禽源致病性沙门氏菌耐药表型与耐药基因的分析

为了解江苏苏中地区禽源沙门氏菌耐药现象及耐药基因的存在情况,采用K-B法对22株沙门氏菌分离株进行12种抗菌药物敏感性试验,并通过PCR方法检测了12种耐药基因的存在情况。结果显示,所有菌株对甲氧胺嘧啶、阿米卡星、庆大霉素、萘啶酮酸、诺氟沙星的耐药率均在72.73%～100%之间;存在着5种耐药表型,每个耐药表型均在4重或4重以上,耐药最多菌株可耐受10种药物;扩增出9种耐药基因,与GenBank中的参考序列同源性均在98%以上;耐药表型与耐药基因的存在基本一致,但无绝对相关性。本研究结果表明苏中地区沙门氏菌耐药现象非常严重,且耐药基因广泛存在。     

新疆猪源沙门氏菌耐药性及耐药基因检测

为了解新疆某规模化养殖场猪源沙门氏菌对临床上常用抗菌药物的耐药情况,以及β-内酰胺酶、16S rRNA甲基化酶和质粒介导的喹诺酮耐药（plasmid mediated quinolone resistance,PMQR）基因的流行情况,本试验通过琼脂稀释法对分离的菌株进行最小抑菌浓度测定,PCR方法进行β-内酰胺酶bla_TEM、bla_CMY-2、bla_CTX-M、bla_LAP-1、bla_KPC、bla_OXA和bla_SHV基因,16S rRNA甲基化酶基因armA和rmtB及PMQR类基因qnrA、qnrB、qnrC、qnrD、qnrS、qepA、oqxA、oqxB和aac（6'）-Ib-cr的检测,确定阳性菌株,分析其携带的基因型与耐药表型之间的关系。分离的猪源沙门氏菌对环丙沙星和安普霉素耐药率最高,均为77.9%（102/131）,耐药菌主要以8耐为主（29.0%,38/131）。从被检基因中检测出11种耐药基因,不同基因共存有24种类型,主要以bla_TEM+bla_OXA+qnrS+aac（6'）-Ib-cr+oqxA+oqxB（27.6%,32/116）;bla_TEM+bla_OXA+qnrS+oqxA+oqxB（24.1%,28/116）;bla_TEM+qnrS（18.0%,21/116）形式共存。该养殖场分离的沙门氏菌耐药现象严重,分离耐药菌株存在β-内酰胺酶、16S rRNA甲基化酶和PMQR类基因共存现象,提示应加强对β-内酰胺酶、16S rRNA甲基化酶和PMQR类因子的监控。     

京津冀地区猪源沙门氏菌耐药性及耐药基因分析

研究旨在为防控沙门氏菌引起的猪疾病以及指导猪场用药提供一定参考。试验对京津冀地区猪饲料厂、猪养殖场、猪屠宰场、超市、农贸市场等采集的饲料、猪肛拭子、水、猪肉等样品中沙门氏菌进行分离鉴定,对50株7类以上药物耐药的菌株进行全基因组测序,分析其耐药基因分布情况。对分离筛选得到沙门氏菌菌株通过抗菌药物浓度梯度法测定其对22种抗菌药物的敏感性,结合全基因组测序结果,分析不同来源沙门氏菌耐药基因。结果显示：5 024份样品中共检出沙门氏菌484株,平均检出率为9.63%;484株沙门氏菌对22种抗菌药物的耐药性检测,耐药率是86.16%。耐药率大于30%的抗菌药物有庆大霉素（30.8%）、阿莫西林（31.6%）、四环素（36.8%）、多西环素（33.5%）、萘啶酸（31.8%）。对耐受7种以上抗菌药物的50株沙门氏菌进行全基因组测序,并对耐药基因进行分析,共检出43种耐药基因,其中aac6’-Iaa、aadA1、aph(3’)-Ia、aph(3’’)-Ib、aph3’-Iia、aac3-Iid、mcr-1、tet(B)、sul1、sul2、gyrA、floR、dfrA12、aac(6’)-Ib-...     

