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1.
本研究旨在初步了解重庆市羊源沙门氏菌的耐药性及耐药基因流行情况。在5个山羊养殖场采集185份山羊粪便样品，经选择性增菌和PCR鉴定分离沙门氏菌，确定了其血清型，测定了分离菌对28种抗菌药物的敏感性，并检测了喹诺酮耐药决定区（QRDR）的耐药突变位点和质粒介导的喹诺酮耐药（PMQR）、超广谱β-内酰胺酶（ESBL）基因。共分离到羊源沙门氏菌11株，其中10株为德尔卑沙门氏菌。分离的菌株对氨苄西林、头孢唑啉、头孢氨苄、头孢噻肟、四环素和培氟沙星耐药严重；9株菌表现为多重耐药，对3~7类药物耐药。所有菌株均存在QRDR耐药突变且携带blaTEM基因；7株菌携带1~5种PMQR基因。本研究分离的羊源沙门氏菌对常用抗菌药物整体耐药较为严重，且广泛存在耐药突变和携带耐药基因。 相似文献
2.
《中国畜牧兽医》2020,(7)
本研究旨在初步了解重庆市羊源沙门氏菌的耐药性及耐药基因流行情况。在5个山羊养殖场采集185份山羊粪便样品,经选择性增菌和PCR鉴定分离沙门氏菌,确定了其血清型,测定了分离菌对28种抗菌药物的敏感性,并检测了喹诺酮耐药决定区(QRDR)的耐药突变位点和质粒介导的喹诺酮耐药(PMQR)、超广谱β-内酰胺酶(ESBL)基因。共分离到羊源沙门氏菌11株,其中10株为德尔卑沙门氏菌。分离的菌株对氨苄西林、头孢唑啉、头孢氨苄、头孢噻肟、四环素和培氟沙星耐药严重;9株菌表现为多重耐药,对3～7类药物耐药。所有菌株均存在QRDR耐药突变且携带bla_(TEM)基因;7株菌携带1～5种PMQR基因。本研究分离的羊源沙门氏菌对常用抗菌药物整体耐药较为严重,且广泛存在耐药突变和携带耐药基因。 相似文献
3.
本研究旨在探讨不同血清型沙门氏菌在环丙沙星抗生素压力下突变频率及在耐药发展过程中靶位基因突变、外排泵及调控基因表达的差异。选取临床分离的印第安纳型、肠炎型和鼠伤寒型沙门氏菌的敏感菌株,在环丙沙星压力下诱导耐药突变,分别获得一系列不同程度的耐药突变株。分别检测不同血清型沙门氏菌突变株的突变频率、靶位基因喹诺酮耐药决定区(QRDRs)和外排泵调控基因ramR-ramA突变及外排泵相关基因的表达水平;同时检测了母株在羰基氰化物间氯苯腙(CCCP)存在情况下环丙沙星药物的蓄积浓度,以确定母株是否存在外排泵的作用。结果表明,在环丙沙星压力下,印第安纳型沙门氏菌较肠炎型和鼠伤寒型有更高的突变频率,易获得耐药株;印第安纳血清型菌株耐药性的获得主要是由于靶位基因gyrA发生单突变,协同外排泵外排作用增强而获得高水平耐药;肠炎型沙门氏菌耐药性获得主要是由于靶位基因gyrA发生83和87位双位点突变,并随着gyrB和parC基因的多位点同时突变而获得高水平耐药,耐药性的发展过程中没有外排泵作用参与;而鼠伤寒沙门氏菌在抗生素压力下不易发展成耐药菌,耐药性发生主要是由于靶位基因gyrB发生突变,而伴随parC基因突变及微弱的外排泵作用导致耐药水平增加。 相似文献
4.
将氯霉素（Cm）抗性基因克隆到pUTmini-Tn5Km2载体中,利用含卡那霉素（Km）和氯霉素（Cm）抗性基因的pUTmini-Tn5Km2（Cm）转座载体将Km和Cm抗性基因随机插入鸡白痢沙门氏菌基因组中,以相应的抗生素进行筛选,获得大量在不同位点插入突变的突变体,从中筛选鸡白痢沙门氏菌某功能缺陷型突变株。通过对突变株的基本特征以及PCR鉴定后,再进行插入基因定位。结果表明,Km和Cm抗性基因已成功转座至鸡白痢沙门氏菌基因组上;基因定位显示突变株均有且只有1个插入位点,插入位点的位置不尽相同。这为研究鸡白痢沙门氏菌功能基因和筛选特定突变株提供了必要的基础。 相似文献
5.
