一株牛源都柏林沙门氏菌的全基因组测序及毒力与耐药性分析

【背景】沙门氏菌(Salmonella spp.)是重要的人畜共患病原菌,其毒力和耐药性的不断增强引起广泛关注。【目的】了解从通辽市一犊牛死亡病例中所分离牛源都柏林沙门氏菌的毒力及耐药性情况。【方法】以病死犊牛肺脏为材料,经细菌分离纯化及16S rRNA基因测序,鉴定病原为沙门氏菌。采用动物试验、药敏试验和PCR方法对分离菌进行毒力、耐药性,以及毒力基因和耐药基因检测,并对其进行全基因组测序分析。【结果】分离菌具有较强毒力,对小鼠半数致死量为2.8×10⁶ CFU/mL。分离菌为多重耐药菌,仅对多粘菌素B和噻孢霉素敏感,对强力霉素和恩诺沙星中度敏感。检测13种沙门氏菌常见毒力基因,检出率为92.3%。对分离菌进行全基因组测序分析,该菌株为都柏林沙门氏菌,基因组大小为4 965 370 bp,GC含量为52.12%,同时携带2个质粒,大小分别为79 524 bp (pTLS-1)和45 301 bp (pTLS-2)。分离菌中共携带996个毒力基因和24个毒力岛;共携带42个耐药基因,其中4个为可水平转移基因,基因组中存在9个可移动遗传元件,包括插入序列和转座子等。【结论】分离牛源都柏林沙门氏菌菌株具有较强毒力且为多重耐药株,携带大量毒力基因及耐药基因。     

PCR-RFLP检测猪源致病性沙门氏菌gyrA基因点突变

四川农业大学谭炳乾、王红宁和叶如俊等先生利用PCR-RFLP法检测猪源致病性沙门氏菌gyrA基因点的突变。他们测定了16株猪源致病性沙门氏菌对5种常用氟喹诺酮类药物的最低抑菌浓度（MIC）。结果表明对此5种药物的耐药率在31．2％～56．2％。分别以质粒和染色体为模板,应用PCR扩增16株沙门氏菌的gyrA基因,以后所有菌株均获得以染色体为模板的扩增产物,还从1株鼠伤寒沙门氏菌获得了以质粒为模板的扩增产物。     

嗜水气单胞菌对喹诺酮类药物耐药的分子机制

摘要:【目的】调查从浙、苏、皖等地水产动物中分离的23 株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011 版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

嗜水气单胞菌对喹诺酮类药物耐药的分子机制

【目的】调查从浙、苏、皖等地水产动物中分离的23株致病性嗜水气单胞菌的耐药谱并探索喹诺酮类抗生素耐药菌株的耐药分子机制。【方法】根据美国临床实验室标准化协会药敏判断标准(2011版)测定23株嗜水气单胞菌耐药谱并筛选喹诺酮类抗生素耐药株;对筛选的耐药菌株和体外诱导耐药菌株的gyrA和parC基因耐药决定区进行分析;用二倍稀释法测定加入多重耐药外排泵抑制剂碳酰氰基-对-氯苯腙前后耐药菌株对恩诺沙星最小抑菌浓度的变化,同时检测喹诺酮类药物相关的外排泵基因qepA、oqxA和mdfA;并检测质粒介导的喹诺酮耐药的qnr家族基因qnrA、qnrB、qnrC、qnrD和qnrS。【结果】所有菌株都存在对5种以上药物的耐药性;39.1%(9/23)的嗜水气单胞菌对喹诺酮类药物耐药,其中55.6%(5/9)对恩诺沙星耐药。耐恩诺沙星的5株菌株均携带qnrS,而不携带qnrA、qnrB、qnrC、qnrD基因,也不携带外排泵基因qepA、oqxA和mdfA。其中耐药菌株AH19同时存在gyrA与parC基因双突变、质粒介导的耐药基因qnrS和主动外排泵三种耐药机制,菌株AH4、AH7和AH20存在gyrA与parC基因双突变和质粒介导的耐药基因qnrS两种耐药机制,AH6存在gyrA基因突变和质粒介导的耐药基因qnrS机制,体外诱导耐药菌株ATCC7966-QR相对于原始菌株ATCC7966发生了gyrA与parC基因双突变。【结论】喹诺酮类药物作用靶位的改变和质粒介导的耐药基因qnrS的存在是本研究中涉及的嗜水气单胞菌对喹诺酮类药物耐药的主要作用机制,主动外排泵机制是个别菌株存在的耐药机制。     

