狐和貉源大肠埃希菌药敏试验及耐药基因检测

为了解秦皇岛昌黎县狐和貉源致病性大肠埃希菌耐药性及耐药基因的情况,以昌黎县分离的15株狐和貉源致病性大肠埃希菌为试验菌株,采用药敏纸片法对抗菌药物进行药敏试验,并对四环素类耐药基因tetA、tetB、tetC、tetD,氨基糖苷类耐药基因strA-strB、aadA1,磺胺类耐药基因sul1、sul2、sul3进行PCR检测。结果显示,15株大肠埃希菌分离菌株对土霉素、多西环素、头孢拉定、新霉素、青霉素、磺胺间甲氧嘧啶、磺胺二甲氧嘧啶的耐药率均为100%;对阿莫西林、庆大霉素、头孢曲松、阿米卡星、大观霉素、链霉素、复方新诺明、氨苄西林、环丙沙星耐药率分别为93.33%、53.33%、33.33%、73.33%、86.66%、86.66%、86.66%、66.67%和66.67%。9种耐药基因tetA、tetB、tetC、tetD、strA-strB、aadA1、sul1、sul2、sul3的检出率分别为0、46.67%、26.67%、13.33%、40%、100%、53.33%、46.67%和53.33%,其中四环素类耐药基因以tetB的检出率最高,氨基糖苷类耐药基因以aadA1的检出率最高,磺胺类耐药基因的检出率相差不大。说明15株狐和貉源大肠埃希菌耐药谱广,耐药性高,耐药基因流行普遍。     

猪源大肠埃希菌磺胺类药物主要耐药基因的检测与分析

为查明辽宁地区猪源大肠埃希菌磺胺类耐药菌株中sul1、sul2及dfrA1 3种基因型的流行及分布情况,科学指导兽医临床合理用药,为猪大肠埃希菌病的防控提供科学依据,利用PCR技术对磺胺类耐药菌株进行了3种耐药基因型的检测。通过对耐药基因的检测发现,27株磺胺类耐药菌株中sul2的检出率最高,为74.07%(20/27);其次为sul1、dfrA1,检出率分别为33.33%(9/27)、22.22%(6/27),同时携带sul1、sul2和sul2、dfrA1两种基因型的菌株均占11.11%(3/27),同时携带sul1、sul2及dfrA1 3种基因型的菌株占14.81%(4/27)。表明辽宁地区猪源大肠埃希菌磺胺类耐药菌株中sul2为主要流行的基因型,并且在辽宁不同地区普遍存在,是介导猪源大肠埃希菌对磺胺类药物耐药性的主要基因型,其次为sul1、dfrA1。     

锦州某奶牛养殖场乳房炎大肠埃希菌药敏试验及耐药基因检测

为了解锦州某奶牛养殖场临床型乳房炎病原菌耐药情况。对该场进行患病乳样采集、主要病原菌分离鉴定、药物敏感性试验和耐药基因筛选。结果表明,该场乳房炎主要病原菌为大肠埃希菌,共得到乳房炎大肠埃希菌分离株36株;药物敏感性试验结果表明,分离株对磺胺类药物高度耐药、对头孢噻呋和头孢唑啉敏感;对分离株进行磺胺类药物耐药基因筛查结果表明,sul1、sul2、sul3基因的阳性率分别为76.0%、17.1%和0%,说明sul1基因为该场大肠埃希菌磺胺类药物主要耐药基因。     

畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌的LAMP检测及其耐药性分析

为深入了解畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌的流行及耐药状况,采用建立的畜禽粪便中金黄色葡萄球菌和大肠埃希氏菌的环介导等温扩增技术（LAMP）检测方法对山东省内牛粪污、猪粪、鸡粪、鸭粪中的金黄色葡萄球菌和大肠埃希氏菌进行检测,进而对检测阳性样品进行分离,并对药敏试验中多重耐药性菌株进行耐药基因预测。结果表明,LAMP方法检测畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌与国家标准方法检测结果符合率为100%,重复性好,特异性高。从80份畜禽粪污样品中分离到的3株金黄色葡萄球菌均对青霉素耐药;34株大肠埃希氏菌对氟苯尼考、利福平的耐药比例高于50%,高于25%以上的还有氨苄西林、四环素、多西环素、磺胺异噁唑、头孢噻吩和头孢噻呋;耐药基因预测结果显示,鸡粪、鸭粪、牛粪和猪粪中的4株大肠埃希氏菌分别携带10、8、20和15种耐药基因;鸡粪中的1株金黄色葡萄球菌携带11种耐药基因。说明山东地区畜禽粪污中金黄色葡萄球菌和大肠埃希氏菌耐药状况严峻,菌株普遍多重耐药,且携带多种耐药基因。     

