鸡舍内外环境中气载大肠杆菌同源性的分子鉴定

采用ANDERSEN-6级空气微生物样品收集器和RCS离心式采样器在5个鸡场舍内空气、舍外上风向和下风向不同距离收集气载大肠杆菌;并收集鸡的粪便,分离大肠杆菌。利用大肠杆菌基因间重复一致序列为引物的聚合酶链式反应（ERIC-PCR）分型技术,扩增不同测量点收集的大肠杆菌的DNA图谱。通过每个采样点的大肠杆菌的浓度变化以及大肠杆菌遗传相似性分析确认动物舍微生物气溶胶向舍外环境的传播。结果显示：5个鸡舍内空气中大肠杆菌的浓度远远高于舍外上风和下风向的大肠杆菌浓度（P〈0.05或P〈0.01）,但是舍外不同距离间的大肠杆菌浓度差异并不显著（P〉0.05）。同样,ERIC-PCR结果表明,从鸡的粪便中分离的大肠杆菌与从舍内空气中分离的部分大肠杆菌（34.1%）,以及从鸡场舍外下风方向分离到的多数大肠杆菌（54.5%）与从舍内空气或粪便中分离的大肠杆菌相似性可达100%。而从鸡舍上风向分离到的大肠杆菌与从舍内空气或粪便中分离的大肠杆菌相似性为73%-92%。从而说明来自动物体的大肠杆菌既能污染舍内空气,又能对其周围的环境构成污染。本研究揭示了微生物气溶胶的传播规律,具有公共卫生及流行病学意义。     

ERIC-PCR和PFGE对大肠埃希菌由舍内向舍外传播的鉴定及两种方法的比较

为了深入认识鸡舍环境微生物气溶胶的产生及其向舍外环境的传播,同时比较ERIC-PCR和PFGE对细菌的基因分型结果,本研究以大肠埃希菌为指示菌,采用Andersen-6级空气微生物样品收集器,分别在两个鸡舍的舍内、舍外上风向10 m和50 m和下风向10、50、100、200、400 m的距离处收集空气样品,同时采集鸡的粪便,分离、鉴定大肠埃希菌。采用ERIC-PCR和PFGE两种方法对不同来源的大肠埃希菌分离株进行基因分型,根据每个采样点大肠埃希菌的相似性指数,确认动物舍内微生物气溶胶向舍外环境传播的模式,并比较这两种方法对大肠埃希菌的基因分型结果,分析这两种分子生物学分型方法各自的优缺点和适应性。本试验共采集到28株大肠埃希菌,ERIC-PCR和PFGE两种方法分别进行同源性鉴定显示,从鸡的粪便中分离到的大肠埃希菌与从舍内空气中分离到的部分大肠埃希菌(62.5%)具有相同来源;从鸡场舍外下风方向分离到的多数大肠埃希菌(20%)与舍内空气或粪便中分离的大肠埃希菌来源相同。而从鸡舍上风没有分离到大肠埃希菌,排除了下风向采得的大肠埃希菌来自上风向的可能。而很多从舍内空气和舍外下风方向分离到的大肠埃希菌与粪便中的具有相同来源,说明粪便中的细菌能够形成气溶胶,并且通过舍内外气体交换传播到舍外,依气象条件传播到舍外不同的距离。造成周边环境的生物污染以及病原微生物的扩散。本研究同时对ERIC-PCR和PFGE两种方法所得的分型结果进行了比较。PFGE的分辨率高于ERIC-PCR,而且PFGE的重复性也很高,不同试验室得出的结果可以相互比较,是细菌基因分型的"金标准"。但是PFGE试验费用高,需要时间长,对试剂、仪器和操作都具有很高的要求;ERIC-PCR虽然分辨率不及PFGE,但是ERIC-PCR简单、快速,成本低,对操作和试剂以及仪器要求都不高,所以ERIC-PCR也可以对肠杆菌进行同源性鉴定,应用于流行病学调查和对气源性微生物传播的鉴定。由于ERIC-PCR和PF-GE是根据DNA中不同的遗传特征进行分类的,所以这两种分型技术结合起来分析菌株的遗传进化关系,意义更大。     

猪场环境大肠杆菌qnrA和aac(6') - Ib基因检测

为了解质粒介导的喹诺酮类抗生素耐药基因qnrA、aac(6’)- Ib - cr在猪场环境大肠杆菌的流行分布及变异情况,从贵州省规模养猪场空气、污水、粪便和土壤样本中分离、鉴定大肠杆菌57株,PCR检测qnrA和aac(6’)- Ib基因,测序分析aac(6’)- Ib - cr基因变异体,并用药敏纸片琼脂扩散法测其对...     

