Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.     

Recognition of mechanisms involved in bile resistance important to halting antimicrobial resistance in nontyphoidal Salmonella

Increasing antimicrobial resistance in nontyphoidal Salmonella (NTS) is a global public health problem that complicates antimicrobial therapy. As an enteric pathogen, Salmonella must endure the presence of bile in the intestinal tract during the course of infection. In this study, we sought to identify Salmonella genes necessary for bile resistance and to investigate their association with antimicrobial resistance. Four genes related to bile resistance were identified, namely rfaP, rfbK, dam and tolC. The first three genes are involved in lipopolysaccharide synthesis, and tolC is associated with an efflux pump. Antimicrobial susceptibility testing showed increased susceptibility to polymyxin B and ciprofloxacin in rfaP and tolC mutants of Salmonella, respectively. Genetic analysis of 45 clinical isolates of NTS revealed that all isolates with reduced susceptibility to fluoroquinolones (minimum inhibitory concentration ≥0.125 mg/L) were associated with point mutations in the quinolone resistance-determining regions of the gyrA and parC genes. The efflux pump also played a role, as evidenced by the reduction in fluoroquinolone resistance when the TolC efflux pump was inhibited by Phe-Arg-β-naphthylamide, a competitive efflux pump inhibitor. Based on these results, we conclude that an intact membrane structure and the efflux pump system provide mechanisms enabling NTS to resist bile. Caution should be taken when using ciprofloxacin and polymyxin B to treat Salmonella enteric infection, as resistance to these agents involves the same mechanisms. Addition of an efflux pump inhibitor to fluoroquinolones may be an effective strategy to deal with the increasing resistance in NTS.     

泵抑制剂对耐喹诺酮类铜绿假单胞菌临床株作用的研究

目的　了解四种泵抑制剂对喹诺酮类耐药铜绿假单胞菌株耐药水平的降低作用与耐药决定区基因突变及耐药表型的关系。方法　采用羰基氰氯苯腙 (CCCP)、利舍平、维拉帕米及奥美拉唑以琼脂稀释法分别加入喹诺酮耐药的菌株中 ,与喹诺酮类抗生素共同作用 ,并同时测定其最低抑菌浓度 (MIC)的变化。对部分耐药株 Gyr A和 Par C区进行 PCR- RFL P分析 ,并行 DNA测序观察突变情况。结果　四种泵抑制剂中CCCP能极大的降低 MIC的值。利舍平也可部分减低 MIC的值 ,但维拉帕米及奥美拉唑作用轻微。高度耐药株存在 gyr A和 par C的突变。结论　寻找理想的泵抑制剂可降低铜绿假单胞菌的耐药水平。     

Contribution of mutations in DNA gyrase and topoisomerase IV genes to ciprofloxacin resistance in Escherichia coli clinical isolates

DNA gyrase (GyrA and GyrB) and topoisomerase IV (ParC and ParE) are the two essential type II topoisomerases in Escherichia coli. These enzymes act via inhibition of DNA replication. Mutations in the quinolone resistance-determining region (QRDR) of the gyrA, gyrB, parC and parE genes from clinical isolates of E. coli were determined by DNA sequencing of 54 ciprofloxacin-resistant clinical isolates from a hospital in Delhi, India. The majority of the E. coli isolates were shown to carry mutations in gyrA, parC and parE. Ciprofloxacin resistance due to accumulation of such a high number of mutations in the QRDR regions of gyrA at positions Ser83 and Asp87 and parC at position Ser80 as well as outside of the QRDR region of parE at Ser458 and Glu460 confers high-level resistance of ciprofloxacin in clinical isolates. The high frequency of occurrence of mutations in the parE gene (44.4% strains) is alarming, as topoisomerase IV is a secondary target of quinolones.     