犬源大肠埃希菌对β-内酰胺类药物的耐药表型和耐药基因检测

为研究犬源大肠埃希菌的耐药情况,对成都地区采集的犬肛门拭子进行细菌分离鉴定,共分离156株大肠埃希菌,同时,进行5种抗生素耐药性检测和β-内酰胺类药物耐药基因的菌株筛选。结果显示,大肠埃希菌对β-内酰胺类耐药率较高,头孢喹肟100%,氨苄西林73.1%,但对美罗培南敏感率高达95.5%。采用PCR方法检测β-内酰胺类药物常见的3种耐药基因blaCTX-M、blaSHV和blaTEM,blaCTX-M检出率最高为89.74%,blaTEM为78.84%,blaSHV无检出。耐药性和耐药基因比较表明,犬源大肠埃希菌β-内酰胺类的耐药性与耐药基因型检出率基本呈正相关,为成都犬源大肠埃希菌β-内酰胺类药物的耐药现状和疾病治疗提供了理论依据。     

广东地区水禽源大肠杆菌耐药性和磺胺类耐药基因检测

为调查广东地区水禽源大肠杆菌磺胺类药物耐药性和磺胺类耐药基因的流行情况,采用琼脂二倍稀释法测定大肠杆菌分离株对磺胺类药物的敏感性,通过PCR方法调查磺胺耐药株携带耐药基因sul1、sul2、sul3的情况。药敏试验结果表明,256株水禽源大肠杆菌分离株对复方磺胺甲恶唑和磺胺二甲嘧啶耐药率分别为91.0%和86.7%;在248株磺胺耐药株中,sul1、sul2和sul3检出率分别为73.8%、74.6%和44.7%。表明sul1和sul2为广东地区主要携带的磺胺类耐药基因,对水禽磺胺类耐药菌株的产生起重要的作用。     

奶牛子宫内膜炎源性大肠埃希菌药敏试验与耐药基因检测

为探索奶牛子宫内膜炎源性大肠埃希菌耐药基因及其耐药性之间的关系。采用K-B纸片法对临床分离的54株奶牛子宫内膜炎源性大肠埃希菌进行12种抗菌药物敏感性测定,应用PCR进行这些抗菌药物相关耐药基因的检测。结果显示,54株大肠埃希菌对复方新诺明、恩诺沙星和四环素的耐药率较高,分别为67%、39%和35%。分离的54株大肠埃希菌中有41株至少含有所检测耐药基因中的1种,CyrA、CyrB、ParC和Tet耐药基因较为普遍,其耐药菌基因检出率依次为96%、83%、100%和95%,13株没有检测到相应的耐药基因。奶牛子宫内膜炎源性大肠埃希菌对常见抗菌药物耐药严重,耐药基因普遍存在于耐药菌株中,除氨基糖苷类耐药菌株,其他3种抗菌药物类型耐药表型与耐药基因检测结果有较高一致性。     

贵州省猪源大肠杆菌对磺胺类抗菌药物耐药性及耐药基因检测

为了解贵州省猪源大肠杆菌对磺胺类抗菌药物的耐药性及其耐药基因的流行情况,采用微量肉汤稀释法测定66株从贵州省6个地区规模化养猪场分离保种的猪源大肠杆菌对5种磺胺类抗菌药物敏感性,并用PCR方法对磺胺类耐药的sul基因进行检测。结果显示,贵州省猪源大肠杆菌对磺胺二甲嘧啶耐药率为93. 94%,其次是磺胺甲噁唑和磺胺噻唑耐药率分别为86. 36%、89. 39%,磺胺间甲氧嘧啶和磺胺嘧啶耐药率分别为84. 85%、80. 30%。66株大肠杆菌sul基因的检出率为100%,其中sul3为100%(66/66),sul1为89. 4%(59/66),sul2为77. 3%(51/66),同时携带sul1、sul2及sul3三种基因型的大肠杆菌占74. 2%(49/66)。结论:贵州省6个地区规模化养猪场大肠杆菌对磺胺类药物耐药形式严峻,磺胺类sul3耐药基因携带率较高。     

