哈尔滨地区猪源大肠杆菌PMQR基因流行性检测

为了解哈尔滨地区猪源大肠杆菌中质粒介导的氟喹诺酮耐药(Plasmid mediated fluoroquinolone resistant,PMQR)基因的流行情况,本文通过对哈尔滨地区规模化养猪场进行采样处理,最终分离得到67株猪源大肠杆菌菌株,采用微量肉汤稀释法测定其对8种药物的最小抑菌浓度,针对PMQR基因设计特异性引物进行PCR扩增,结果显示,对8种兽医临床常用抗菌药物耐药率较高,多表现为多重耐药。oqx AB,qnr S,aac(6′)-Ib-cr,qnr B,qep A的检出率分别为70.15%,64.18%,22.39%,13.43%,11.94%,并没有检测到qnr A、qnr C、qnr D基因,这表明,PMQR基因在哈尔滨地区猪群中存在很高的检出率,这势必会加剧临床用药的难度,并且还为基因的水平传播提供可能。     

广东地区患病食品动物源大肠杆菌ESBLs和AmpC酶流行分布调查

质粒介导的超广谱β-内酰胺酶（ESBLs）和AmpC酶广泛传播和扩散,导致全球范围内肠杆菌科细菌对超广谱头孢菌素类的耐药性发展迅速,引起人们高度重视。食品动物源大肠杆菌可作为耐药基因储库,对细菌耐药性在动物、环境和人之间的传播起着非常重要的作用。本研究从2009年患病的食品动物,包括鸡、鸭、鹅和猪,分离鉴定了315株大肠杆菌,调查ESBLs和AmpC酶在患病食品动物中的流行分布。经双纸片协同扩散实验筛选出61株ESBL阳性菌株,PCR检测共检测出8种blaCTX-M基因,分别为blaCTX-M-14/14b、blaCTX-M-79、blaCTX-M-65、blaCTX-M-27、blaCTX-M-15、blaCTX-M-24、blaCTX-M-98和blaCTX-M-13,其中检出率最高的为blaCTX-M-79,其次为blaCTX-M-14/14b。本研究还检测出1株新的SHV型酶,命名为SHV-135;PCR共检测出5株大肠杆菌携带质粒源CMY-型AmpC酶,其中2株鹅源大肠杆菌携带1个新型CMY酶,命名为CMY-64。本研究表明患病动物分离的大肠杆菌中ESBLs和AmpC酶存在复杂性和多样性,而且新型β-内酰胺酶检出日益增多,提示兽医临床应慎用β-内酰胺类抗生素。     

食品动物源沙门菌ESBLs和PMQR耐药基因流行特征分析

本研究对从猪、鸭和鹅分离的31株沙门菌进行耐药性分析,调查ESBLs基因和PMQR基因在沙门菌的流行分布特征.结果显示,31株沙门菌中只检测到CTX-M型ESBLs,3株携带blaCTX-M-14,3株携blaCTX-M-27基因;有13株携带blaOXA型均为窄谱blaOXA-1,6株携带blaTEM型也为窄谱blaTEM-1b.blaSHV、blaPER、blaVEB、blaGES等各型β-内酰胺酶基因均未检出.PMQR基因oqxAB和aac(6′)-Ib-cr检出率最高分别为39%和26%.4株鸭源和1株猪源沙门菌中都检测到qnrA基因,而qnrD基因仅在一种猪源沙门菌中检出.qnrB、qnrC、qnrS、qepA等基因均未检出.研究发现有19株沙门菌同时携带β-内酰胺酶和PMQR,其中一株鸭源沙门菌同时携带CTX-M-14 ESBLs和CMY-2 AmpC酶;有7株同时携带4-5种β-内酰胺酶和PMQR基因.     