沙门氏菌1,4,[5],12:i:-耐药性和遗传特征研究进展

近10年来,一种与鼠伤寒沙门氏菌具有相似抗原的非典型沙门氏菌血清型1,4,[5],12:i:-在全球多个国家检出率大幅度增加,目前已被列为引起人类沙门氏菌病的主要血清型之一。沙门氏菌1,4,[5],12:i:-菌株具多重耐药特性,存在分别以质粒和染色体介导的两种主要的多重耐药菌株(西班牙株系和欧洲株系)。多个抗生素抗性基因(blaTEM、blaCTX-M-1、aac(3)-IV、aadA2、cmlA1、sul1、sul2、dfrA12、strA-strB、tet(A)和tet(B))在1,4,[5],12:i:-耐药菌株中检出。遗传特征分析显示,沙门氏菌1,4,[5],12:i:-与鼠伤寒沙门氏菌具有很高的遗传相似度,并且推测是由于多个独立的遗传事件的改变,导致鼠伤寒沙门氏菌变异而产生不同的1,4,[5],12:i:-株系。对于沙门氏菌1,4,[5],12:i:-,相关快速、准确检测方法的建立及致病、耐药机制研究将是今后的重要方向。     

PhoQ基因重组鼠伤寒沙门氏菌株的构建及毒力研究

应用PCR技术从鼠伤寒沙门氏菌基因组DNA中克隆phoQ基因片段,构建原核表达pUC18重组质粒,测定序列(GenBank登录号为DQ787014),并转入鼠伤寒沙门氏菌,经异丙基硫代半乳糖苷(IPTG)诱导,进行高效表达。对重组菌株、野生菌株进行毒力检测对比实验,通过口腔注入45日龄健康无菌KM小鼠,测定其半数致死量(LD50)。结果发现：重组菌株与野生菌株的毒力存在显著差异,其半致死量分别为3.981×107 cf u/ mL and 5.012×102 cf u/ mL,PhoQ基因重组菌株的毒力远远低于非重组菌株。说明phoQ基因是调节鼠伤寒沙门氏菌致病机制中一个重要的调节因子。     

重庆市北碚区宠物源沙门氏菌耐药性监测及ESBL和PMQR基因检测

【背景】沙门氏菌是一种常见的人兽共患病病原菌。随着饲养宠物的人越来越多,宠物肠道内沙门氏菌对公共卫生的威胁逐渐显现,但鲜见宠物携带沙门氏菌的流行病学及宠物源沙门氏菌耐药性的报道。【目的】了解重庆市北碚区宠物源沙门氏菌的流行情况,以及其对常用抗菌药物的敏感性及超广谱β-内酰胺酶(Extended-Spectrumβ-Lactamases,ESBL)基因和质粒介导的喹诺酮耐药(Plasmid-Mediated Quinolone Resistance,PMQR)基因的携带情况。【方法】共采集北碚区宠物粪便样品1 038份,通过样品前增菌、选择性增菌、选择性平板筛选和PCR鉴定沙门氏菌特异性invA基因分离鉴定沙门氏菌;接着测定了分离菌株对28种抗菌药物的敏感性,检测了10种ESBL基因和10种PMQR基因。【结果】共分离到沙门氏菌41株,分离率为3.95%。这些菌株对磺胺异噁唑、氨苄西林、四环素、多西环素的耐药率都超过50%,而且82.92%为多重耐药菌株,对阿米卡星、氧氟沙星、依诺沙星、加替沙星完全敏感;85.37%的分离菌携带ESBL基因,以blaTEM基因最为流行;46.34%携带PMQR基因,以qnrS最为流行;48.57%的ESBL阳性菌株携带至少一种PMQR基因。【结论】北碚区宠物体内的沙门氏菌对一些抗菌药物存在不同程度的耐药性,而且耐药性可能是通过耐药质粒介导的。     