为了解牛结核分枝杆菌临床分离菌株对利福平(RFP)、异烟肼(INH)的分子耐药机制,本研究采用噬茵体生物扩增法对分离并鉴定的25株牛结核分枝杆茵进行药敏分析.筛选出9株耐利福平、异烟肼菌株.PCR扩增耐药株rpoB基因片段(213bp)和KatG基因片段(458bp)并测序.通过对耐药基因的测序分析,5株利福平耐药株rpoB基因在53l位(RNA聚合酶β-亚基编码基因起始密码子起,下同)、526位点、511位点发生突变,其中有3株rpoB基因531位点发生突变,1株rpoB基因526位点突变;1株rpoB基因在531位点和511位点发生了较为少见的联合突变;4株异烟肼耐药菌株,其中2株在315位点(KatG蛋白编码基因起始密码子起,下同)发生碱基突变,2株在463位点发生碱基突变. 相似文献
6.
错配PCR方法快速检测耐FQs沙门氏菌基因突变的初步研究 总被引：2，自引：0，他引：2
根据耐药沙门氏菌基因突变位点碱基变化情况,设计合适的引物进行错配PCR,鉴别突变位点,使其结果与测序结果相吻合,以建立错配PCR方法快速检测耐氟喹诺酮类药物沙门氏菌基因突变,判断菌株是否耐药.错配PCR检测方法条件优化成熟之后,对经过测序的菌株进行耐药突变位点的快速检测,检测结果与测序结果符合率高达96%. 相似文献
7.
《中国预防兽医学报》2018,(11)
为研究副猪嗜血杆菌(HPS)链霉素(SM)抗性的分子基础,本实验采用甲基磺酸乙酯(EMS)间断传代诱导HPS SC1401菌株,构建SM抗性突变株,测定其对SM的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),并对其耐药基因rpsL和rrs进行测序;通过对比分析筛选出rpsL基因中两个引起SM完全耐药的突变位点;为了进一步验证突变位点与抗性的关联性,设计位点突变引物以构建突变型质粒,并分别转化到亲本株中,获得单一位点突变菌株,测定其对SM的MIC和MBC。结果显示:EMS诱导的SM抗性突变菌株rpsL基因第43号(AAA→AGA)或第88号密码子(AAA→AGA)发生了突变,rrs基因未检测到突变;该抗性突变菌株与单一位点突变菌株1401D43和1401D88对SM的MIC和MBC均大于8 192μg/mL,远高于亲本株SC1401的SM抗性水平。实验结果表明,HPS的SM抗性主要由rpsL基因特定位点突变引起,其中,rpsL基因第43号和第88号密码子为主要突变位点。本实验为完善HPS耐药分子机制研究,特别是对氨基糖苷类药物产生耐药的分子基础提供一定的参考。 相似文献
8.
9.
嗜水气单胞菌gyrA氟喹诺酮抗性决定区的克隆与分析 总被引：1，自引：0，他引：1
研究了从病鱼体内分离的嗜水气单胞菌对氟喹诺酮类药物耐药性的分子机理。以4株对诺氟沙星等氟喹诺酮类药物耐药菌株及2株敏感菌株为模板，参照杀鲑气单胞菌gyrA基因序列，设计了1对引物，进行gyrA基因氟喹诺酮抗性决定区PCR扩增，将扩增产物克隆入pMD18-T载体，转化入大肠埃希氏菌DH5α中，小提质粒，酶切鉴定，测序并分析比较耐药菌和敏感菌的氨基酸残基序列。发现耐药菌有5个氨基酸突变位点，分别是83位点的Ser→Ile,92位点的Leu→Met,174位点的Ile→Phe,202位点的Asn→Asp,203位点的Leu→Arg，耐药菌突变后的氨基酸残基均比敏感菌正常的相对应氨基酸残基分子量大。83位点的突变与大肠埃希氏菌的耐药性突变一致。122位点具有保守的Try。 相似文献
10.