山东临沂地区鸡源致病性大肠杆菌体外耐药性测定

采用纸片扩散法对从山东省临沂地区分离鉴定的鸡源致病性大肠杆菌进行了药物敏感性测定,结果发现:耐药率较高的为青霉素、苯唑青霉素、氨苄青霉素、复合磺胺、万古霉素、红霉素、链霉素、环丙沙星、四环素、氟哌酸等,前6种均高达100%,后4种分别为93.33%、88.89%、84.62%、83.33%;敏感率较高的为头孢他啶、头孢哌酮、头孢三嗪、头孢噻肟、头孢呋新和左旋氧氟沙星,其极敏和高敏的比率之和分别高达100%、92.86%、87.50%、82.35%、78.57%和71.43%;呋喃妥因高敏和中敏的比率之和高达100%,由此可知这7种药是目前山东临沂地区鸡大肠杆菌病的首选药物。     

内蒙古地区奶牛源致病性沙门氏菌耐药性分析及ESBLs基因检测

为研究内蒙古地区奶牛源致病性沙门氏菌的耐药特性及ESBLs基因流行特征,试验采用微量肉汤稀释法测定了22种兽医临床常用抗菌药物对临床分离沙门氏菌的最低抑菌浓度(MICs),并对其多重耐药特性进行了分析;采用PCR法对具有β-内酰胺类药物耐药表型的菌株进行了8种动物源沙门氏菌常见ESBLs基因的检测。结果显示,内蒙古地区奶牛源致病性沙门氏菌对甲氧苄啶(97.4%)及磺胺甲基噁唑(94.7%)的耐药率最高,对多数β-内酰胺类、氨基糖甙类、氯霉素类及四环素类药物的耐药性也较为严重(耐药率为40%~80%),所有菌株对氟喹诺酮类药物均敏感;菌株的多重耐药率为94.7%,有29株菌(76.3%)同时对6类抗菌药物具有耐药性;35株对β-内酰胺类药物耐药的菌株中,有30株(85.7%)为ESBLs基因阳性,共检出6种ESBLs基因,其中CTX-M型基因检出率最高(40.0%),未检出SHV和PSE型基因;共发现15种ESBLs基因型,9种ESBLs基因型组合,有16株菌(45.7%)同时携带两种或两种以上ESBLs基因。结果表明,内蒙古地区奶牛源致病性沙门氏菌对兽医临床常用抗菌药物的耐药性较为严重,且ESBLs基因的流行模式较为复杂,提示兽医临床抗菌药物不合理使用可能是造成该地区奶牛源沙门氏菌耐药性产生的原因。     

鸡源大肠杆菌ESBLs基因型检测及耐药性分析研究

为调查哈尔滨市周边地区鸡源大肠杆菌中超光谱β-内酰胺酶(ESBLs)基因型的流行情况,探索携带ESBLs对鸡源大肠杆菌耐药性的影响,从哈尔滨市周边地区多个养殖场分离了137株鸡源大肠杆菌,用微量肉汤稀释法测定137株菌对18种抗菌药物的最小抑菌浓度,采用CLSI推荐的表型筛选和确证试验检测大肠杆菌携带ESBLs情况,用PCR方法检测ESBLs基因(blaTEM、blaSHV、blaCTX-M、blaOXA)的流行分布情况。结果显示:109株大肠杆菌为产ESBLs菌株,占79.6%;137株鸡源大肠杆菌对临床常用18种抗菌药物产生不同程度耐药,且产ESBLs大肠杆菌的耐药性比非产ESBLs大肠杆菌的耐药性严重;blaTEM、blaCTX-M基因阳性率分别为40.9%、71.5%,所有菌株均未扩增出blaSHV和blaOXA基因。提示,blaTEM和blaCTX-M基因为哈尔滨地区鸡源产ESBLs大肠杆菌的流行基因型,且鸡源产ESBLs大肠杆菌菌株分布广泛,应加强当地用药监管。     