鼠伤寒沙门氏菌对三甲氧苄二氨嘧啶耐药机制的研究

对临床分离的鼠伤寒沙门氏菌进行了三甲氧苄二氨嘧啶(TMP)耐药机制的研究。结果表明,50株鼠伤寒沙门氏菌对TMP的耐药率为76％,其中7株菌对TMP高度耐药。7株耐药菌中有4株含有不同的质粒,TMP耐药质粒可以在种内和种间转移,而且8％的。SDS可以有效地消除R质粒。比较不同TMP耐药菌和对照菌的二氢叶酸还原酶(DHFR)活力和特性得出,鼠伤寒沙门氏菌TMP耐药机制为：无质粒菌的TMP耐药是由于染色体编码的DHFR过量产生所致,TMP对该酶的产生起诱导作用含质粒菌是由于R质粒编码产生了Ia型抗TMP的DHFR和R质粒编码产生了另一种新型的抗TMP的DHFR,后者目前尚无报道。此研究结果为临床合理用药及控制致病菌的耐药性发展和传播提供了理论依据。     

肺炎克雷伯杆菌对氟喹诺酮耐药的机制研究

目的研究肺炎克雷伯杆菌对氟喹诺酮类药物（FQNs）的耐药机制。方法筛选临床分离的对环丙沙星耐药的肺炎克雷伯杆菌共10株,采用微量肉汤稀释法检测菌株对5种氟喹诺酮类药物的MIC值;采用PCR方法检测菌株染色体和质粒携带的喹诺酮耐药基因（gyrA基因、parC基因和qnr基因）并测序;质粒接合试验验证qnr基因的转移性。结果 10株肺炎克雷伯杆菌对5种氟喹诺酮类药物均产生耐药性。扩增产物经测序发现10株肺炎克雷伯杆菌染色体的gyrA基因和parC基因均有突变;有2株菌株（K79和K107）携带qnrA基因,这2株菌的接合菌对喹诺酮抗菌药的MIC值上升了5～30倍;未检测到qnrB阳性的菌株。结论 gyrA和parC基因突变是肺炎克雷菌对氟喹诺酮类产生耐药机制的主要原因,质粒上qnrA基因的存在,也是产生喹诺酮耐药的一个重要因素。     

鸡蛋生产链中沙门氏菌对抗生素及消毒剂的耐药性研究

为研究鸡蛋生产链中沙门氏菌的污染情况及抗生素、消毒剂耐药情况,本文鉴定了鸡蛋生产链中分离得到的111株沙门氏菌(Salmonella)血清型,并测定了抗生素和消毒剂对沙门氏菌的最小抑菌浓度(Minimum inhibitory concentrations, MICs),检测了其对抗生素和消毒剂的耐药基因的表达情况。研究结果表明,沙门氏菌对甲氧苄啶(Trimethoprim, TMP)耐药率最高(N=100,P=90.09%),对阿莫西林/克拉维酸(Amoxicillin and clavulanate, AMC)、头孢噻呋钠(Sodium ceftiofur, CFS)、庆大霉素(Gentamicin, CN)敏感。沙门氏菌共产生6种不同的耐药谱型,TMP是最主要的耐药谱型(N=36,P=32.43%),52.25%的菌株(N=58)具有多重耐药性。苯扎氯铵(Benzalkonium chloride, BC)与氯化十六烷基吡啶(Cetylpyridinium chloride, CPC)对沙门氏菌的MIC的范围分别为：8~128 μg/mL、8~256 μg/mL。相对于质控菌株Escherichia coli ATCC10536,101株沙门氏菌对BC和CPC同时具有较高的耐药性(P=90.99%),109株沙门氏菌对抗生素和消毒剂具有共同耐药性(P=98.20%)。抗生素耐药基因检出率最高为blaTEM(N=49, P=44.14%),未检测出qnrA、qnrB、qepA基因,仅检测出qacEΔ1消毒剂耐药基因(N=63, P=56.76%)。抗生素耐药基因sul1和消毒剂耐药基因qacEΔ1具有显著相关性(P<0.01)。S. Derby对TMP、土霉素(Oxytetracycline, OTC)、阿莫西林(Amoxicillin, AML)、环丙沙星(Ciprofloxacin, CIP)同时表现较高的耐药性,S. Derby检出了11种抗生素耐药基因,消毒剂耐药基因qacEΔ1的检出率为81.25%(N=52)。鸡场中养殖内环境沙门氏菌对抗生素和消毒剂的耐药率以及耐药基因检出率均高于养殖外环境,鸡蛋包装、储存及销售等环节中沙门氏菌耐药率及耐药基因检出率均较高。由此可见,鸡蛋生产链中沙门氏菌对抗生素、消毒剂耐药性较严重,且存在共同耐药的现象。因此,需要进一步规范防控鸡场中沙门氏菌,规范抗生素和消毒剂的使用以及加强鸡蛋生产链条中卫生安全的监管。     

