectrum agents

The in vitro activity of cefepime was compared to that of a range of other broad-spectrum agents, using a gradient diffusion MIC method, against 995 recent clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, other nonfermentative gram-negative bacilli, staphylococci (except oxacillin-resistant Staphylococcus aureus), streptococci, enterococci, and aerobic gram-positive bacilli. Cefepime had excellent activity against Enterobacteriaceae, including eight presumptive extended-spectrum beta-lactamase producers and 33 stably derepressed mutants of natural cephalosporinases. Activity against Pseudomonas aeruginosa was similar to ceftazidime and superior to cefpirome. Its activity against gram-positive cocci was also good, being more active against staphylococci and only slightly less active against streptococci than ceftriaxone. Cefepime maintained activity against bacteria resistant to aminoglycosides and ciprofloxacin. Enterococci, Bacillus species, Burkholderia cepacia and Stenotrophomonas maltophilia were predicably resistant. Cefepime has a spectrum of activity almost as broad as that of the carbapenems.     

Fluoroquinolone resistance mechanisms in urinary tract pathogenic Escherichia coli isolated during rapidly increasing fluoroquinolone consumption in a low-use country

Resistance to ciprofloxacin in Escherichia coli from urinary tract infections (UTI) in Denmark is increasing parallel to increased use of fluoroquinolones both in Denmark and in other European countries. The objective was to investigate the occurrence of ciprofloxacin resistance mechanisms, phenotypic coresistance, and if ciprofloxacin resistance was caused by clonal spread or to individual mutational events in a collection of consecutively obtained E. coli submitted to a clinical microbiology department at a Danish hospital. One hundred four UTI-related E. coli resistant toward nalidixic acid by disc diffusion were typed by Pulsed Field Gel Electrophoresis (PFGE) with XbaI. One isolate representing each PFGE type and only one patient (n = 77) were investigated for point mutations in sequenced PCR amplicons of the four topoisomerase genes; qnr genes by use of PCR; aac(6')-Ib-cr by BtsCI restriction of PCR products; and efflux using efflux pump inhibitors in a broth dilution assay. Minimal inhibitory concentration (MIC) was determined for 21 antibacterial agents, including ciprofloxacin. Of the 77 isolates, the majority were resistant to ciprofloxacin (91%) and multiresistant (resistant to ≥ 3 antimicrobial classes, 83%). Ciprofloxacin-resistant isolates showed at least one target mutation. A significant, positive correlation was found regarding MIC of ciprofloxacin and the number of target mutations. Efflux was found as a resistance mechanism in 77% of isolates tested (n = 60). The aac(6')-Ib-cr gene was detected on plasmids from five isolates showing ciprofloxacin MICs >512 mg/L. No overall clonal relationship among isolates was found according to PFGE. Target modification is the dominating fluoroquinolone resistance mechanism often found in combination with efflux and sometimes aac(6')-Ib-cr. In Denmark, increasing ciprofloxacin resistance in E. coli is mainly due to mutational events and not to spread of clones.     

Pseudomonas aeruginosa--a significant hospital pathogen and resistance to carbapenem

Pseudomonas aeruginosa (P. aeruginosa) is an etiologic agent of nosocomial infections of various localizations. The frequency of infections is a consequence of an increased number of immunocompromised patients, large surgical interventions, long-term hospital care and virulence factors of the bacterium. The use of carbapenems in the treatment of infections caused by P. aeruginosa and other gram-negative bacteria entail an unfortunate consequence of creating resistance to carbapenems. P. aeruginosa exhibits numerous mechanisms of resistance and is singularly problematic for combining intrinsic resistance and acquired resistance due to multiple mutations. It is also a carrier of the multiple-resistance plasmide. Carbapenems are a class of beta-lactam antibiotics with broad-spectrum activity to gram-positive, gram-negative and strictly anaerobic bacteria. The resistance of P. aeruginosa to carbapenems is complex and heterogeneous. It is determined by weakening the penetration through the outer membrane due to the loss of porin D2, decrease in the accumulation of antibiotic in the cell consequentially to active efflux due to hyperproduction of proteins, hydrolysis of carbapenem by specific metallo-beta-carbapenemases, and alteration in the target area of antibiotic activity on the bacterial cell. The loss of porin D2 results in resistance to imipenem with MIC values of 8.0-32.0 microg/mL. Selective low-level resistance to meropenem in the hyperproduction of the efflux system results in MIC values of 2.0-4.0 microg/mL. A high-level resistance to carbapenems with MIC values above 128 microg/mL for imipenem and meropenem is a consequence of the secretion of IMP and VIM series beta-carbapenemases. The frequency of resistance to P. aeruginosa to carbapenems varies worldwide from 15% to > or = 35%. In Croatia, resistance to carbapenems in the year 2003 was estimated to 12%. The problems in clinical practice are the increased resistance of P. aeruginosa to carbapenems, the presence of beta-carbapenemases and acquisition of these enzymes in certain types of Enterobacteriacea. The problems in laboratory practice are those of precise determination of the level and phenotype resistance of P. aeruginosa to carbapenems.     

