鸡源金黄色葡萄球菌耐药基因定位的研究

从北京郊区鸡场分离到１０株耐 药性金黄色葡萄球菌,对这些菌株进行质粒消除,根据质粒消除前后的耐药性检测,发现耐各种药物的抗性基因大多数位于质粒上,少部分抗性基因位于染色体上,由质粒介导的耐药基因占７２．５％,由染色体编码的抗性基因占２７．５％,说明质粒在决定对抗生素的抵抗性中起主要作用。     

大肠杆菌耐药基因定位及耐药质粒消除

对采集的9株野生型大肠杆菌在药敏试验的基础上,进行了耐药基因定位。结果表明,4个菌株的氨苄青霉素耐药基因位于质粒上,不同畜群来源菌株之间耐氨苄青霉素质粒存在差异。用中草药艾叶的乙醇提取物和蒸馏提取物对大肠杆菌庆大霉素耐药性及耐药质粒进行了消除试验,结果艾叶乙醇提取物的消除率最高可达69.4%。     

食品动物源沙门氏菌质粒介导喹诺酮类耐药基因的检测与分析

为了解食品动物源沙门氏菌质粒介导喹诺酮类耐药性(Plasmid-mediated quinolone resistance,PMQR),采用微量肉汤稀释法和PCR方法,检测了316株食品动物源沙门氏菌对20种抗菌药物的敏感性,以及菌株中PMQR基因的携带率.结果显示:316株沙门氏菌对20种抗菌药物呈不同程度的耐药性,95.57％菌株为多重耐药菌;316株菌中未检出qnrA、qnrC、qnrD、qnrS和qepA基因,7.91％菌株检出qnrB基因,15.19％菌株检出aac(6 ′ )-Ib-cr基因,7.91％菌株检出oqxA基因,8.86％菌株检出oqxB基因,这是首次在沙门氏菌中发现oqxAB基因;98.11％PMQR阳性菌同时携带2种及以上的耐药基因,呈8～17耐的多重耐药性,其中以qnrB和aac(6′)-Ibcr基因型为主;53株PMQR阳性菌分属于5种不同的基因型,耐药表型或耐药基因型不同的菌株却有相同的PFGE谱型.本次检测的316株食品动物源沙门氏菌耐药较为严重;菌株主要携带qnrB、aac(6 ′ )-Ib-cr及oqxAB基因;不同来源菌株存在同一耐药克隆株的流行.     

中草药消除大肠埃希氏菌耐药性及耐药质粒的研究

在系统鉴定和药敏试验的基础上,对９株大肠埃希氏菌多重耐药菌株进行而药基因定位,结果表明有４株菌株的氨苄青霉素基因位于质粒上。通过对中草药煎煮、水蒸气蒸馏等方法提取其有效成分,对耐药大肠埃希氏菌进行耐药性消除试验,结果发展大蒜油等对大肠埃希氏菌氨苄青霉素耐药性有消除作用;鱼腥草、紫草等对大肠埃希氏菌庆大霉素而药性及耐药质粒有消除作用,消除率分别为２．９８％和４．２０％。     

鸡源性多重耐药沙门菌质粒与耐药性相关性分析

为了解鸡源性沙门菌临床分离株多重耐药情况与质粒的携带关系,采用K-B纸片法对13株鸡源沙门菌分离菌株进行10种抗菌药物敏感性试验,通过质粒图谱检测耐药菌株质粒携带情况,分析质粒图谱和耐药谱的关系,采用0.5%SDS和43°C对分离菌进行质粒消除,根据质粒消除前后耐药谱的变化,初步对耐药基因进行定位。13株分离菌均表现为多重耐药,大部分菌株为5重及以上耐药,最主要的多重耐药谱是AMP-CIP-OFL-NOR-SXT-TET,分离菌均检测到了质粒的存在。质粒消除后,耐药谱明显变窄,耐药菌株恢复了对大多数药物的敏感性。结果表明,鸡源性多重耐药沙门菌的耐药性与质粒的携带关系密切,初步推断编码四环素、环丙沙星、氧氟沙星和诺氟沙星的耐药基因主要存在于质粒上。     