Rapid detection of gyrA and parC mutations in quinolone-resistant Salmonella enterica using Pyrosequencing technology

Fluoroquinolones are broad-spectrum antimicrobials highly effective in the treatment of a wide variety of clinical infections. Salmonella gastroenteritis is usually only treated with fluoroquinolones when the patient is elderly or immunocompromised. Fluoroquinolones are also used for the treatment of systemic Salmonella infection or for long-term salmonella carriage. Resistance to quinolones is commonly mediated by point mutations within the topoisomerase genes gyrA and parC. Pyrosequencing technology is a DNA sequencing method using 'sequencing by synthesis' and is suitable for the rapid detection of single nucleotide polymorphisms (SNPs). One hundred and ten Salmonella enterica isolates, representing 18 different serotypes, were used in this study. One hundred and four isolates had ciprofloxacin MICs of 0.25-32 microg/mL; the remaining six were ciprofloxacin-sensitive (ciprofloxacin MIC相似文献   

Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography

The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae.     

Fluoroquinolone resistance and gyrA and parC mutations of Escherichia coli isolated from chicken

Escherichia coli is a common inhabitant of the intestinal tracts of animals and humans. The intestines of animals also represent an ideal environment for the selection and transfer of antimicrobial resistance genes. The aim of this study was to investigate the resistance of E. coli isolated from chicken fecal samples to fluoroquinolones and to analyze the characterization of mutations in its gyrA and parC gene related resistance. One hundred and twenty-eight E. coil isolates showed a high resistance to ciprofloxacin (CIP; 60.2%), enrofloxacin (ENO; 73.4%) and norfloxacin (NOR; 60.2%). Missense mutation in gyrA was only found in the amino acid codons of Ser-83 or Asp-87. A high percentage of isolates (60.2%) showed mutations at both amino acid codons. Missense mutation in parC was found in the amino acid codon of Ser-80 or Glu-84, and seven isolates showed mutations at both amino acid codons. Isolates with a single mutation in gyrA showed minimal inhibitory concentrations (MIC) for CIP (相似文献   

Molecular cloning of the DNA gyrase genes from Methylovorus sp. strain SS1 and the mechanism of intrinsic quinolone resistance in methylotrophic bacteria

The genes encoding the DNA gyrase A (GyrA) and B subunits (GyrB) of Methylovorus sp. strain SS1 were cloned and sequenced. gyrA and gyrB coded for proteins of 846 and 799 amino acids with calculated molecular weights of 94,328 and 88,714, respectively, and complemented Escherichia coli gyrA and gyrB temperature sensitive (ts) mutants. To analyze the role of type II topoisomerases in the intrinsic quinolone resistance of methylotrophic bacteria, the sequences of the quinolone resistance-determining regions (QRDRs) in the A subunit of DNA gyrase and the C subunit (ParC) of topoisomerase IV (Topo IV) of Methylovorus sp. strain SS1, Methylobacterium extorquens AM1 NCIB 9133, Methylobacillus sp, strain SK1 DSM 8269, and Methylophilus methylotrophus NCIB 10515 were determined. The deduced amino acid sequences of the QRDRs of the ParCs in the four methylotrophic bacteria were identical to that of E. coli ParC. The sequences of the QRDR in GyrA were also identical to those in E. coli GyrA except for the amino acids at positions 83, 87, or 95. The Ser83 to Thr substitution in Methylovorus sp. strain SS1, and the Ser83 to Leu and Asp87 to Asn substitutions in the three other methylotrophs, agreed well with the minimal inhibitory concentrations of quinolones in the four bacteria, suggesting that these residues play a role in the intrinsic susceptibility of methylotrophic bacteria to quinolones.     