小肠结肠炎耶尔森菌对磺胺类和β-内酰胺类抗生素的耐药性研究

为了探讨12株小肠结肠炎耶尔森菌对磺胺类和β-内酰胺类抗生素的耐药性,试验采用标准K-B纸片扩散法检测该菌对磺胺类和β-内酰胺类抗生素的耐药表型,并采用PCR法检测磺胺类耐药基因(sul1、sul2、sul3基因)和β-内酰胺类耐药基因(TEM基因)。结果表明:头孢噻吩、复方新诺明和磺胺异唑治疗效果较差,耐药率分别高达58.3%、66.7%、58.3%,但先锋霉素Ⅴ抑菌效果较好,头孢菌素次之;在菌株中检测出磺胺类耐药基因sul1、sul2和β-内酰胺类耐药基因TEM,其检出率分别为91.7%、100%、75.0%,而sul3基因未检出。     

不同地区猪源大肠埃希菌磺胺类药物耐药性研究

对不同地区猪源大肠埃希菌磺胺药物耐药性进行系统性调查研究.对从山东、内蒙古、山西分离到的161株猪源大肠埃希菌测定磺胺药物对其最低抑菌浓度(MIC值),结果表明大肠埃希菌对磺胺药物高度耐药,对磺胺异恶唑(SF)和新诺明(SMZ)的耐药率分别为91.30%和90.06%.同时根据GenBank上已有的二氢叶酸合成酶基因S...     

Detection and Analysis of Virulence Genes and Drug Resistance Genes in E. coli Strains Isolated from Dairy Cows with Clinical Mastitis in Liaoning Region

In order to understand the virulence genes and drug resistance genes carried by E.coli strains from dairy cows with mastitis in large-scale dairy farms in Liaoning region, and also provide improved programs for control and treatment of mastitis to dairy farmers, 66 strains of E.coli isolated from milk of dairy cows with clinical mastitis in several large-scale farms in Liaoning region were examined to detect 4 virulence genes and 4 drug resistance genes using PCR methods. The results showed that none of the target genes was detected in only one strain, while at least 2 and up to 7 target genes were detected in the rest of 65 strains. The detection rates of the virulence genes stx2e, eaeA, K99 and astA were 56.1%, 47.0%, 34.8% and 31.8%, respectively. In addition, the detection rate of dual virulence gene reached 43.9%, in which the genotype with the highest detection rate was eaeA/stx2e. The detection rates of the drug resistance genes sul3, sul1, cmlA and aacA4 were 87.9%, 83.3%, 40.9% and 28.8%, respectively. And the dual resistance gene detection rate was 36.4%, in which the highest detection rate was sul1/sul3 genotype; Triple drug resistance gene detection rate was 37.9%, and cmlA/sul1/sul3 presented the highest detection rate. These results confirmed that the E.coli isolated from dairy cow mastitis in large-scale dairy farms in Liaoning region had high detecting rates of sulfonamide resistance genes and chloramphenicol resistance genes, which was directly related to the drug resistance of the E.coli. These results provided important guiding significance for the prevention and control of dairy cow mastitis in Liaoning region as well as the safety of public health.     