质粒介导的喹诺酮耐药基因在动物源金黄色葡萄球菌的流行分布

为了解广东省动物源金黄色葡萄球菌耐药现状、分析耐喹诺酮类金黄色葡萄球菌耐药分子特征,本研究从2010—2011年广东33个养殖场的不同动物来源的样本中分离鉴定出46株金黄色葡萄球菌,采用二倍琼脂稀释法测定对14种抗菌药物的敏感性;采用聚合酶链式反应(PCR)检测耐喹诺酮金黄色葡萄球菌grlA、grlB、gyrA、gyrB基因在喹诺酮耐药决定区(QRDR)的突变特征、质粒介导喹诺酮类耐药(PMQR)基因(qnr、aac6′-Ib-cr、oqxA和qepA)的流行分布特征,同时还检测了β-内酰胺酶编码基因(blaCTX-M、blaCMY和blaSHV)、质粒介导金黄色葡萄球菌耐氟苯尼考的基因(cfr和fexA)和万古霉素耐药基因(vanA、vanB和vanC)。结果表明,46株金黄色葡萄球菌对克林霉素耐药率最高为67%,对环丙沙星和氟苯尼考耐药率为52%,对青霉素、氨苄西林、庆大霉素、红霉素、四环素和复方新诺明的耐药率在40%⁵0%之间,对苯唑西林和氯霉素耐药率为28%,对妥布霉素耐药率为13%,对头孢噻肟的耐药率在10%左右,有2株耐万古霉素。20株耐环丙沙星金黄色葡萄球菌的grlA和gyrA基因的QRDR发生了碱基突变并导致编码的氨基酸发生改变,GrlA氨基酸只有1种突变模式,Ser80→Phe,而GyrA氨基酸存在3种突变模式:Ser84→Leu,Ala或Phe。PMQR基因中,oqxA检出率最高(80%),其次是aac6′-Ib-cr占13%,qnrS1检出3株、qnrD检出2株。检测到2株菌株携带超广谱β-内酰胺酶编码基因blaSHV-12;23株氟苯尼考耐药菌株全部携带fexA基因,其中3株同时携带cfr基因;2株耐万古霉素菌株未检测到相关耐药基因。广东省动物源金黄色葡萄球菌多重耐药现象较为普遍,本研究首次在动物源金黄色葡萄球菌中检测到超广谱质粒编码的喹诺酮耐药基因流行分布广泛,大多数菌株同时携带2种以上质粒编码的耐药基因,提示养殖过程或治疗过程中使用任何一种药物(尤其是动物促生长剂)都可以筛选出耐药菌株。     

致病性大肠杆菌耐药性与其质粒相关性研究

对本地临床分离的20株致病性大肠杆菌进行耐药性检测和质粒检测。耐药性检测结果显示,受检20株致病性大肠杆菌对临床常用5种抗菌药物均表现为耐药,大肠杆菌耐药程度及多重耐药现象较为严重。质粒图谱分析结果表明,在大肠杆菌耐药性菌株中,尤其是耐药程度较高且呈多重耐药的菌株中,耐药质粒的检出率较高。实验结果提示:致病性大肠杆菌耐药性与其耐药质粒存在密切相关。     