Mutations in the gyrB, parC, and parE genes of quinolone-resistant isolates and mutants of Edwardsiella tarda

The full length genes gyrB (2,415 bp), parC (2,277 bp), and parE (1,896 bp) in Edwardsiella tarda were cloned by PCR with degenerate primers based on the sequence of the respective quinolone resistance-determining region (QRDR), followed by elongation of 5' and 3' ends using cassette ligation-mediated PCR (CLMP). Analysis of the cloned genes revealed open reading frames (ORFs) encoding proteins of 804 (GyrB), 758 (ParC), and 631 (ParE) amino acids with conserved gyrase/topoisomerase features and motifs important for enzymatic function. The ORFs were preceded by putative promoters, ribosome binding sites, and inverted repeats with the potential to form cruciform structures for binding of DNA-binding proteins. When comparing the deduced amino acid sequences of E. tarda GyrB, ParC, and ParE with those of the corresponding proteins in other bacteria, they were found to be most closely related to Escherichia coli GyrB (87.6% identity), Klebsiella pneumoniae ParC (78.8% identity) and Salmonella typhimurium ParE (89.5% identity), respectively. The two topoisomerase genes, parC and parE, were found to be contiguous on the E. tarda chromosome. All 18 quinoloneresistant isolates obtained from Korea thus far did not contain subunit alternations apart from a substitution in GyrA (Ser83→Arg). However, an alteration in the QRDR of ParC (Ser84→Ile) following an amino acid substitution in GyrA (Asp87→Gly) was detected in E. tarda mutants selected in vitro at 8 microng/ml ciprofloxacin (CIP). A mutant with a GyrB (Ser464→Leu) and GyrA (Asp87→Gly) substitution did not show a significant increase in the minimum inhibitory concentration (MIC) of CIP. None of the in vitro mutants exhibited mutations in parE. Thus, gyrA and parC should be considered to be the primary and secondary targets, respectively, of quinolones in E. tarda.     

Role of mutations in parC and gyrA in forming resistance of Mycoplasma hominis to fluoroquinolones

The set of the laboratory strain M. hominis H-34 mutants resistant to fluoroquinolones (ciprofloxacin-Cfl, lomefloxacin-Lfl, ofloxacin-Ofl) was obtained by selection in broth medium. The mutation was found in the quinolone resistance-determining region (QRDR) of A subunit of topoisomerase IV gene (parC) and new mutations were found in QRDR of genes encoding the A subunit of DNA gyrase (gyrA) in M. hominis mutants resistant to various concentrations of the Cfl, Lfl and Ofl. After multistep selection of the obtained mutants at constant concentrations of Cfl additional mutation Ser83 to Trp was revealed. No mutations in parE and gyrB were found. Mutations in parC for laboratory strain M. hominis H34 appeared at lower antibiotic concentrations than in gyrA. All mutations in gyr A were associated with mutations in parC. This confirms the previous data that topoisomerase IV is the primary target of Cfl and Ofl and suggests that it is the primary target of Lfl. Some M. hominis mutants selected at Ofl without any substitution in QRDRs were shown to be insensitive to Cfl and of Lfl. Studies of cross-resistance of the selected M. hominis mutants showed that their resistance to various fluoroquinolone concentrations could not depend on any mutations in QRDR of topoisomerase IV and DNA gyrase genes and suggests involvement of other unknown molecular mechanisms specific for Mycoplasmas.     