规模化猪场猪源沙门氏菌的耐药性及相关耐药基因检测

为了解贵州省某规模化猪场猪源沙门氏菌分离株的药物敏感性和耐药基因的存在情况,也为进一步研究细菌耐药的分子机制和临床用药提供资料,利用K-B法检测了6株猪源沙门氏菌对15种药物的敏感性,采用PCR方法检测了9种常见耐药基因在分离株中的分布情况。结果显示,所有菌株对青霉素、氨苄西林和吡哌酸的耐药率最高均为100%,对头孢克洛、链霉素和庆大霉素的耐药率最低。从9种常见耐药基因中扩增到3种β-内酰胺类耐药、3种氨基糖苷类、2种氟喹诺酮耐药基因。研究表明,沙门氏菌极易产生耐药性,耐药基因广泛存在于耐药菌株中,但耐药表型与耐药基因之间无绝对相关性。     

鸡源性沙门氏菌耐药基因检测与耐药相关性分析

为研究沙门氏菌耐药基因与其耐药性之间的关系,本研究以K-B纸片法对临床分离的29株鸡源性沙门氏菌进行10种抗菌药物敏感性测定,应用PCR技术进行这些抗菌药物相关耐药基因检测.并通过质粒接合试验证明了耐药性及耐药基因的转移.结果显示:29株分离株对氨苄西林耐药率为31.0％,对头孢唑啉耐药率为10.3％,对四环素耐药率为44.8％,对复合磺胺耐药率为41.4％,对氯霉素耐药率为3.4％,对环丙沙星、氧氟沙星、诺氟沙星的耐药率均为37.9％,但对庆大霉素和卡那霉素均敏感.本实验共检测到8种耐药基因,其中blatem-1阳性率为31.0％,tetA和tetB阳性率为44.8％,sull和sul2阳性率为41.3％,aadAl阳性率为3.4％,dfrA1阳性率为10.3％,qnr阳性率为37.9％,而tetG、blaCMY-2和catI均未检出.此外,通过与非耐药菌接合后绝大部分接合子均获得了供体菌质粒编码的相应抗性表型,耐药基因在接合过程也发生了转移,赋予被接合细菌新的耐药性.以上结果表明沙门氏菌的耐药性与其相关耐药基因的检出率基本一致,而且耐药性通过质粒进行转移.     

猪源致病性沙门氏菌四环素耐药基因tetC的PCR扩增及序列分析

四环素类抗生素作为一类广谱抗生素,现已产生严重的耐药性.作为人兽共患病原菌的沙门氏菌,也存在大量这样的耐药菌株,使得本病的发病率和死亡率都不断上升.本研究对规模化猪场分离的经动物试验和生化试验鉴定的致病性沙门氏菌16株、药敏质控菌ATCC25922和沙门氏菌标准株C79-13对进行了四环素、金霉素、土霉素的药敏试验,结果表明16株致病性沙门氏菌对四环素类抗生素表现出普遍耐受性,耐药率达100%.其中MIC值＞128μg/mL的高耐药菌株13株,高耐药率81.25%;MIC值为32μg/mL的低耐药菌株3株,低耐药率18.75%.设计沙门氏菌四环素抗性基因tetC的引物,对16株致病性沙门氏菌的tetC基因做PCR扩增,结果12株菌获得以质粒为模板长约400bp的特异性扩增产物,未能从染色体扩增到产物.分别对临床分离的1株低耐药菌株(DY1)和1株高耐药菌株(SL1-3)的tetC基因扩增产物进行序列分析,结果表明菌株DY1和SL1-3的tetC的核苷酸同源率为97.7%;菌株DY1与质粒PBR322中tetC的核苷酸同源率为98.7%;菌株SL1-3与质粒pBR322中tetC的核苷酸同源率为98.4%,说明对于耐药程度和地方来源各不相同的菌株,在核苷酸序列上同源率很高.本文用PCR技术对规模化猪场分离的致病性沙门氏菌的四环素耐药基因进行了研究,以期对四环素耐药性的分子流行病学进行监测,克服了药敏试验只能检测耐药表型的缺点,为有效控制沙门氏菌感染提供理论基础和科学依据,在兽医、食品卫生和公共卫生等方面具有重要的意义.     