陕西地区羊源致病性大肠杆菌耐药性分析与毒力基因检测

旨在了解陕西省部分地区腹泻羊源致病性大肠杆菌(E. coli)耐药性及毒力基因携带情况,本研究从10个养殖场采集54份腹泻羊拭子样品,经分离纯化、生化鉴定及16S rRNA基因序列分析,共分离得到50株E. coli,对分离菌进行药敏试验、耐药基因及毒力基因检测。结果显示,分离菌对氨苄西林、氟苯尼考和磺胺异噁唑耐药率达90%以上,且98%(49/50)为多重耐药菌,对8~11种抗生素耐药的菌株占68%(34/50),仅对美罗培南敏感。所有菌株均携带1~6种不同的耐药基因,其中,Sul1(64%)、TetA(34%)、bla_CTX-M(32%)携带率较高,未检测到bla_SHV。有5株产ESBLs的E. coli携带mcr-1耐药基因。毒力基因检测结果显示,98%(49/50)的菌株携带毒力基因,其中,etrA检出率最高,为80%(40/50)。综上表明,陕西省羊源E. coli多重耐药情况严峻,β-内酰胺类耐药基因与耐药表型不符,提示可能存在其他耐药机制,同时,分离菌具有复杂的毒力谱。本研究为陕西省羊源致病性E. coli感染的防控提供科学依据。     

不同地区动物源致病性大肠杆菌的分子特性与表型分析

为进一步了解致病性大肠杆菌流行状况,减轻大肠杆菌对畜牧业和公共卫生的危害,对不同时间国内外报道的动物中分离的大肠杆菌,进行毒力、血清型、耐药性和基因型等方面的比较,对比分析大肠杆菌分子特性和表型差异,从而为临床医学和公共卫生控制提供指导。     

贵州部分地区猪源大肠杆菌耐药性分析及ESBLs基因型检测

为了解贵州地区规模养猪场大肠杆菌菌株耐药情况和ESBLs基因型的流行情况,试验采用CLSI推荐的方法对采自贵州省4个地区规模养猪场的164株大肠杆菌进行药物敏感性试验和产ESBLs菌的检测,并用PCR方法对TEM、SHV、OXA-1和CTX-M-1 4种常见ESBLs基因进行检测。结果显示,164株大肠杆菌对10种常用抗菌药物耐药率分别是头孢噻呋93.29%、氨苄西林87.19%、四环素86.59%、庆大霉素81.10%、链霉素53.66%、多黏菌素51.83%、环丙沙星53.05%、卡那霉素47.56%、金霉素34.76%和氟苯尼考21.95%,且大多为多重耐药,其中检测出ESBLs阳性菌株137株,阳性率为83.54%,各个地区的检出率不同;137株产ESBLs大肠杆菌中,TEM、SHV、OXA-1和CTX-M-1基因的检出率分别为90.51%、70.07%、51.82%和43.07%,且多为复合基因型耐药菌株,各地区的各种基因检出率不同。试验结果表明,贵州部分地区的猪源大肠杆菌耐药现象严重,ESBLs菌株的检出率很高,耐药基因的检出率也极高,且多为复合基因型耐药菌株,应加强当地产酶耐药菌的监测和研究,有效防制此类细菌引发的疾病。     

动物源耐氟喹诺酮类药物致病性大肠杆菌gyrA基因的突变研究

用微量稀释法测定盐酸环丙沙星等5种氟喹诺酮类抗菌药物对20株耐氟喹诺酮类药物的动物源致病性大肠杆菌的最低抑菌浓度(MIC),PCR扩增gyrA基因的喹诺酮耐药决定区(quinolone resistance determining regions,QRDR),扩增的片断长度为496 bp,PCR产物直接测序,用DNAStar软件分析氨基酸序列。结果显示,盐酸环丙沙星、恩诺沙星、氧氟沙星、甲磺酸培氟沙星和烟酸诺氟沙星的MIC范围分别为64～>512μg/mL、16～256μg/mL、16～128μg/mL、64～>512μg/mL和64～>512μg/mL。gyrA基因的突变位点均位于83和87位氨基酸位点,主要的变异方式为Ser83→Leu(15/20),其次是Asp87→Asn(13/20),其他的为Asp87→Tyr(6/20),Ser83→Trp(4/20),Ser83→Ala(1/20)和Asp87→Gly(1/20)。说明试验用菌株对氟喹诺酮类药物均表现为多重耐药,其gyrA基因QRDR的突变表现为多种形式。     

Prevalence of ESBLs and PMQR genes in fecal Escherichia coli isolated from the non-human primates in six zoos in China