High prevalence of plasmid-mediated quinolone resistance determinant aac(6')-Ib-cr amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China

In this study, the antimicrobial susceptibilities and prevalence of plasmid-mediated quinolone resistance determinants amongst Salmonella enterica serotype Typhimurium isolates from hospitalised paediatric patients with diarrhoea in China were investigated. In total, 40 (64.5%) of 62 S. Typhimurium isolates were resistant to ciprofloxacin (minimum inhibitory concentration ≥0.5 μg/mL), comprising 28 isolates with low-level resistance and 12 isolates with high-level resistance. All ciprofloxacin-resistant isolates were multiresistant to other antimicrobial agents. Four pulsed-field gel electrophoresis (PFGE) clusters were found amongst the 40 ciprofloxacin-resistant isolates, amongst which PFGE clusters A, B, E and D accounted for 7, 4, 1 and 28 isolates, respectively. Two isolates with high-level ciprofloxacin resistance had two mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The remaining ciprofloxacin-resistant isolates had only one mutation in the QRDR of gyrA. All 62 S. Typhimurium isolates were negative for qnr genes and qepA and 23 (37.1%) of the isolates were positive for aac(6')-Ib-cr. Nineteen isolates harbouring aac(6')-Ib-cr belonged to PFGE cluster D. A high prevalence of ciprofloxacin resistance and aac(6')-Ib-cr was found amongst S. Typhimurium isolates in China from hospitalised paediatric patients with diarrhoea not receiving quinolones. A single mutation in the QRDR of gyrA as well as production of AAC(6')-Ib-cr contributed to ciprofloxacin resistance. Clonal spread was responsible for the dissemination of aac(6')-Ib-cr amongst S. Typhimurium isolates.     

Update on fluoroquinolone resistance in Helicobacter pylori: new mutations leading to resistance and first description of a gyrA polymorphism associated with hypersusceptibility

Helicobacter pylori eradication by standard therapy is decreasing due to clarithromycin and metronidazole resistance. Fluoroquinolones are valuable drugs for alternative therapy, but their activity needs to be updated. We determined minimum inhibitory concentrations (MICs) of the newly marketed fluoroquinolones (levofloxacin, moxifloxacin and gatifloxacin) and assessed the prevalence of resistance in 128 H. pylori strains isolated in 2004-2005. The quinolone resistance-determining region (QRDR) of gyrA was sequenced for all strains. Gatifloxacin MICs (MIC(50) = 0.25 mg/L) were two- to four-fold lower than those of the other fluoroquinolones. The prevalence of resistance (ciprofloxacin MIC > 1 mg/L) was 17.2% (22 strains). All resistant strains harboured one gyrA mutation at codons 86, 87 or 91, including three new mutations (Asp86Asn, Thr87Ile and Asn87Tyr). Ciprofloxacin-susceptible strains were devoid of such gyrA mutations, but harboured a polymorphism at codon 87 that distinguished 18 isolates (17%) with a Thr87 like the reference strain J99 from 88 strains with Asn87 like the reference strain 26695. Strains with Thr87 were four-fold more susceptible to nalidixic acid, pefloxacin, ciprofloxacin and levofloxacin and were equally susceptible to moxifloxacin and gatifloxacin. The high rate of quinolone resistance in H. pylori requires the use/implication of a 'test and treat' strategy that can confidently rely on QRDR gyrA sequencing.     

Trends in antimicrobial drug resistance in Salmonella enterica serotypes Typhi and Paratyphi A isolated in Europe, 1999-2001

Results of antimicrobial sensitivity tests for strains of Salmonella enterica serotypes Typhi and Paratyphi A isolated from patients in ten European countries between 1999 and 2001 have been transferred electronically to the Enter-net surveillance hub. For Typhi between 22 and 29% of isolates were multiresistant (to four drugs or more) with decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) increasing from 20% in 1999 to 26% in 2001. Nineteen of 169 (11%) strains with decreased ciprofloxacin susceptibility were sensitive to nalidixic acid. For Paratyphi A multiple resistance increased from 9% in 1999 to 25% in 2001 and decreased ciprofloxacin susceptibility from 6 to 17%. Clinicians should be aware of the possibility of treatment failures when fluoroquinolones are used as the first-line drug for infections with Typhi and Paratyphi A, particularly for patients recently returning from areas where drug-resistant strains are endemic.     