鸡源性多重耐药沙门氏菌 类整合子与耐药基因研究

本试验旨在了解鸡源性沙门氏菌分离株多重耐药与Ⅰ类整合子及耐药基因的携带关系。采用K-B纸片法对29株鸡源性沙门氏菌分离菌株进行10种抗菌药物敏感试验;应用PCR技术对分离菌株进行Ⅰ类整合子及耐药基因检测。29株分离株中有13株对2种以上抗菌药物耐药,属于多重耐药株,氨苄西林-四环素-头孢唑啉-复合磺胺是主要多重耐药谱;13株多重耐药菌中有8株携带Ⅰ类整合子,blatem-1、tetA和tetB基因检出最高。结果表明沙门氏菌多重耐药性与整合子的携带之间关系密切,耐药表型测定结果与耐药基因检测结果基本一致。     

四环素耐药基因在鸡源沙门氏菌中的分布和传播

试验分析了84株鸡源沙门氏菌分离株的四环素耐药性,用PCR方法检测四环素耐药基因在分离株中的分布情况。结果显示,四环素耐药率为49%(41/84),鸡白痢和鸡伤寒沙门氏菌仅携带tet(A)基因(23/23),肠炎沙门氏菌和德尔卑沙门氏菌携带tet(A)(8/18)、tet(B)(17/18)或tet(G)(10/18)三种基因,tetC基因在这些沙门氏菌中都没有检测到。该类基因多数位于结合性质粒上,但是不在整合子范围内。     

贵州省猪源沙门氏菌对β-内酰胺类药耐药性及耐药基因分析

为了解贵州省猪源沙门氏菌对β-内酰胺类抗菌药物耐药性及其耐药基因的流行情况,本试验从贵州省9个地区规模养猪场中分离鉴定130株沙门氏菌,采用微量肉汤稀释法测定其对常用的8种β-内酰胺类抗菌药物的敏感性,并用PCR法对β-内酰胺酶耐药基因进行检测。结果显示,沙门氏菌对常用的β-内酰胺类抗菌药物耐药性十分严重,其中对头孢他啶的耐药率为100%,其次是氨苄西林和阿莫西林,耐药率分别为80.77%和76.15%,耐药率最低的是头孢噻呋和头孢氨苄,均为46.15%。所有菌株均为多重耐药,其中最少为二重,占总数的2.31%,最多为八重,占总数的4.62%,多重耐药主要集中在四至七重,占总数的88.46%。PCR结果显示,SHV耐药基因未检出,TEM、OXA、CTX-M 3种基因检出率分别是85%、75%和46%,细菌的耐药性与相关耐药基因的检出率基本呈正相关。结果表明,猪源沙门氏菌对β-内酰胺类药物具有普遍耐药性,其中头孢他啶尤为严重。TEM、OXA、CTX-M基因是贵州省猪源沙门氏菌主要耐药基因,临床日益严重的耐药现象与耐药基因的普遍存在有很大的关系。     

PCR和核酸探针检测猪源沙门氏菌四环素耐药基因tetC的研究

对沙门氏菌的四环素耐药性进行了检测，结果表明，沙门氏菌对四环素类抗生素产生了广泛的耐药性，耐药率达100％，对其四环素耐药基因tetC进行了扩增，结果获得以质粒为模板的特异性产物，与药敏试验结果阳性符合率75％，具有较高的检出率。用光生物素对PcR产物进行标记，制备核酸探针，采用菌落原位杂交的方法对沙门氏菌进行检测，结果表明13株阳性、3株阴性，杂交结果与PCR结果阳性符合率为93．75％，具有较高的特异性。通过条件的优化，建立的四环素耐药基因tetC的PCR和核酸探针检测技术，为四环素耐药性的分子流行病学监测提供了有效的途径。     

Detection of Resistance and Resistance Genes of Salmonella from Swine in Xinjiang