麻旺鸭源大肠杆菌耐药表型分析及β-内酰胺酶、PMQR基因检测

本研究旨在了解麻旺鸭源大肠杆菌对β-内酰胺类、氨基糖苷类、四环素类、酰氨醇类、氟喹诺酮类和磺胺类药物的耐药情况以及β-内酰胺酶基因和质粒介导的喹诺酮耐药(plasmid-mediated quinolone resistance, PMQR)基因流行情况。从重庆市酉阳县4个麻旺鸭养殖场采集146份泄殖腔拭子样品,经选择性增菌和PCR扩增大肠杆菌特异性phoA基因分离鉴定大肠杆菌。采用纸片扩散法测定分离菌对25种抗菌药物的耐药性并检测分离菌株中的β-内酰胺酶基因和PMQR基因。结果显示：共分离到大肠杆菌146株。这些菌株对氨苄西林、头孢唑啉耐药严重,耐药率超过50%;对庆大霉素、阿米卡星、氧氟沙星完全敏感。分别有111株(76.0%)和83株(56.8%)菌携带至少1种β-内酰胺酶基因和PMQR基因,其中bla_TEM(74.0%,108/146)和qnrS(54.8%,80/146)基因最为流行。63株菌同时携带β-内酰胺酶基因和PMQR基因。麻旺鸭源大肠杆菌对常用抗菌药物存在耐药,且广泛携带β-内酰胺酶基因和PMQR基因。     

猪场环境大肠杆菌qnrA和aac(6’)-Ib基因检测

为了解质粒介导的喹诺酮类抗生素耐药基因qnrA、aac(6’)-Ib-cr在猪场环境大肠杆菌的流行分布及变异情况,从贵州省规模养猪场空气、污水、粪便和土壤样本中分离、鉴定大肠杆菌57株,PCR检测qnrA和aac(6’)-Ib基因,测序分析aac(6’)-Ib-cr基因变异体,并用药敏纸片琼脂扩散法测其对喹诺酮类和氨基糖苷类的耐药性。结果表明:57株大肠杆菌均未携带qnrA基因;8株检出aac(6’)-Ib基因,分别来自污水、粪便和土壤样本,且介导对喹诺酮类和氨基糖苷类抗生素的广泛耐药性。E.coli3和E.coli4在第75(A)、76(G)或488位(A)碱基缺失;E.coli5在第222位(A→T)变异,E.coli1、E.coli2、E.coli3、E.coli4、E.coli5、E.coli7和E.coli8在223位(T→C)和454位(G→T)变异;E.coli3、E.coli4在第492位有A、C碱基增添。说明贵州省规模养猪场肠杆菌科细菌qnrA基因携带率较低,但存在aac(6’)-Ib-cr基因。     

牛源大肠杆菌质粒介导喹诺酮类耐药基因的检测分析

为研究近年来新疆地区牛源大肠杆菌中质粒介导喹诺酮类药物耐药基因的分布及其对喹诺酮类抗生素的耐药情况,本研究于2016-2018年从新疆石河子、沙湾、奎屯、玛纳斯和伊犁5个地区12个规模化奶牛场分离出116株牛源大肠杆菌,药敏试验检测其耐药性,同时利用PCR扩增PMQR耐药基因。药敏试验结果显示,62.93%的菌株对氨苄西林耐药,耐药率最高。对链霉素、四环素、卡那霉素和恩诺沙星的耐药率依次为56.90%、54.31%、43.10%和42.24%。对头孢他啶和头孢噻肟的耐药率较低,分别为7.76%和11.21%。分离菌主要携带qnrA、qnrS和aac（6'）-Ⅰb-cr 3种耐药基因;116株大肠杆菌中有31株携带PMQR的耐药基因,检出阳性率为26.72%,其中26株仅携带1种PMQR耐药基因,占所有菌株的22.41%,4株携带2种PMQR耐药基因,占所有菌株的3.45%,1株携带3种PMQR耐药基因,占所有菌株的0.86%。综上所述,新疆地区牛源大肠杆菌质粒介导喹诺酮类药物基因主要为qnrA、qnrS和aac（6'）-Ⅰb-cr 3种,且对恩诺沙星、诺氟沙星、环丙沙星、左氧氟沙星均产生不同程度的耐药性。     