大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮耐药的相关性

目的:研究大肠埃希菌gyrA、parC和marOR基因突变与喹诺酮类耐药的相关性。方法:采用微量稀释法进行常规药敏试验,筛选3株萘啶酸敏感大肠埃希菌和37株萘啶酸耐药大肠埃希菌株;PCR扩增大肠埃希菌喹诺酮耐药决定区(QRDR)相关gyrA、parC基因,进行聚合酶链反应-单链构象多态性(PCR-SSCP)分析,同时PCR扩增marOR基因;在耐药株选取部分菌株对gyrA、parC及marOR基因进行测序,检测其突变情况,其结果与体外药敏试验结果进行比较,研究其相关性。结果:37株耐药株均出现gyrA基因突变,但对环丙沙星低耐株最低抑菌浓度(MIC)=2mg/L只出现gyrA单位点突变,而parC基因未发生突变;环丙沙星高耐株(MIC=64mg/L)gyrA基因出现3个位点突变,parC基因出现单位点突变;在环丙沙星高耐株(MIC=256mg/L),并伴有其他类抗菌药物的多重耐药时,除了出现gyrA和parC基因双位点突变,同时检测到marOR基因的多位点突变。结论:gyrA和parC基因突变在大肠埃希菌对喹诺酮耐药中起着重要作用,gyrA和parC基因突变的程度与大肠埃希菌耐药水平有关,marOR基因多位点突变在多重耐药机制中具有一定的作用。     

淋病奈瑟菌的耐药性与喹诺酮耐药基因突变的相关性分析

目的 分析本地区淋病奈瑟菌临床菌株gyrA基因突变与喹诺酮类药物耐药的相关性.方法 采用琼脂稀释法检测37株淋病奈瑟菌临床菌株对6种抗生素的最小抑菌浓度(MIC),PCR法扩增淋病奈瑟菌临床菌株gyrA基因片段并测序.结果37株淋病奈瑟菌临床菌株对大砚霉素、头孢曲松、四环素、青霉素、氧氟沙星和环丙沙星的耐药率分别为0、...     

Complete nucleotide sequence of the gyrA gene of Helicobacter pullorum and identification of a point mutation leading to ciprofloxacin resistance in poultry isolates

To assess the molecular basis of nalidixic acid and ciprofloxacin resistance in Helicobacter pullorum, the gyrA gene of H. pullorum CIP 104787T was sequenced. In addition, 9 isolates (2 susceptible to ciprofloxacin and resistant to nalidixic acid, 3 susceptible and 4 resistant to both antibiotics) were selected from 44 poultry isolates and the nucleotide sequences of their quinolone resistance-determining regions (QRDRs) were compared. The 2490 bp gyrA gene showed an open reading frame encoding a polypeptide of 829 amino acids. The deduced amino acid sequence of gyrA showed>or=72% identity to Helicobacter hepaticus, Helicobacter pylori and Wolinella succinogenes. Moreover, >or=98% amino acid sequence identity was found comparing the QRDR of the H. pullorum type strain with the QRDRs of the aforementioned bacterial species. All ciprofloxacin-resistant poultry isolates showed an ACA-->ATA (Thr-->Ile) substitution at codon 84 of gyrA, corresponding to codons 86, 87 and 83 of Campylobacter jejuni, H. pylori and Escherichia coli gyrA genes, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin-resistant phenotype of poultry isolates. This is the first report describing the complete 2490 bp nucleotide sequence of H. pullorum gyrA and confirming the involvement of the Thr84Ile substitution of GyrA in ciprofloxacin resistance of H. pullorum.     

High levels of multidrug resistance in clinical isolates of Gram-negative pathogens from Nigeria

In Nigeria, quinolones and β-lactam antibiotics are widely used to treat bacterial infections. This study aimed to identify the prevalence of resistance to these drugs and to determine the mechanisms of resistance to these agents. In total, 134 non-duplicate, Gram-negative enteric isolates of 13 species from different hospitals were investigated for susceptibility to a panel of antibiotics, carriage of plasmid-mediated quinolone and β-lactam resistance genes, production of extended-spectrum β-lactamases (ESBLs), and mutations within topoisomerase genes. The level of resistance to all antibiotics tested was extremely high, with minimum inhibitory concentrations for 90% of the organisms (MIC(90) values) of ≥ 256 μg/mL for all drugs. Of the 134 isolates, 92 had mutations within the quinolone resistance-determining region (QRDR) of gyrA or within gyrA and parC. In addition, the plasmid-mediated quinolone resistance genes qnrA, qnrB, aac(6')-Ib-cr and qepA were identified. The qnrD allele, which has previously only been found in Salmonella isolates from China, was identified in two Proteus isolates and one Pseudomonas isolate. Of the 134 isolates, 23 (17.2%) carried aac(6')-Ib-cr, 11 (8.2%) carried a qnr variant and 5 (3.7%) were positive for qepA. Twenty-eight isolates (20.9%) produced ESBL variants, with a CTX-M variant being carried by 25 isolates (18.7%). In addition, six isolates (4.5%) carried ampC variants [ACT-1 (1 isolate), DHA-1 (4 isolates) and CMY-2 (1 isolate)]. This study demonstrates a very high level of multidrug resistance amongst Gram-negative enteric bacilli isolated from different sites from patients in Nigerian hospitals as well as the presence of a variety of plasmid-associated resistance genes, including some identified from Africa for the first time.     

Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates

To understand the relationship between mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene and drug resistance to ofloxacin, 85 laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis mutant strains and 110 M. tuberculosis clinical isolates, screened by denaturing high-performance liquid chromatography to contain mutations, were analysed for their mutation patterns by sequencing as well as their ofloxacin minimal inhibitory concentrations (MICs). All mutations detected occurred at the codons Ala74, Ala90, Ser91 and Asp94 in all strains. One of the five different forms of missense mutation in Asp94 occurred in 60% of the laboratory-selected strains and 78% of the clinical isolates. However, 53 clinical isolates (48%) and only 2 laboratory-selected strains (2.4%) harboured double point mutations. The mutation Ala74Ser occurred only in the clinical isolates and only in combination with the Asp94Gly mutation. The ofloxacin MIC for the clinical isolates ranged from 0.5microg/mL to 20microg/mL, whilst the MICs for the laboratory-selected strains were > or =10microg/mL. The differences in gyrA gene mutation patterns and MICs between the laboratory-selected resistant strains and clinically isolated resistant strains identified here might help to understand the mechanisms involved in fluoroquinolone resistance.     

Emergence of multidrug-resistant Salmonella enterica serovar Typhi associated with a class 1 integron carrying the dfrA7 gene cassette in Nepal

A total of 121 Salmonella enterica serovars Typhi and Paratyphi A isolated from enteric fever patients at a university hospital in Nepal between February 2004 and January 2006 were tested for their antimicrobial susceptibility. The occurrence and cassette content of integrons as well as the molecular mechanisms of resistance among the multidrug-resistant (MDR) S. Typhi were evaluated. Thirty-nine percent of the isolates were susceptible to all the antimicrobial agents tested. Seven of the S. Typhi strains were MDR. None of the 121 S. enterica isolates were resistant to ciprofloxacin, cefazolin, rifampicin or kanamycin. All MDR S. Typhi isolates contained a class 1 integron with a single cassette, dfrA7, conferring resistance to trimethoprim. Pulsed-field gel electrophoresis (PFGE) of XbaI-generated genomic restriction fragments yielded 12 different patterns. Five of the seven MDR isolates containing class 1 integrons had an identical PFGE pattern. Resistance to sulfamethoxazole, streptomycin, ampicillin, tetracycline and chloramphenicol was mediated by sul1, strA-strB, blaTEM-like, tetB and catA genes, respectively. To the best of our knowledge, this is the first report of integron-associated multidrug resistance as well as the first molecular characterisation of the mechanism of resistance of S. Typhi isolated from Nepal. This study indicates the spread of integron-associated multidrug resistance in S. Typhi in Nepal.     

In vitro fluoroquinolone-resistant mutants of Salmonella enterica serotype Enteritidis: analysis of mechanisms involved in resistance

This study analysed the mechanisms involved in the acquisition of resistance to quinolones in mutants obtained in vitro of Salmonella enterica serotype Enteritidis. Two nalidixic acid-resistant (minimal inhibitory concentrations, MIC>256 mg/l), ciprofloxacin-susceptible (MIC 0.5 mg/l) clinical isolates of Salmonella Enteritidis with a mutation at amino acid codon Ser-83 of the gyrA gene were grown on plates containing increasing concentrations of ciprofloxacin. The increase in MIC to ciprofloxacin, sparfloxacin and trovafloxacin was totally or partially associated with over-expression of an AcrAB-like efflux pump. In addition, unidentified mechanism(s) may have been involved in the increased MIC to these antimicrobials. This study demonstrated that AcrAB-like efflux pumps appear to play a relevant role in the increase in MIC to some quinolones although, other, as yet undefined, mechanisms may be involved.     