铜绿假单胞菌parC基因突变与耐氟喹诺酮的关系

研究了成都地区临床分离的铜绿假单胞菌拓扑异构酶ⅣparC基因突变与耐氟喹诺酮类药物的关系。测定临床分离的55株铜绿假单胞菌的MIC值,从中筛选出1株敏感菌和8株耐药菌,以标准敏感菌株ATCC27853作为质控菌株。用PCR反应扩增parC基因的喹诺酮耐药决定区(QRDR),扩增产物片段长度为396bp,同时对上述10株菌的PCR产物进行测序分析。临床分离敏感菌和标准菌株ATCC27853的parC基因序列与国外报道的序列相同,而R25,R42,R43,R44等4株耐药菌株在87位(TCGCG→TTG)均有突变,该单位点突变引起氨基酸由Ser→Leu的改变,此外,新发现在所有耐药菌株115位有一静止突变(GCT→GCG),该突变未引起氨基酸的改变。拓扑异构酶ⅣparC基因突变是铜绿假单胞菌对氟喹诺酮类药物产生耐药性的机制之一,以87位的突变最为常见。     

氟喹诺酮类药物体外诱导肺炎克雷伯菌耐药后GyrA和ParC的变异

目的了解3种氟喹诺酮类（FQS）体外诱导肺炎克雷伯菌（Klebsiella pneumoniae,Kpn）耐药性的差异,研究肺炎克雷伯菌DNA旋转酶A亚单位（GyrA）和拓扑异构酶ⅣC亚基（ParC）的变异与其耐喹诺酮类药物的关系。方法采用环丙沙星（CW）、左氧氟沙星（LEX）和加替沙星（GAT）对从临床分离8株Kpn进行体外分步诱导,采用琼脂平板二倍稀释法测定CIP、LVF及GAT对Kpn诱导前、后的最低抑菌浓度（MIC）,并对诱导成功的17株Kpn的GyrA的基因（gyrA）和ParC的基因（parC）进行PCR扩增,选取其中8株KpnDNA测序并进行序列分析比较。结果8株耐FQS菌株都存在GyrA变异,同FQS耐药性相关的变异有Ser83（TCC）→Phe（TTC）、Ile（ATC）和Tyr（TAC）,Asp87（GAC）→Ala（GCC）、ma（GCC）和Glu（GAA）,5株Kpn同时存在ParC的变异：丝氨酸Ser80（AGC）→Ile（ATC）。结论本研究体外实验证实了Kpn可在长期低剂量的接触抗菌药物后形成耐药菌株。在高度耐FQS的Kpn中同时存在GyrA和ParC变异。     

Molecular cloning of the gyrA gene and characterization of its mutation in clinical isolates of quinolone-resistant Edwardsiella tarda

Knowing the entire sequence of the gene encoding the DNA gyrase Subunit A (gyrA) of Edwardsiella tarda could be very useful for confirming the role of gyrA in quinolone resistance. Degenerate primers for the amplification of gyrA were designed from consensus nucleotide sequences of gyrA from 9 different Gram-negative bacteria, including Escherichia coli. With these primers, DNA segments of the predicted size were amplified from the genomic DNA of E. tarda and then the flanking sequences were determined by cassette ligation-mediated polymerase chain reaction. The nucleotide sequence of gyrA was highly homologous to those of other bacterial species, in both the whole open-reading frame and the quinolone-resistance-determining region (QRDR). The 2637-bp gyrA gene encodes a protein of 878 amino acids, preceded by a putative promoter, ribosome binding site and inverted repeated sequences for cruciform structures of DNA. However, the nucleotide sequence of the flanking region did not show any homologies with those of other bacterial DNA gyrase Subunit B genes (gyrB) and suggested the gyrase genes, gyrA and gyrB, are non-continuous on the chromosome of E. tarda. All of the 12 quinolone-resistant isolates examined have an alteration within the QRDR, Ser83 --> Arg, suggesting that, in E. tarda, resistance to quinolones is primarily related to alterations in gyrA. Transformation with the full sequence of E. tarda gyrA bearing the Ser83 --> Arg mutation was able to complement the sequence of the gyrA temperature-sensitive mutation in the E. coli KNK453 strain and to induce increased resistance to quinolone antibiotics at 42 degrees C.