猪圈及其周边社区环境大肠埃希菌气溶胶产生及其传播

目的是以大肠埃希菌为指示菌研究猪舍环境中微生物气溶胶产生及向其舍外环境的传播。采用Andersen-6级空气微生物样品收集器和RCS-离心式采样器分别在4个猪场舍内、舍外不同距离收集大肠埃希菌气溶胶,计算每个采样点大肠埃希菌的浓度;同时,采集猪的粪便,分离大肠埃希菌。利用ERIC-PCR技术,扩增其DNA条带,形成聚类图谱。通过每一个采样点大肠埃希菌遗传相似性分析及其浓度变化的比较,确认动物舍微生物气溶胶向舍外环境的传播模式。结果表明,4个猪场舍内空气中大肠埃希菌的浓度远远高于舍外大肠埃希菌浓度(P0.05)。52.4%粪便大肠埃希菌与舍内空气中的大肠埃希菌相似性可达90%以上;从猪场舍外下风方向分离到的多数大肠埃希菌(55%)与舍内空气或粪便中分离的大肠埃希菌相似性可达90%以上。说明源于猪舍的微生物气溶胶能够传播到舍外环境或居民社区,具有流行病学及公共卫生意义。     

潍坊市肉鸭源大肠杆菌喹诺酮类药物耐药性的检测

为研究近年来山东省肉鸭源致病性大肠杆菌对喹诺酮类药物的耐药性,本研究将2010年以来分离自山东省潍坊市发病肉鸭的232株大肠杆菌进行了洛美沙星、培氟沙星、氧氟沙星、诺氟沙星等4种喹诺酮类药物的药敏试验,并对其进行了质粒介导喹诺酮类药物耐药基因的PCR检测。结果表明,山东省潍坊市肉鸭源大肠杆菌对4种喹诺酮类抗生素均产生了较高耐药性(54.31%~82.760%),质粒介导喹诺酮类耐药(PMQR)基因携带率达到58.19%(135/232),26.29%(61/232)的菌株携带两种产PMQR基因,1.72%(4/232)的菌株携带三种PMQR基因。未检测到qnr A、qnr B、qnr C、qnr D与Qep A基因,qnr S、oqx A和oqx B基因在山东省禽源致病性大肠杆菌中分布较为广泛,其检出率依次为19.83%(46/232)、41.81%(97/232)和26.29%(61/232)。     

重庆部分羊场沙门氏菌的分离鉴定、耐药表型分析及耐药基因检测

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。     

山东省养鸡场大肠杆菌多黏菌素耐药基因mcr-1的流行情况

2017-2018年,从山东省8个大型集约化养鸡场中共采集720份新鲜粪便样品,通过细菌分离鉴定共分离到697株非重复大肠杆菌。采用琼脂稀释法对所有菌株进行药敏试验;利用PCR扩增多黏菌素耐药基因(mcr-1);并对mcr-1阳性菌株扩增β-内酰胺类耐药基因(ESBL)和喹诺酮类耐药基因(PMQR)。结果显示,从697株大肠杆菌中共检测到83株(11.9%)携带mcr-1基因,其中mcr-1阳性菌株对氨苄西林的耐药率最高(82/83,98.8%),其次是阿莫西林(81/83,97.6%)和氟苯尼考(79/83,95.2%),但对阿莫西林/克拉维酸的耐药率较低(18/83,21.7%)。从mcr-1阳性菌株中共检测到5种ESBL基因,其中所有菌株均携带bla_TEM(83/83,100%),其次为bla_CTX-M(67/83,80.7%);检测到4种PMQR基因,其中aac(6′)-Ib-cr检出率最高(34/83,40.9%),其次为qnrA(9/83,10.8%)。结果表明,山东省禽源大肠杆菌是mcr-1基因的储存库,可能对人类公共健康造成威...     

Study on Aerotransmission of Escherichia coli Carrying the Plasmid-mediated Quinolone Resistance Genes of Swine

To investigate the aerotransmission of Escherichia coli (E.coli) carrying the plasmid-mediated quinolone resistance (PMQR) genes,airborne E.coli were isolated from indoor air,upwind air and downwind air samples in five swine farms.Fecal samples from swine houses were randomly collected to isolate the E.coli.The sensitivities of the E.coli strains against 14 antibiotics were tested.The E.coli carrying the PMQR genes (qnr,aac(6')-Ib-cr,qepA) were identified by ERIC-PCR,and then the genetic fingerprints of E.coli were established to analyze its origins and spread toward the outside surroundings.The results showed that E.coli isolated from five swine farms showed high resistance against 12 antibiotics,such as gentamicin,kanamycin,tetracycline,streptomycin,nalidixic acid and sulfamethoxazole,and presented multi-drug resistant.Results of ERIC-PCR showed that 46.34% (19/41) of strains isolated from indoor air samples had the same origin with fecal-obtained strains,and 73.68% (14/19) of them shared the same PMQR genes with fecal-obtained strains.68.42% (26/38) of strains isolated from downwind air samples had the same origin with fecal-obtained or indoor air-obtained strains,and 65.38% (17/26) of them shared the same PMQR genes with fecal-obtained or indoor air-obtained strains.This indicated that E.coli carrying PMQR genes and originating from feces in swine houses could form aerosols to pollute the indoor air and then spread to the downwind air through air exchange (≥400 m),which could be a potential threaten to public environment and human health.     