辽宁地区奶牛乳房炎源大肠杆菌毒力基因及耐药基因检测分析

为了掌握辽宁地区规模奶牛场乳房炎源大肠杆菌携带的毒力基因和耐药基因,为奶牛养殖业提供更好的乳房炎防制方案,本研究采用PCR检测方法对辽宁地区多个规模奶牛场临床奶牛乳房炎奶样中分离的66株大肠杆菌进行了4种毒力基因和4种耐药基因的检测分析。结果发现,66株大肠杆菌中仅有1株未检出相关目的基因,其余65株中最少检出2种目的基因,最多检出7种目的基因。其中,毒力基因stx2e、eaeA、K99和astA的检出率分别为56.1%、47.0%、34.8%和31.8%,双重毒力基因的检出率达到43.9%,以eaeA/stx2e基因型的检出率最高;耐药基因sul3、sul1、cmlA及aacA4的检出率分别为87.9%、83.3%、40.9%和28.8%,双重耐药基因的检出率为36.4%,以sul1/sul3基因型检出率最高;三重耐药基因的检出率为37.9%,以cmlA/sul1/sul3检出率最高。本研究结果证实,辽宁地区奶牛乳房炎源大肠杆菌携带磺胺类耐药基因和氯霉素类耐药基因的比率较高,与大肠杆菌的耐药性有较直接的关系,该结果对于辽宁地区奶牛乳房炎的防制具有重要的指导意义,更具有重要的公共卫生意义。     

牛肉源大肠杆菌的耐药性检测及相关耐药基因分布

为了解牛肉源大肠杆菌的耐药性并检测其相关耐药基因分布,本研究选取了117株牛肉源大肠杆菌,经药敏纸片法对11种抗菌药物进行了药敏检测,并根据耐药表型利用普通和(或)多重PCR技术对耐四环素菌中tet(A)、tet(B)和tet(C)基因,耐氨苄西林菌中blaTEM1、blaPSE1和blaOXA1基因,耐链霉素菌中strA-strB、aadA1基因,耐磺胺类药菌中sul1、sul2和sul3基因进行了调查分析。结果显示,117株大肠杆菌对四环素、氨苄西林、链霉素和磺胺异恶唑的耐药率较高,分别为89%、42%、38%和22%。tet(A)基因是所有四环素耐药基因中最为流行的一种基因(55%);在检测的3个β-内酰胺类药物耐药基因中,最流行的为blaTEM1基因(73%);链霉素的耐药性主要由strA-strB基因(38%)编码;sul2基因在耐磺胺异恶唑菌中的检出率最高(77%)。结果表明本次分离的牛肉源大肠杆菌耐药非常严重,进一步肯定了肉牛业生产中抗菌药的使用对大肠杆菌耐药性的产生和发展所发挥的重要作用,提示食品动物养殖应严格控制饲料中抗菌药的滥用。     

Prevalence and characterization of plasmids carrying sulfonamide resistance genes among Escherichia coli from pigs,pig carcasses and human

Background

Sulfonamide resistance is very common in Escherichia coli. The aim of this study was to characterize plasmids carrying sulfonamide resistance genes (sul1, sul2 and sul3) in E. coli isolated from pigs and humans with a specific objective to assess the genetic diversity of plasmids involved in the mobility of sul genes.

Methods

A total of 501 E. coli isolates from pig feces, pig carcasses and human stools were tested for their susceptibility to selected antimicrobial. Multiplex PCR was conducted to detect the presence of three sul genes among the sulfonamide-resistant E. coli isolates. Fifty-seven sulfonamide-resistant E. coli were selected based on presence of sul resistance genes and subjected to conjugation and/or transformation experiments. S1 nuclease digestion followed by pulsed-field gel electrophoresis was used to visualize and determine the size of plasmids. Plasmids carrying sul genes were characterized by PCR-based replicon typing to allow a comparison of the types of sul genes, the reservoir and plasmid present.

Results

A total of 109/501 isolates exhibited sulfonamide resistance. The relative prevalences of sul genes from the three reservoirs (pigs, pig carcasses and humans) were 65%, 45% and 12% for sul2, sul1, and sul3, respectively. Transfer of resistance through conjugation was observed in 42/57 isolates. Resistances to streptomycin, ampicillin and trimethoprim were co-transferred in most strains. Class 1 integrons were present in 80% of sul1-carrying plasmids and 100% of sul3-carrying plasmids, but only in 5% of sul2-carrying plasmids. The sul plasmids ranged from 33 to 160-kb in size and belonged to nine different incompatibility (Inc) groups: FII, FIB, I1, FIA, B/O, FIC, N, HI1 and X1. IncFII was the dominant type in sul2-carrying plasmids (52%), while IncI1 was the most common type in sul1 and sul3-carrying plasmids (33% and 45%, respectively). Multireplicons were found associated with all three sul genes.