禽致病性大肠杆菌ST95流行菌株耐药表型及耐药基因分析

为探究禽致病性大肠杆菌(APEC)ST95类群菌株的耐药表型和耐药基因分布情况,对37株ST95 APEC菌株进行血清型、耐药性测定以及耐药基因检测。结果显示:37株ST95 APEC的优势血清型为O2:K1和O1:K1,所有禽源ST95菌株均为多重耐药(MDR)特性,其至少对12种抗生素耐药,ST95菌株对头孢类抗生素的耐药比率很高,对β-内酰胺酶抑制剂耐药率也达到35%,对非头孢类抗生素如环丙沙星、庆大霉素等同样具有广泛耐药性。PCR检测发现30株APEC菌株含有β-内酰胺酶基因,在这些ST95菌株中检测出多种质粒编码的抗性基因,如链霉素/壮观霉素抗性基因strA和strB等。研究表明ST95 APEC菌株呈现明显的广谱耐药特性,质粒携带的耐药基因广泛分布于ST95菌株。     

仔猪源大肠杆菌耐药性分析

为了解河北省仔猪源大肠杆菌耐药现状及耐药质粒的分布情况,运用K-B法对分离自全省10个不同市县的已经过系统鉴定和致病性检验的40株仔猪源大肠杆菌进行了22种常见抗菌药的药敏试验,并提取质粒,进行质粒谱型分析,初步探讨大肠杆菌耐药性与质粒图谱之间的关系。结果显示:40株均为多重耐药菌株,6耐和7耐的居多,占了总数的42.5%,其中对氨苄西林、阿莫西林、四环素、复方新诺明耐药率较高,分别达到了95.0%,87.5%,85.0%和72.5%;对卡那霉素、庆大霉素和强力霉素耐药率也达到了40.0%以上。     

河北省保定地区雏鸡鸡白痢沙门菌的分离鉴定及耐药性检测

为了有效防控雏鸡白痢沙门菌病,试验自保定某蛋种鸡场疑似为鸡沙门菌病雏鸡采取肝脏病料进行细菌的分离培养、生化鉴定、鸡白痢沙门菌多价血清学鉴定及沙门菌inv A基因PCR鉴定,并对分离菌进行致病性检测及对7种抗菌药物的耐药性检测。结果表明,10株分离菌的形态特征、生化特性、血清学鉴定结果符合鸡白痢沙门菌的特征,说明从病死雏鸡组织中分离出的细菌为鸡白痢沙门菌。沙门菌inv A基因PCR扩增出373 bp目的片段,进一步证实分离菌为鸡沙门菌。用分离菌株接种雏鸡,其死亡率高达90%,表明该菌株有很高的致病性。10株分离菌株对阿莫西林和青霉素耐药率达100%,对阿齐霉素的耐药率达90%,而对阿米卡星、头孢唑林和庆大霉素耐药性较弱、对氧氟沙星较为敏感。3耐菌株多重耐药比例为42.86%,4耐菌株多重耐药比例为57.14%,5耐菌株多重耐药比例为71.43%,说明雏鸡沙门菌分离株对所试抗菌药物均表现出不同程度的耐药性,且表现出多重耐药现象。本试验为鸡白痢沙门菌病的防治提供参考。     

猪源大肠杆菌耐药性质粒的监测研究

在１１５株猪大肠杆菌耐药性监测的基础上,提取耐药类型最普遍的５株耐药菌的质粒进行质粒指纹图谱分析。结果表明：同一来源、耐药类型相同的菌株有相似的质粒图谱,来源不同者,酶切图谱有提示出它们之间的同源性,说明质粒指纹图谱可作为流行病学调查的工具。     

大肠埃希菌多重耐药性的形成机制

细菌耐药(尤其多重耐药)现象的出现,给我国养殖业带来巨大的经济损失,大肠埃希菌耐药性可分为原发性和获得性.大肠埃希菌外排泵功能增强、膜通透性下降均可以导致多重耐药的产生,而质粒、整合子/基因盒系统等可移动的遗传因子在多重耐药性的传播上有着重要作用.此外,细菌生物被膜的形成使细菌对多种抗菌药物产生强大的抵抗力.文章从上述几个方面阐述大肠埃希菌产生多重耐药的形成机制,并提出相应的防控对策.     