《中国预防兽医学报》2020,(9)
为探究双组分系统arcB基因对鼠伤寒沙门氏菌耐药性的影响,本研究利用Red同源重组系统构建arcB基因缺失株和arcB基因回复株以及通过无缝克隆和重组技术构建3株arcB基因关键位点点突变株。采用微量稀释法测定亲本株、arcB缺失株、回复株及点突变株药物敏感性和体外生长速率,初步分析双组分系统arcB基因对鼠伤寒沙门氏菌耐药性的影响。结果显示,经PCR测序鉴定结果表明构建了鼠伤寒沙门氏菌CVCC541ΔarcB缺失株及其回复株、arcB基因点突变株(arcB~(His-292-Ala)、arcB~(Asp-576-Ala)、arcB~(His-717-Ala))。生长曲线测定结果显示:缺失株、点突变株和亲本株相比生长速率降低,回复株生长速率回复,与亲本株生长曲线一致;MIC测定结果显示:与亲本株相比,arcB基因缺失株对β-内酰胺类、大环内酯类、四环素类、氯霉素类抗生素的敏感性无变化,但对氨基糖苷类的链霉素、阿米卡星、庆大霉素的耐药性分别增加16、8、4倍;基于该结果测定的各菌株对氨基糖苷类抗生素的MIC结果显示:与亲本株相比,arcB基因缺失株对链霉素、妥布霉素、庆大霉素的耐药性增加8倍,对阿米卡星、新霉素、奈替米星、卡那霉素的耐药性增加4倍,对大观霉素的敏感性未发生变化;而回复株与亲本株药物敏感性相似;arcB基因关键位点突变株的药物敏感性与arcB基因缺失株一致。以上结果表明,arcB基因参与鼠伤寒沙门氏菌对氨基糖苷类抗生素耐药性的调控。上述实验结果为深入研究双组分系统对鼠伤寒沙门氏菌的耐药机制提供实验依据。 相似文献
11.
旨在了解猪链球菌对氟喹诺酮类药物耐药性与parC、gyrA基因突变的相关性,通过微量稀释法测定34株猪链球菌对4种氟喹诺酮类药物的MIC值,采用PCR方法扩增并测序分析了临床分离的猪链球菌对氟唪诺酮类约物10株耐药株和9株敏感株的parC和gyrA基因喹诺酮耐药决定区(QRDRs).在氟喹诺酮类药物耐药菌株parC基因QRDRs发生Ser79→Phe、Arg 87→Leu的氨基酸突变,在4株高度耐药菌株gyrA基因QRDRs发生Arg66→Ser,Ser81→Arg氨基酸突变;当菌株对氟喹诺酮类药物敏感时,parC和gyrA基因的QRDR区均未有突变;而当MIC≥32 μg·L-1 时,parC的氨基酸发生了 Ser79→Phe的突变,同时发生gyrA氨基酸Arg66→Ser,Set81→Arg突变.结果表明,猪链球菌对氟喹诺酮类药物低水平类耐药是由parC单一位点突变引起,而高水平耐药是由parC和gyrA双位点突变引起. 相似文献
12.
通过药敏实验检测了鸡鲍氏、人鲍氏和人福氏三株志贺菌对12种常用抗生素的耐药情况,发现三者均未产生对喹诺酮类药物的耐药性。用聚合酶链反应(PCR)分别扩增三株志贺菌的DNA旋转酶A亚单位(gyrA)和拓扑异构酶ⅣC亚单位(parC)基因,并对其中的耐喹诺酮类决定区(QRDR)进行了分析。发现鸡和人鲍氏志贺菌QRDR区的序列没有发生任何碱基突变,这与药敏试验结果相吻合。人福氏志贺菌虽然也对喹诺酮类无耐药性,但其gyrA基因存在83位Ser83(TCG)→Leu(TTG)的突变,由于只有83位点一处突变,所以尚未表现出耐药性表征,parC基因中存在58位Ser58(AGC)→Ile(ATC)的突变,该突变点与常见的80、84位氯基酸的改变有所不同,却仍在QRDR区域之中,虽然暂时还无法根据这一结果判断该突变与喹诺酮类耐药性之间的关系,但至少表明该菌株正处在耐药性演变进程之中,其在志贺菌耐药性中的确切作用还有待进一步研究。 相似文献
13.