Clinically significant borderline resistance of sequential clinical isolates of Klebsiella pneumoniae

Two sequential clinical isolates of Klebsiella pneumoniae (Kpn) were isolated from bronchoalveolar lavage fluid (Kpn#1) and sputum (Kpn#2) of a patient with pneumonia, complicated by anatomical and immunosuppressive problems due to Wegener's granulomatosis. Despite 4 weeks of systemic treatment with ciprofloxacin (CIP) Kpn#2 was isolated thereafter. A fluoroquinolone-resistant mutant (Kpn#1-SEL) was derived from Kpn#1 in vitro by selecting on agar plates supplemented with ofloxacin. Kpn#1, Kpn#1-SEL and Kpn#2 had an identical pattern in PFGE. CIP MICs were 0.25, 2 and 4 mg/l for Kpn#1, Kpn#2 and Kpn#1-SEL, respectively. Kpn ATCC 10031 (CIP MIC 0.002 mg/l) served as control. We analyzed mechanisms of fluoroquinolone resistance by determining antibiotic susceptibility, organic solvent tolerance, accumulation of fluoroquinolones, dominance testing with wild-type topoisomerase genes (gyrA/B, parC/E), sequencing of the quinolone resistance determining regions of gyrA/B, parC/E and marR and Northern blotting of marR and acrAB genes. Compared with Kpn ATCC 10031, elevated MICs to fluoroquinolones and unrelated antibiotics in Kpn#1 was presumably due to a primary efflux pump other than AcrAB and increased the CIP MIC 125-fold. Although Kpn#1 tested sensitive according to NCCLS breakpoints, the elevated CIP MIC of 0.25 mg/l presumably rendered this isolate clinically resistant and lead to therapeutic failure in this case. Further increase of MIC to fluoroquinolones in vivo and in vitro was distinct. Kpn#1-SEL, selected in vitro, acquired a GyrA target mutation, whereas in Kpn#2 no known resistance mechanism could be detected.     

Ofloxacin resistance mechanism in PA150 and PA300-clinical isolates ofPseudomonas aeruginosa in Korea

Five hundred and seventy clinical strains ofPseudomonas aeruginosa were isolated from August 1993 to August 1994 in Korea and screened for their resistance to ciprofloxacin, norfloxacin, and ofloxacin. Among these, twoP. aeruginosa strains (PA150 and PA300) were selected based on their strong resistance (MICs>50 μg/ml) to all three quinolones. The susceptible strain as well as two resistant strains had proton gradient-dependent efflux system. Efflux system in PA300 showed different specificities to ofloxacin and ciprofloxacin while PA 150 had less permeability for ofloxacin. Ofloxacin had a less inhibitory action on DNA synthesis in permeabilized cells of PA150 and PA300 than 1771M. When quinolone resistance determining region (QRDR) ingyrA was sequenced, PA300 had one missense mutation, Asn 116Tyr, which was newly reported in this work. The results showed that PA150 became ofloxacin resistant by reduced ofloxacin accumulation due to the existence of efflux system and low permeability, while resistance of PA300 was due to the efflux system and a mutation in QRDR ofgyrA-the target site of quinolone.     

Sensitivities of ciprofloxacin-resistant Mycobacterium tuberculosis clinical isolates to fluoroquinolones: role of mutant DNA gyrase subunits in drug resistance

Minimum inhibitory concentrations of sitafloxacin, gatifloxacin, moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin against 59 ciprofloxacin-resistant clinical isolates of Mycobacterium tuberculosis from Japan were determined. The isolates were most susceptible to sitafloxacin and gatifloxacin. To understand better the basis for drug resistance, nucleotide sequences encoding the gyrA and gyrB quinolone resistance-determining region were determined. Predicted amino acid sequences revealed distinct mutational patterns likely to be responsible for fluoroquinolone resistance. Double gyrA mutations as well as mutations in both gyrA and gyrB correlated with increased resistance to all fluoroquinolones.     