The aim of this study is to characterize the prevalence of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated quinolone resistance (PMQR) genes in Escherichia coli from captive non-human primates. A total of 206 E. coli isolates were collected from primates in six zoos in China in 2009 and their susceptibility to 10 antimicrobials were tested by broth microdilution. The susceptibility patterns of E. coli strains varied greatly among different zoos reflecting different backgrounds of antimicrobial usage. Both the ESBL-encoding genes and the PMQR genes were detected by PCR. Of the 206 strains, 65 (32%) were confirmed as phenotypic ESBL producers with bla(CTX-M) (27%, bla(CTX-M-15), n=31, bla(CTX-M-3), n=23 and bla(CTX-M-14), n=2) mainly mediating the ESBL phenotype. qnrS1 (18%, n=36) and oqxAB (15%, n=31) were the predominant PMQR genes and the prevalence of PMQR genes was much higher among phenotypic ESBL producers than that among phenotypic non-ESBL producers from any zoo. Notably, the PMQR genes qnrS1 and oqxAB and β-lactamase genes bla(TEM-1) and bla(CTX-M-3) were found together in 23 E. coli isolates in two zoos in Shanghai. PFGE analysis of these 23 isolates demonstrated nearly identical PFGE profiles (similarity matrix >97%) indicating this specific E. coli genotype was prevalent in these two zoos. To the best of our knowledge, this is the first report of these four genes coexisting in an E. coli genotype and the first report of antimicrobial resistance profiles in E. coli isolated from primates in China.     

猪和野生动物源大肠杆菌及沙门菌中磺胺类药物耐药基因的检测

对从四川省10个规模化猪场和16种野生动物的粪样中分离鉴定出的67株大肠杆菌、57株沙门菌进行了药敏试验。结果，猪源和野生动物源分离菌对磺胺类药物的耐药率分别为72．0％（72／100）和29．2％（7／24）。根据GenBank中登录的序列，设计了3对特异性引物，对磺胺类药物的耐药基因Sul1、Sul2、Sul3进行了三重PCR检测。在这124株细菌中，Sul1基因的检出率最高，为47．6％（59／124），Sul2基因的检出率为17．7％（22／124），Sul3基因的检出率为18．5％（23／124）。药敏试验结果与基因检测结果的符合率为89．9％。     

Differentiation of pathogenic and nonpathogenic Escherichia coli isolated from poultry

Twenty-one isolates of Escherichia coli recovered from chickens and turkeys were evaluated for pathogenicity in 1-week-old chicks. Fifteen produced coli-septicemia (pathogenic) and six were innocuous (nonpathogenic). Both pathogenic and nonpathogenic E. coli were tested for their ability to selectively absorb Congo red (CR) dye incorporated into agar medium. Eight of 15 pathogenic E. coli (somatic antigen types O1, O78, O11, O88, and OX9) absorbed the dye and produced red colonies (CR+) between 48 to 72 hours of incubation. All serotypes of E. coli with homologous somatic antigen O78 were CR+, while those of O2 antigen were CR- (white colonies). Five of six nonpathogenic E. coli also were CR+. In contrast to pathogenic E. coli, however, nonpathogenic isolates absorbed CR early, between 18 to 24 hours of incubation. Although CR dye binding did not correlate well with pathogenicity, it may be an identifiable property of some serotypes of E. coli.     

宠物源大肠杆菌的血清型和毒力基因及耐药性调查

为研究宠物源致病性大肠杆菌(Escherichia coli)的血清型、毒力基因和耐药性之间的相关性,本研究由健康和患病犬、猫直肠拭子样品中分离177株E.coli,并采用玻片凝集法鉴定其血清型,结果显示分离株中定型菌株135株,分别属于20个不同的血清型,其中O1、O2和O8为主要的流行血清型。PCR方法检测11种毒力相关基因,并采用琼脂稀释法测定分离菌株对14种抗菌药的敏感性,结果表明3个血清型的菌株拥有相似的耐药表型,但毒力基因谱不同。62%的分离菌株携带fimH基因,并且毒力基因组合iroN+hlyF、iroN+fimH和traT+sitA比较流行。55%的O1血清型携带fimH基因,并且多数耐受四环素、多西环素、头孢噻吩和庆大霉素;20.8%的O2血清型菌株携带traT和sitA基因;50%的O8血清型携带traT和fimH基因。3个血清型分离菌株多数对安普霉素和阿米卡星敏感。     