Sonia Jurado José A Orden Pilar Horcajo Ricardo De La Fuente José A Ruiz-Santa-Quiteria Susana Martínez-Pulgarín Gustavo Domínguez-Bernal 《Journal of veterinary diagnostic investigation》2008,20(3):342-345
Thirty-seven fluoroquinolone-resistant Escherichia coli strains from ruminants (according to Clinical and Laboratory Standards Institute guidelines) were screened by molecular methods for mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes and for the presence of the qnrA gene. One of the strains studied was an enterohemorrhagic E. coli (EHEC) strain potentially pathogenic for humans. Three E. coli strains resistant to enrofloxacin (minimal inhibitory concentration [MIC] = 2 microg/ml) but not to ciprofloxacin (MIC = 1 microg/ml) presented single mutations in the gyrA and parC genes, while 34 strains resistant to both fluoroquinolones presented double and single mutations in gyrA and parC, respectively (31 strains), or double mutations in gyrA and parC (3 strains). The EHEC strain presented a double amino acid substitution in the GyrA protein (Ser-83-->Leu and Asp-87-->Gly) and a double amino acid substitution in the ParC protein (Gly-78-->Cys and Ser-80-->Arg), one of which has not been previously described. The present study shows that most of the mutations in the QRDR of the gyrA and parC genes of fluoroquinolone-resistant E. coli strains from ruminants are the same as those seen in E. coli strains from other animal species and humans and that there are no differences in mutation patterns in the QRDR of E. coli strains from healthy ruminants and those with diarrhea. No strains carried qnrA, which indicates that this gene does not play an important role in the selection of fluoroquinolone-resistant E. coli strains from ruminants. 相似文献
14.
取临床分离的、对5种氟喹诺酮类药物（环丙沙星、氧氟沙星、恩诺沙星、单诺沙星和沙拉沙星）均耐药的9株鸡源性沙门氏菌耐药株,提取其染色体DNA。设计引物gyrAF和gyrAR、gyrBF和gyrBR,分别扩增菌株DNA旋转酶gyrA基因和gyrB基因的氟喹诺酮类耐药决定区（QRDR）,对PCR扩增产物进行测序及序列分析。与质控菌株相比,9株临床分离耐药株中只有菌株38和60的gyrA基因发生单碱基突变,菌株38的gyrA基因第371位碱基发生C→T突变,菌株60的gyrA基因第350位碱基发生A→C突变,两处突变均位于QRDR内,其余菌株的核苷酸未发生任何突变。菌株38的碱基突变导致gyrA基因第99位氨基酸发生R→C取代,即Arg→Cys;菌株60的碱基突变导致gyrA基因第92位氨基酸发生M→L取代,即Met→Leu。9株临床分离鸡源性沙门氏菌氟喹诺酮类耐药株gyrB基因QRDR的核苷酸序列与质控菌株完全相同;只有菌株42的gyrB基因第1592位碱基发生C→A突变,但其位于gyrB基因QRDR之外,且菌株42的gyrB基因的碱基突变并没有导致相应氨基酸的改变。上述结果提示,DNA旋转酶gyrA基因和gyrB基因QRDR突变可能并非沙门氏菌耐药性产生的主要原因。 相似文献
15.
Lukkana M Wongtavatchai J Chuanchuen R 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2012,74(4):435-440
The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants. 相似文献
16.
探讨不同禽源大肠埃希菌中喹诺酮类药物的耐药情况及耐药基因gyrA的分布和突变特征。采用K-B药敏纸片法、gyrA基因的PCR扩增,对9株大肠埃希菌进行喹诺酮类药物试验,并将gyrA基因的PCR产物测序,对测序结果采用DNA MAN、DNA Star、MEGA6等软件分析。药敏试验结果表明,C1、C2、C3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星敏感,D1、D2、D3、B1、B2和B3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星均表现为耐药和中介;gyrA基因的测序结果表明,除B1菌株有1处核苷酸突变位点和B2菌株有14处核苷酸突变位点;B2菌株gyrA基因的氨基酸突变发生在87位Ile→Val替代、101位Leu→Met替代、102位Ala→Ser替代、129位Lys→Gln替代。9株禽源大肠埃希菌的同源性和进化树分析表明,不同禽源耐氟喹诺酮类药物的大肠埃希菌菌株中B2菌株gyrA基因与其他9株菌株相比,同源性在90%左右,进化树不在一个分支上,研究中的B2菌株将为大肠埃希菌的氟喹诺酮类耐药机制的研究提供候选菌株。 相似文献
17.