The objective of this study was to investigate the resistance of Salmonella and prevalence of resistance genes isolated from a pig farm in Xinjiang, and their coexistence with the major β-lactamases, 16S rRNA methylation enzyme genes and PMQR. The minimum inhibitory concentrations of Salmonella isolated from the pig farms were determined by agar dilution method, PCR was used to detect bla_TEM, bla_CMY-2, bla_CTX-M, bla_LAP-1, bla_KPC, bla_OXA and bla_SHV genes, 16S rRNA methylation enzyme genes armA and rmtB, and PMQR including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, oqxA, oqxB and aac(6')-Ib genes. The positive strains were performed by using DNA sequencing to determine the purpose of the belt. The result showed that resistant rate of Salmonella isolates from swine were highest to ciprofloxacin and apramycin sulfate (77.9%, 102/131). Resistant mainly based on 8 kinds of drug resistance (29.0%, 38/131), 11 kinds of resistance genes were detection, the coexistence of different genotypes had 24 types. The main types were bla_TEM+bla_OXA+qnrS+aac(6')-Ib-cr+oqxA+oqxB (27.6%, 32/116), bla_TEM+bla_OXA+qnrS+oqxA+oqxB (24.1%, 28/116) and bla_TEM+qnrS (18.0%, 21/116). The Salmonella isolated from the farm had phenomenon seriously resistance, it coexisted with the main β-lactamase, 16S rRNA methylation enzyme genes and PMQR factors. The result suggested that it should strengthen monitoring to the β-lactamase enzymes, 16S rRNA methylation enzyme genes and PMQR factors.     

Analysis of Antimicrobial Resistance and Detection of ESBLs Genes of Pathogenic Salmonella Isolates from Dairy Cows in Inner Mongolia

The aim of the present study was to investigate the characteristics of antimicrobial resistance and prevalence of ESBLs genes of Salmonella isolates from dairy cows in Inner Mongolia.Micro-dilution broth method recommended by CLSI was used to determine the MICs of 22 antimicrobial agents commonly employed in veterinary clinic against the 38 Salmonella isolates.PCR analysis was carried out to detect the presence of 8 ESBLs genes mostly observed in animal origin Salmonella in the isolates with resistance to at least one β-lactam antibiotics, and the features of multi-resistance of the isolates were also analyzed.The results showed that most Salmonella isolates were resistant to trimethoprim(97.4%), sulfamethoxazole(94.7%), and to most of the β-lactam, aminoglycosides, chloramphenicol and tetracycline antibiotics with the resistance rate ranging from 40% to 80%, however, all of the isolates were sensitive to fluoroquinolone drugs.76.3%(n＝29) of the isolates were simultaneously resistant to 6 categories antibiotics and the multi-resistance rate was 94.7%. 85.7%(n＝30) of the β-lactam antibiotics resistant isolates were ESBLs genes positive.Of the six genotype of ESBLs genes found in this study, CTX-M (40.0%) was the mostly encountered ESBLs gene, however, the SHV and PSE types gene were absent in the isolates.A total of fifteen genotypes of the ESBLs genes and nine ESBLs genes combinations were observed in this study, and 16 isolates (45.7%) simultaneously contained two or more than two ESBLs genes.It was indicated that the current situation of the resistance of the Salmonella isolates from dairy cows in Inner Mongolia to the commonly used antimicrobial agents in veterinary clinic was serious, and the prevalence pattern of the ESBLs genes in the isolates was complex, suggesting that the irrational employment of the antibiotic in the treatment of animal infectious diseases was the crucial reason causing the antimicrobial resistance of the isolates.     