Characters of Escherichia coli 078 isolated from septicaemic animals

Twenty-one Escherichia coli isolates of serogroup 078 from animal septicaemia were obtained from laboratories in France, England and Canada. The bacteria were compared for outer membrane protein (OMP) patterns, lipopolysaccharide patterns, surface proteins of fimbrial types, biotypes, antibiotypes, colicin production, hydroxamate production and virulence in mice. Sixteen isolates from bovine, ovine, porcine and avian species in France and England had a similar OMP pattern. This characteristic associated with minor properties like surface proteins type, colicin V production and virulence in mice made these 16,078 E. coli isolates from 4 animal species, good candidates for the same clonal grouping. The five other bovine isolates with "Vir" or "31a" phenotypes were heterogeneous for most of the characteristics studied.     

Prevalence of eae and shiga toxin genes among Escherichia coli strains isolated from healthy calves

Strains of Escherichia coli (n = 390) isolated from 132 healthy, 4-8-week old calves, were tested by polymerase chain reaction (PCR) for the eae (intimin) gene and shiga toxin genes (stx1 and stx2). All strains were also analysed for F5, F17 and F41 fimbriae and for the heat-labile (LT) and heat-stable (STI and STII) genetic markers. Overall, the eae gene was detected in 84 (21.5%) of the strains tested. Only 21 (5.4%) isolates were positive for stx1 (18 strains) or stx2 (three strains); nine of the stx1-positive isolates also possessed the eae gene. A high percentage (29.2%) of the isolates tested expressed F17 but no enterotoxin genes were detected. None of the eae- or stx-positive strains belonged to the O157 serogroup.     

河南猪源致病性大肠杆菌分离、鉴定及其生物学特性

从河南省不同地区采集腹泻死亡仔猪进行病原分离鉴定,通过对细菌形态观察、PCR检测、生化试验以及小鼠毒力试验鉴定出了8株具有致病性大肠杆菌。通过WHO推荐的Kirby-Bauer(K-B)法测定8株大肠杆菌对21种抗菌药物的耐药性,并进一步对分离的8株大肠杆菌进行运动性以及生物被膜形成特性进行比较。生化特性结果表明8株大肠杆菌对各种糖的分解能力无明显差异;耐药性结果表明8株大肠杆菌均是多重耐药菌株,并且8株大肠杆菌在半固体培养基上均具有运动性,形成较明显的运动圈;生物被膜表型检测结果表明:有4株大肠杆菌具有较强的生物膜形成能力。本试验为进一步研究河南地区大肠杆菌的流行病学和探讨生物被膜和致病性、毒力之间的相关性奠定基础。     

Antimicrobial susceptibility of pathogenic Escherichia coli isolated from pigs in Korea

The in vitro susceptibilities of 285 isolates of Escherichia coli from preweaned and postweaned pigs with diarrhea and edema disease were tested with the 15 commonly used antimicrobial drugs by an agar dilution minimal inhibitory concentration procedure according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines. All E. coli isolates tested in this study belonged to enterotoxigenic E. coli, attaching and effacing E. coli, or Shiga toxin-producing E. coli. Field isolates had low MIC90 for ceftiofur (1 microg/ml). No correlation in antimicrobial resistance was found in three types of E. coli.     

Penetration of drug resistance in Escherichia coli isolated from laboratory animals

A total of 713 strains of fecal Escherichia coli (E. coli) isolated from laboratory animals in the colonies of 4 research laboratories and 4 commercial breeders in Japan in 1994 were examined in regard to resistance to 8 antibacterial agents. The incidence of resistance to sulfadimethoxine (Su), streptomycin (Sm), ampicillin, cephaloridine, tetracycline, chloramphenicol, kanamycin, and gentamicin was 99.9%, 32.5%, 6.7%, 0.7%, 7.0%, 2.6%, 6.6% and 0.7%, respectively. These results indicated that Su and Sm resistance are penetrating into normal E. coli strains isolated from laboratory animals.