Resistance to fluoroquinolones linked to gyrA and par C mutations and overexpression of acr AB efflux pump in Salmonella enterica serotype Choleraesuis

Between 2000 and 2002, 60 clinical isolates of Salmonella enterica serotype Choleraesuis were collected to investigate the mechanism of fluoroquinolone resistance. PCR and sequencing were performed to identify mutations in gyrA, gyrB, par C, the Acr AB-TolC efflux pump regulator, acr R, and the global regulons mar RAB and sox RS. All resistant strains showed mutations in the target genes leading to amino acid changes of Ser 83 Phe and Asp 87 Asn in GyrA and Ser 80 Ile in Par C. A mutation in gyrB was linked to the serotype genetic diversity but not to fluoroquinolone resistance. An efflux pump inhibitor, Phe-Arg-beta-naphthylamide, caused fourfold lower MIC of ciprofloxacin in the resistant isolates, indicating that efflux systems are involved in fluoroquinolone resistance. Western blot analysis showed moderate overproduction of Acr A in fluoroquinolone- resistant isolates. A mutation in acr R gave rise to an internal stop codon in both ciprofloxacin-resistant and -susceptible isolates, suggesting another serotype genetic diversity. No mutations were detected in mar RAB and sox RS among the isolates examined. Cross-resistance to three fluoroquinolones was observed, but gatifloxacin demonstrated relatively lower MICs than those of ciprofloxacin and levofloxacin. Fluoroquinolone resistance in S. Choleraesuis appears to be the combination effect of multiple mutations in various target genes and overexpression of the Acr AB-TolC efflux pump.     

Patterns of quinolone susceptibility in Campylobacter jejuni associated with different gyrA mutations

AIMS: To investigate the diversity of genetic mutation in the quinolone resistance-determining region (QRDR) of gyrA in clinical isolates and laboratory-derived mutants of Campylobacter jejuni resistant to ciprofloxacin (CipR) and to determine the influence of this mutation on the susceptibility of the organisms to different quinolone antibiotics. METHODS: Laboratory-derived CipR mutants were obtained from C. jejuni NCTC 11 168 and six quinolone-sensitive faecal isolates (parent prototypes) grown in sub-inhibitory concentrations of ciprofloxacin. Initial mutants found to be CipR were designated 'primary mutants' and subjected to a repeat of this process to select 'secondary mutants' with increased resistance. The susceptibility of the mutants and an additional six clinical isolates of CipR C. jejuni to seven quinolone antibiotics was determined by measuring their MICs. The QRDR of gyrA in all strains was amplified by PCR, sequenced and compared with that of the L04566 C. jejuni gyrA gene. RESULTS: All six CipR clinical isolates contained a Thr-86-Ile mutation. This was also the commonest mutation found amongst the laboratory derived CipR strains. Other derived mutations in the in vitro derived CipR group included Asp-90-Asn, Thr-86-Ala, and a previously unreported double mutation, Asp-85-Tyr and Thr-86-Ile. Strains with the Thr-86-Ile mutation had the highest MICs to seven different quinolones. CipR strains with other single mutations had a lower range of MICs. There were no additional QRDR mutational changes detected in secondary mutants even where MICs to the fluoroquinolones were higher than in primary mutants. CONCLUSIONS: Thr-86-Ile mutations were common in both clinical and laboratory derived CipR strains. Other mutations found amongst the latter strains were more sensitive to the fluoroquinolones. Different QRDR changes in gyrA differentially affected the susceptibility of CipR C. jejuni to the various fluoroquinolones.