Conclusions

Sul genes were distributed widely in E. coli isolated from pigs and humans with sul2 being most prevalent. Sul-carrying plasmids belonged to diverse replicon types, but most of detected plasmids were conjugative enabling horizontal transfer. IncFII seems to be the dominant replicon type in sul2-carrying plasmids from all three sources.     

河北省犊牛腹泻大肠杆菌致病性及耐药性分析

为确定河北省犊牛腹泻大肠杆菌的致病性与耐药性,本研究对2018年1月至2019年6月期间从河北省石家庄、保定、承德、唐山、廊坊的部分肉牛场腹泻犊牛样品中分离到的50株大肠杆菌进行了致病性试验、药物敏感性试验、毒力基因和耐药基因检测。结果显示:50株分离菌均对小鼠具有致病性,致病菌占比100%(50/50)。毒力基因fyuA、irp2、eaeA、ler检出率分别为68.0%、66.0%、34.0%、34.0%。50株分离菌均对15种抗生素中的2种及以上表现为耐药,对10种及以上抗生素耐药的菌株占比达34%(17/50);分离菌对土霉素、磺胺对甲氧嘧啶、磺胺间甲氧嘧啶、替米考星4种药物表现为高度耐药,耐药率分别为90%、90%、94%、100%。除四环素类tetD基因检出率为0外,其它耐药基因均有检出,其中四环素类tetC、氨基糖苷类aadA1、喹诺酮类gyrA、gyrB基因检测率高达100%。试验的50株大肠杆菌均具有较强的毒力和多重耐药性。本研究为河北省大肠杆菌所致犊牛腹泻病的防治提供了实验依据。     

内蒙古地区羊源大肠杆菌耐药性研究

为确定内蒙古地区羊源大肠杆菌的耐药表型及其耐药基因的流行情况,本研究采用微量稀释法测定了内蒙古地区108株羊源大肠杆菌对13种临床常用抗菌药物的最小抑菌浓度。结果显示,分离菌株对阿莫西林、头孢噻吩、磺胺甲唑、黏菌素的耐药率最高,均达100.0%,对阿莫西林/克拉维酸、四环素、环丙沙星的耐药率在50%~80%之间,对头孢噻肟、美洛培南、新霉素的耐药率均低于10%,较为敏感。108株羊源大肠杆菌中耐7种以上药物的菌株占94.4%,其中15.6%菌株对13种抗菌药物耐药,只有1株菌对所有抗菌药物敏感。采用PCR方法对羊源大肠杆菌分离株所携带的6种相关耐药基因进行检测,结果显示,6种耐药基因中的4种耐药基因bla_TEM、proP-2、sul-Ⅰ、ampG检出率超过50%,耐药基因aph（3'）-Ⅰ携带率达40%,只有耐药基因aac（3）-Ⅱ检出率仅为5.5%。由此可见,内蒙古地区羊源大肠杆菌对13种抗菌药物产生了不同程度的耐药性,且存在严重的多重耐药情况,羊源大肠杆菌分离株携带aph（3'）-Ⅰ、sul-Ⅰ、ampG、bla_TEM、proP-2、aac（3）-Ⅱ耐药基因。     

Study on the Drug-resistance of Escherichia coli Isolated from Sheep in Inner Mongolia