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。     

活禽产业链中大肠杆菌耐药性及耐药基因调查

为了解家庭农场供货模式下"养殖-销售"活禽产业链中各环节大肠杆菌耐药性及耐药基因流行情况,本研究共采集132份来自自贡市沿滩区某一农贸市场及其活禽供货的33家家庭农场的样品,通过细菌分离纯化、形态学鉴定、特异性引物PCR检测、系统发育群PCR检测、药敏试验及耐药基因鉴定等方法对细菌特性进行分析。研究结果显示,83株分离株在伊红美蓝培养基上形成带有金属光泽的黑色菌落,镜检显示为革兰阴性短杆菌,且特异性引物检测结果均显示阳性,因此,确定83株分离株均为大肠杆菌。系统发育群鉴定结果发现,83株分离株系统发育群集中于A群（53.02%）及B1群（20.48%）;药敏试验结果显示,83株菌株均为多重耐药菌,其中硫酸黏杆菌素、四环素耐药率最高,均为100%,氨苄西林、头孢呋辛、氨曲南、氟苯尼考耐药率均超过90%;耐药基因结果显示,mcr-1、mcr-3、bla_TEM、bla_SHV、bla_{CTX-M-group 1}、bla_{CTX-M-group 9}、qnrS、aac（6'）-Ⅰb-cr、tetA和tetB等耐药基因均有检出,检出率均低于对应抗菌药的耐药率,符合预期结果。此外,统计结果显示,家庭农场细菌耐药率和耐药基因携带率低于农贸市场。本研究结果表明,农贸市场成为活禽产业链中耐药菌及耐药基因的集散地,可为指导禽大肠杆菌临床检测、诊断及合理用药提供试验依据,同时为评估活禽产业链耐药性传播风险提供可靠数据。     

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.     

空肠弯曲杆菌耐药性及其主要相关基因初步分析

从307份鸡盲肠内容物样品中分离到47株空肠弯曲杆菌,并测定这些空肠弯曲杆菌耐药性,结果显示对喹诺酮类药(环丙沙星)耐药性为31.9%;对氨苄青霉素,克林霉素和红霉素耐药性较高,分别达到85.1%、83.0%和83.0%;而对痢特灵和庆大霉素则较为敏感,敏感度分别为85.1%和74.5%。有89.4%的菌株显示多重耐药性。利用MAMAPCR技术,对获得的47株空肠弯曲杆菌进行检测,结果显示对环丙沙星耐药的15株空肠弯曲杆菌均检测出其在gyrA基因257位发生点突变,30株对喹诺酮药物敏感的菌株均未检出。另外2株对环丙沙星耐药的菌株,有1株检测出点突变。     

马乳源大肠杆菌的毒力基因检测和耐药性分析

[目的] 了解新疆伊犁地区某马场乳源大肠杆菌的毒力基因携带情况和药物敏感性。[方法] 对采集到的85份马乳进行大肠杆菌的分离纯化、染色镜检、特异性基因phoA的扩增和16S rDNA测序;采用K-B纸片扩散法药敏试验分析马乳源大肠杆菌的耐药性;采用PCR技术检测马乳源大肠杆菌携带的毒力基因、耐药基因及鉴定其所属系统发育群,对大肠杆菌进行生物被膜形成能力检测。[结果] 从85份马乳中分离得到6株大肠杆菌,其中3株为A群,3株为B1群;6株大肠杆菌均对青霉素和替米考星耐药;均携带ibeB、yijP、mat、sodA和csgA毒力基因;均未检测到耐药基因;有4株具有生物膜形成能力。[结论] 对新疆伊犁地区某马场马乳源大肠杆菌进行初步的毒力基因检测和耐药性分析,发现其对青霉素和替米考星耐药严重,并携带多种毒力基因,具有一定的潜在致病风险。     

Antimicrobial Resistance Genes in Pigeons from Public Parks in Costa Rica

Antimicrobial resistance is known to be an emerging problem, but the extent of the issue remains incomplete. The aim of this study was to determine the presence or absence of nine resistance genes (bla_TEM, catI, mecA, qnrS, sulI, sulII, tet(A), tet(Q), vanA) in the faeces of 141 pigeons from four urban parks in Alajuela, Guadalupe, Tres Ríos and San José in Costa Rica. The genes were identified by real‐time PCR directly from enema samples. About 30% of the samples were positive for genes catI and sulI; between 13% and 17% were positive for qnrS, sulII, tet(A) and tet(Q); and 4% were positive for bla_TEM. The mecA and vanA genes were not detected. The average of antimicrobial resistance genes detected per pigeon was 2. Eight different patterns of resistance were identified, without differences in the sampling areas, being the most common pattern 2 (sulII positive samples). During rainy season, the genes more frequently found were sulI and tet(A). In conclusion, the urban inhabiting pigeons tested are currently carrying antimicrobial resistance genes, potentially acting as reservoirs of resistant bacteria and vectors to humans. To the authors’ knowledge, this is the first study carried out on direct detection of resistance genes in the digestive metagenomes of pigeons.