OBJECTIVE: To investigate the development of enrofloxacin resistance among Escherichia coli isolates obtained from chickens by determining mutant-prevention concentrations (MPCs) and sequence the quinolone resistance-determining regions (QRDRs) of gyrA and parC genes in selected isolates. SAMPLE POPULATION: 15 chicken-derived E coli isolates. PROCEDURES: For all isolates, MPC and minimal inhibition concentration (MIC) of enrofloxacin were determined. The MPCs and maximum serum drug concentrations attained with enrofloxacin doses recommended for treatment of E coli infections in chickens were compared. Mutation frequencies and QRDR sequence changes in gyrA and parC were also determined. RESULTS: In 2 of 15 E coli strains, MPCs were low (0.016 and 0.062 microg/mL), MPC:MIC ratios were 2 and 4, and the GyrA and ParC proteins had no mutations. In 9 susceptible isolates with a GyrA point mutation, MPCs ranged from 2 to 16 microg/mL. For isolates with double mutations in GyrA and a single mutation in ParC, MPCs were > 32 microg/mL (several fold greater than the maximal plasma concentration of enrofloxacin in chickens); mutation frequencies were also much lower, compared with frequencies for single-mutation isolates. CONCLUSIONS AND CLINICAL RELEVANCE: For E coli infections of chickens, MPC appears to be useful for determining enrofloxacin-dosing strategies. The high MPC:MIC ratio may result in enrofloxacin-treatment failure in chickens infected with some wild-type gyrA E coli isolates despite the isolates' enrofloxacin susceptibility (MICs 0.125 to 1 microg/mL). For infections involving isolates with high MPCs, especially those containing mutations in gyrA and parC genes, treatment with combinations of antimicrobials should be adopted. 相似文献
18.
Prevalence of plasmid-mediated quinolone resistance qnr genes in poultry and swine clinical isolates of Escherichia coli 总被引：4，自引：0，他引：4
Yue L Jiang HX Liao XP Liu JH Li SJ Chen XY Chen CX Lü DH Liu YH 《Veterinary microbiology》2008,132(3-4):414-420
The prevalence of qnr genes was investigated in veterinary clinical isolates of Escherichia coli in Guangdong province, China, and the aac (6')-Ib gene and the mutations in QRDRs of gyrase and topoisomerase IV were examined in qnr-positive strains. A total of 232 E. coli strains isolated from pig and poultry were screened for the presence of the qnrA, qnrB and qnrS genes by PCR and sequencing. The aac (6')-Ib gene was detected in qnr-bearing strains by PCR and sequencing. For all strains carrying qnr, MICs for six quinolones were determined. Mutations within the gyrase and topoisomerase were analyzed by PCR and sequencing for all the QRDRs of gyrA, gyrB, parC and parE. Among 232 E. coli isolates, 14 (6%) isolates were positive for the qnr gene, including one for qnrB, 13 for qnrS, but no qnrA was identified in this population. Detection of the aac (6')-Ib gene showed that one qnrS-positive isolate from pig and one qnrB-positive isolate from duck carried aac (6')-Ib gene, and both were the cr variant allele of aac (6')-Ib. All of the 14 isolates had MICs of ciprofloxacin more than 0.25 mg/L. Mutations in the QRDR of gyrA mutations were observed in 5 (35.7%) of the 14 strains. Three fluoroquinolone-resisting strains showed one mutation S83L of gyrA, while one S83I. One high-level resistance strains harboured gyrA S83L and A87N of gyrA. A singe mutation in site 58 of parC was detected in 3 (21.4%) strains. None mutations were found in QRDRs of gyrB and parE. The emergence of qnr genes in veterinary clinical E. coli isolates is described for the first time. This is also the first report of aac (6')-Ib-cr gene in E. coli isolates from food-producing animals. 相似文献
19.
M Cengiz S Sonal E Buyukcangaz A Sen E Arslan B Mat P Sahinturk H Gocmen 《The Veterinary record》2012,171(6):155
The aim of this study were to detect the gyrA, parC and marR mutations and qnr genes (qnrA, qnrB and qnrS) in 120 strains of Escherichia coli isolated from animals. European Committee on Antimicrobial Susceptibility Testing and Clinical Laboratory Standards Institute disc diffusion and minimum inhibitory concentration (MIC) tests, respectively, were used to determine fluoroquinolone (FQ) resistance, and molecular methods were used to detect the mutations and the genes. E coli isolates with an MIC of ≥8 mg/l had mutation at Ser-80 in parC in addition to mutations at Ser-83, Asp-87 or both in gyrA. The nucleotide change was detected in marR (Ser-3?→?Asn, Ala-53?→?Glu, Gly-103?→?Ser, Tyr-137?→?His). Only four E coli isolates (3.3 per cent) contained qnrA and qnrS, and qnrB was not detected. Two E coli isolates from healthy calves also contained qnrA and qnrS. The MICs of enrofloxacin and danofloxacin for qnr-containing E coli isolates ranged from 32 mg/l to 256 mg/l. The results of this study indicated that the FQ-resistant E coli isolates presented an alteration in gyrA (Ser-83?→?Leu, Asp-87?→?Asn) and parC (Ser-80?→?Ile) with high MICs (8-256 mg/l), and there was a low prevalence of qnr genes among E coli isolated from animals. 相似文献