荧光报告质粒构建及其在布鲁菌细胞侵袭试验中的应用

采取质粒shuffling方法,将含GFPmut1的pPS858和能在布鲁菌中复制的质粒pBBRlMCS-2经KpnI酶切后连接,转化DH5a,利用双抗性筛选重组质粒pBBR1-GFP。将荧光质粒转入布鲁菌,获得荧光标记布鲁菌,并用构建好的荧光标记布鲁菌侵染巨噬细胞J774A．1,探讨其用于细胞侵袭试验的可行性。结果显示,成功构建布鲁菌荧光报告质粒pBBR1-GFP,用构建好的荧光标记布鲁菌侵染巨噬细胞后,在细胞内观察到带荧光的布鲁菌,且荧光强度与胞内细菌数成正比。细胞侵袭动态试验结果表明,细菌与细胞共孵育45min后,胞内细菌达到饱和。结果表明,成功构建了布鲁菌的荧光质粒报告系统,可用于布鲁菌细胞侵袭试验。     

Plasmid-mediated quinolone resistance gene detected in Escherichia coli from cattle

Fluoroquinolones resistance in bacteria can be due to chromosomal and plasmid-mediated mechanisms. Of growing concern is the acquisition of genes encoding quinolone resistance in combination with other resistance mechanisms such as extended-spectrum beta-lactamases. In this study we describe the identification of an isolate of Escherichia coli from cattle which carried qnrS1 in combination with a blaCTX-M gene, although they were not co-localised on the same plasmid. In addition, using a DNA array it was possible to identify several other antimicrobial resistance genes in this isolate. This is the first report of a qnr gene in E. coli from cattle in the UK and highlights the need for surveillance of these emerging resistance mechanisms.     

Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate

Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for iss's location in the bacterial genome. Thus far, iss in an avian E coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E coli; intI 1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugant's resistance to complement but not its virulence, in spite of the plasmid's possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipient's impairment for virulence, or an inability of the embryo assay used here to detect this plasmid's contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.     

Linkage of genes for heat-stable enterotoxin, drug resistance, K99 antigen, and colicin in bovine and porcine strains of enterotoxigenic Escherichia coli

Linkages among genes for production of Escherichia coli heat-stable enterotoxin (ST), drug resistance, K99 antigen, and colicin were investigated in 2 bovine and 3 porcine strains of enterotoxigenic E coli. In conjugation experiments, all 5 isolates transferred enterotoxigenicity and the ability to produce K99; 4 of the 5 isolates also transferred antibiotic resistance markers, and 3 colicinogenic strains transmitted the ability to produce colicin. In 2 of the 3 colicin-producing strains, the genes for colicin were located along with those for K99 and ST on a single plasmid. One of these 2 strains transferred the genes for tetracycline resistance and production of both mouse-active ST (STa) and mouse-inactive ST, whereas the other transmitted the gene(s) for STa only. Transformation studies with the 3rd strain revealed that the K99 determinant resided on a 22-megadalton (Mdal)R plasmid, and that STa and colicin production were on a 65-Mdal plasmid. Analysis of the plasmids from the transformation experiments revealed that the larger plasmid was conjugative and the smaller plasmid was nonconjugative; stable cointegrate formation occurred between these 2 plasmids. Genetic information coding for the production of STa and K99 were also present on a single plasmid of approximately 80 Mdal in both noncolicinogenic strains.     

大肠埃希菌耐药质粒消除剂研究进展

质粒消除剂是指通过抑制质粒的拷贝或复制使子代细茵中的质粒消失,并使其介导的耐药性也随之丧失的一些物质.自从质粒消除剂概念提出以来,对此研究越来越深入,并取得了一定的进展.论文就大肠埃希茵耐药质粒特征、耐药质粒消除方法及其作用机制的研究进展进行了综述,旨在探讨一种有效逆转耐药性的方法.     