In order to determine the drug resistance phenotype and prevalence of drug resistance genes in Escherichia coli (E.coli) isolated from sheep in Inner Mongolia, the minimum inhibitory concentrations (MICs) of the isolates to antibiotics commonly used in veterinary clinical were detected by micro-dilution method in vitro. The results showed that the highest resistance rates of the isolates to amoxicillin,cephalothin,sulfamethoxazole and polymyxin were up to 100.0%,respectively.To amoxicillin-clavulanic, tetracycline and ciprofloxacin were 50% to 80%. These isolates were sensitive to cefotaxime, meropenem trihydrate, neomycin, and their resistance rates were all less than 10%. Among the 108 strains of E.coli from sheep, 94.4% of them were resistant to more than 7 antimicrobial agents,15.6% of them were resistant to 13 antimicrobial agents, only one strain was sensitive to all antimicrobial agents. Six kinds of resistance genes among the 108 E.coli isolates were detected by PCR method.The results showed that detection rates of 4 kinds of drug resistance genes including bla_TEM, proP-2, sul-Ⅰ and ampG were all over 50%,the detection rate of resistance gene aph (3')-Ⅰ was up to 40%, only resistance gene aac(3)-Ⅱ detection rate was 5.5%. Thus, the sheep E.coli isolates in Inner Mongolia produced various degrees of resistance to 13 kinds of antibiotics, and their multi-drug resistances were very serious. They carried aph(3')-Ⅰ, sul-Ⅰ, ampG, bla_TEM, proP-2 and aac(3)-Ⅱ resistance genes.     

鸡源致病性大肠埃希菌中氨基糖苷类抗生素耐药基因的检测

为了解鸡源致病性大肠埃希菌对氨基糖苷类抗生素的耐药性变化和钝化酶耐药基因的携带情况及耐药基因与耐药性的相关性,从陕西、河南、河北、山西、宁夏和甘肃6省(区)的部分规模化养鸡场的病、死鸡中分离鉴定320株致病性大肠埃希菌。采用K-B药敏纸片法检测分离菌对6种氨基糖苷类药物的敏感性,PCR方法检测6种氨基糖苷类钝化酶耐药基因,用DNA Star软件对获得的耐药基因序列与GenBank中的相关序列进行比对。结果显示,鸡源致病性大肠埃希菌分离株对庆大霉素、链霉素、妥布霉素、卡那霉素、新霉素和阿米卡星的耐药率分别为53.4%、49.3%、37.5%、34.7%、22.8%和5.3%,对妥布霉素和卡那霉素耐药率呈上升趋势,而对庆大霉素的耐药率虽呈下降趋势,但仍维持在40%以上。3重以上耐药菌株占80%(256/320)。氨基糖苷类钝化酶基因aac(3)-Ⅱ、aph(3′)-Ⅰ和aac(6′)-Ⅰ的检出率分别为50.9%、25.9%和3.1%,未检测到aac(3)-Ⅳ、ant(3′′)-Ⅰ和aph(3′)-Ⅱ基因。研究表明,分离的鸡源致病性大肠埃希菌对氨基糖苷类抗生素的耐药性普遍存在,以多重耐药为主,且对妥布霉素和卡那霉素的耐药性不断上升。耐药基因aac(3)-Ⅱ和aph(3′)-Ⅰ的检出率与其耐药性呈正相关。     

鸭源多杀性巴氏杆菌对磺胺类药物的耐药性检测及耐药基因分析

为明确鸭源多杀性巴氏杆菌对磺胺类药物的耐药性及耐药基因的分布特征,试验采用琼脂二倍稀释法对分离自福建、广东和江西等地的89株鸭源多杀性巴氏杆菌进行甲氧苄啶/磺胺甲噁唑的耐受性检测,用PCR法检测各耐药菌株的磺胺类耐药基因(sul1、sul2和sul3),并利用多位点序列分型(MLST)分析耐药菌株间的亲缘关系。结果显示,33株鸭源多杀性巴氏杆菌对甲氧苄啶/磺胺甲噁唑耐受,耐药率为37.1%(33/89);在耐药菌株中,sul1、sul2和sul3基因检出率分别为100%(33/33)、97.0%(32/33)和21.2%(7/33),其中sul1和sul2基因同时检出率为97.0%(32/33),sul1、sul2和sul3基因同时检出率为21.2%(7/33);所有耐药菌株均属于ST129型。以上结果表明,鸭源多杀性巴氏杆菌对磺胺类药物具有一定的耐药性,其中的耐药菌株都属于同一序列型ST129,均携带有sul1耐药基因,且绝大多数耐药菌株同时携带sul1与sul2两种耐药基因,提示在福建、广东和江西等地需慎用磺胺类药物来防治该细菌的感染。