Molecular study of quinolone resistance mechanisms and clonal relationship of Salmonella enterica clinical isolates

In the last few years, the number of Salmonella enterica strains resistant to nalidixic acid has steadily increased. In a previous study, the quinolone susceptibility phenotype and genotype of 38 S. enterica clinical isolates (19 S. enterica serovar Typhimurium and 19 S. enterica serovar Enteritidis) were determined. Forty-two percent of the isolates showed nalidixic acid resistance associated with a mutation in gyrA together with putative overexpression of efflux pump(s). In this study, mutations in the quinolone resistance-determining region (QRDR) of parE and the regulators of AcrAB (acrR, marRAB, soxRS and ramR) were analysed. Intracellular accumulation of ciprofloxacin and nalidixic acid was determined. Gene expression of the efflux pump components acrB, tolC, acrF and emrB was also assessed. In addition, an epidemiological study of the isolates by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) was performed. No mutations were detected in parE, whereas two amino acid substitutions were found in two susceptible strains in MarR (I84L) and AcrR (N214T) in one strain each, although both were suggested to be polymorphisms. No changes in the gene expression of acrB, tolC, acrF and emrB were detected between nalidixic-acid-resistant and -susceptible strains. Intracellular accumulation was not useful to reveal differences. Epidemiological analysis showed an important clonal relatedness among the S. Enteritidis isolates, whereas major divergence was seen for S. Typhimurium. Altogether, these results suggest the presence of previously undiscovered drug efflux pump(s) and confirm the high clonality of S. Enteritidis and the genetic divergence of S. Typhimurium.     

铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系研究

目的:研究铜绿假单胞菌临床分离株gyrA基因突变与喹诺酮类药物耐药关系,并对聚合酶联反应(PCR)-限制性片段长度多态性(RFLP)-DNA单链构象多态性(SSCP)分析铜绿假单胞菌临床分离株gyrA基因突变的可行性进行评估。方法:以铜绿假单胞菌临床分离株gyrA基因序列为靶序列,用PCR、PCR-RFLP、PCR-SSCP、DNA测序等方法对铜绿假单胞菌ATCC27853及16株临床分离株gyrA基因突变进行对比研究。结果:在8株耐环丙沙星铜绿假单胞菌中,有6株gyrA基因的83位表现出单点突变,其突变方式全为ACC→ATC,导致氨基酸苏氨酸→异亮氨酸的改变;gyrA基因的PCR扩增产物SacⅡ酶切片段与测序结果一致;SSCP分析结果显示,16株细菌中仅2株gyrA带型与ATCC27853相同,其它菌株gyrA带型与ATCC27853均不同。结论:临床分离的铜绿假单胞菌对喹诺酮类药物耐药的分子机制主要表现为gyrA基因83位氨基酸密码子突变,应用PCR-RFLP-SSCP系统可快速、准确地检测耐喹诺酮类药物的铜绿假单胞菌gyrA中碱基的变异。     

TARGETed surveillance: susceptibility of Streptococcus pneumoniae isolated from community-acquired respiratory tract infections in 2003 to fluoroquinolones and other agents

We assessed antibiotic resistance in Streptococcus pneumoniae collected worldwide in 2003. Resistance to clarithromycin was the highest overall (34.1%) followed by penicillin G (22.1%). Patient age and/or country of origin had the greatest effect on susceptibility. Resistance was highest in children<6 years of age and in patients from South Africa or France. Resistance to penicillin or amoxicillin/clavulanic acid decreased in adults and was low in Germany. Fluoroquinolone resistance was very low overall, but 3.0% levofloxacin resistance (2.6% gatifloxacin and 0.4% moxifloxacin) was observed in Italy. Interestingly, many isolates with minimum inhibitory concentrations (MICs) at the top of the fluoroquinolone susceptibility breakpoints possessed single quinolone resistance-determining region (QRDR) mutations. Care should be taken when treating fluoroquinolone-susceptible isolates with a higher MIC, which are likely to harbour QRDR mutations and may become fully resistant and cause treatment failure. We concur with the conclusions of other recent studies that suggest fluoroquinolone breakpoints should be lowered to ensure these isolates are categorised as resistant. Fluoroquinolones would still remain an important alternative treatment for respiratory tract infections (albeit for adults only), with moxifloxacin being the most potent fluoroquinolone tested in this study.