大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体构建

合成胸膜肺炎放线杆菌质粒pKMA2425上长度为126bp及2214bp的2个DNA片段,克隆到大肠埃希氏菌质粒pGSI中,获得重组质粒pGSIA。PCR扩增pTKRED上的大观霉素抗性基因及线性化的pGSIA（126bpDNA片段-pGSI-2214bpDNA片段）,连接,电转化胸膜肺炎放线杆菌,在大观霉素的选择压力下筛选鉴定到连接正确的质粒,命名为pGSIAS。pGSIAS测序结果符合预期。在大观霉素的选择压力下,含有pGSIAS的胸膜肺炎放线杆菌血清1-10型菌株均生长良好;在氨苄青霉素的选择压力下,含有pGSIAS的大肠埃希氏菌生长良好。结果表明本研究构建的大肠埃希氏菌-胸膜肺炎放线杆菌穿梭载体pGSIAS序列正确,在胸膜肺炎放线杆菌及大肠埃希氏菌中均能复制并表达质粒上的相关基因。     

Molecular characterization of plasmids with antimicrobial resistant genes in avian isolates of Pasteurella multocida

The complete nucleotide sequences of two plasmids from avian isolates of Pasteurella multocida that caused outbreaks of fowl cholera in Taiwan were determined. The entire sequences of the two plasmids, designated as pJR1 and pJR2, were 6792 bp and 5252 bp. Sequence analysis showed that the plasmid pJR1 contained six major genes: the first gene (sulII) encoded a type II sulfonamide resistant dihydropteroate synthase, the second gene (tetG) encoded a tetracycline resistance protein, the third gene (catB2) encoded a chloramphenicol acetyltransferase, the fourth gene (rep) encoded a replication protein, and the fifth and sixth genes (mbeCy and deltambeAy) encoded proteins involved in the mobilization of plasmid. The plasmid pJR2 contained five major genes: the first gene (deltaintI1) encoded a truncated form of a type I integrase, the second gene (aadA1) encoded an aminoglycoside adenylyltransferase that confers resistance to streptomycin and spectinomycin, the third gene (blaP1) encoded a beta-lactamase that confers resistance to ampicillin and carbenicillin, and the fourth and fifth genes might encode proteins involved in the plasmid replication or segregation. Sequence comparisons showed that the antibiotic resistance genes found in pJR1 and pJR2 exhibited a high degree of sequence homology to the corresponding genes found in a great variety of gram-negative bacteria, including Escherichia coli, Salmonella enterica Typhimurium DT104, Psedomonas spp., P. multocida, Mannheimia spp., and Actinobacills pleuropneumoniae, which suggests that these resistance genes were disseminated in these bacteria. Although sulII and tetG genes were found previously in P. multocida or Mannheimia spp., this is the first report on the presence of catB2, aadA1, and blaP1 genes in bacteria of the family Pasturellaceae. Moreover, the aadA1 and blaP1 genes found in pJR2 were organized into an integron structure, which is a site-specific recombination system capable of capturing and mobilizing antibiotic resistance genes. This is also the first report on the presence of an integron in bacteria of the family Pasteurellaceae. The presence of a P. multocida integron might facilitate the spreading of antibiotic resistance genes between P. multocida and other gram-negative bacteria.     

一株鸭致病性大肠杆菌的分离鉴定及耐药分析

对江西省樟树市某鸭养殖场送检的病死鸭进行病理剖检,通过细菌分离、生化鉴定得到1株致病性大肠杆菌。药敏试验结果表明,该菌株对头孢拉定、头孢唑啉、头孢曲松和阿莫西林等β-内酰胺类抗生素产生了严重的耐药。通过质粒提取和接合转移试验对分离菌株进行耐药性分析;提取得到一10 kb左右的质粒;质粒接合转移试验表明,成功将该质粒转化入感受态E.coli JM109,并能使E.coli JM109获得对β-内酰胺类抗生素耐药且与分离菌株的耐药谱一致。推测该菌株产β-内酰胺酶的基因存在于质粒,并且该耐药基因能随质粒的转移转化入敏感菌E.coli JM109。