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。     

东南沿海地区水禽源大肠杆菌耐药表型及耐药基因型的调查

为研究水禽中大肠杆菌的耐药表型及耐药基因型,本研究从中国广东、福建、浙江、江苏和山东5个省市的水禽养殖场及周边环境中采集鸭粪便样品、水样以及土样1 505份,采用选择性培养基和基质辅助激光解吸电离飞行时间(MALDI-TOF-MS)质谱分离鉴定大肠杆菌,采用微量肉汤稀释法检测耐药表型,采用PCR方法筛选大肠杆菌中的25种耐药基因。结果显示,1 505份样品中共分离鉴定出449株大肠杆菌,分离率为29.8%,其中335份来源于粪便,52份来源于土壤,62份来源于水,这些样品中浙江(43.3%)和广东(43.2%)的分离率最高,其次是福建(28.1%)和江苏(23.8%),山东(18.3%)的分离率最低。449株大肠杆菌具有多重耐药性,对氨苄西林、环丙沙星、金霉素、氟苯尼考和磺胺类药物的耐药率分别为90.2%、50.3%、96.7%、87.3%和90.6%,而对替加环素和美罗培南则比较敏感,来源不同省份的大肠杆菌对不同抗菌药呈现出不同的耐药率。PCR检测结果发现,449株大肠杆菌中耐药基因tet(A)检出率最高(88.2%),随后是floR(58.13%)、sul2(57.02%)、tet(B)(53.9%)、cmlA(39.2%)和sul1(36.75%),未检测到qnrA、tet(M)和tet(X)耐药基因的大肠杆菌。在这5个省份中,上述耐药基因在山东省样品中分布均较高,而在浙江省和江苏省样品中分布较低。综上所述,本研究调查了中国东南沿海五省水禽及环境中大肠杆菌的耐药现状,为养殖业抗生素的规范使用提供理论依据。     

Study on Drug Resistance of Escherichia coli Isolated from Chicken and Swine in Organic Farm

To determine the drug resistance of E.coli strains from swine and chicken in organic farm,collection of stool specimens was conducted in one organic farm in Conghua city,Guangdong province in 2014,in which 118 E.coli strains were separated,including 55 from chicken and 63 from swine.The sensitivity of the E.coli strains from swine and chicken to 18 kinds of drugs was detected through agar dilution method.Similar properties in drug resistance were observed both in E.coli strains from swine and chicken in the same organic farm.In consideration of the resistance rate,samples were relatively sensitive to ceftazidime,cefquinome,cefoxitin,amikacin,apramycin,gentamicin,neomycin,florfenicol,chloramphenicol,imipenem,ciprofloxacin and olaquindox (chicken 0 to 21.8%;swine 0 to 14.3%).These isolates showed moderate resistance to ampicillin and streptomycin (chicken 29.1% to 38.2%;swine 23.8% to 27.0%).About 56.4% chicken isolates and 47.6% swine isolates showed resistant to more than 3 kinds of drugs.However,severe resistance was observed in trimethoprim-sulphamethoxazole,tetracyclineand and doxycycline (chicken 45.5%,76.4% and 72.7%;swine 50.8%,81.0% and 68.3%).The results also indicated that under organic farming mode,the drug resistance rates of E.coli strains from swine and chicken were relatively low in general.Multi-drug resistance existed but was relatively slight.Although the drug resistance rate to several specific antimicrobials was high,it was still lower than that in conventional farms.Since most studies focused on the drug resistance of the bacteria under conventional farming model,this study filled the blank of the research of drug resistance under organic farming model.     

有机农场鸡、猪源大肠杆菌耐药性研究

为了解有机养殖模式下鸡、猪源大肠杆菌的耐药情况,2014年从广东从化某有机农场鸡、猪粪便中采集并分离出118株大肠杆菌,包括55株鸡源大肠杆菌和63株猪源大肠杆菌,采用琼脂稀释法测定大肠杆菌对18种抗菌药的敏感性。结果发现,同一有机农场中,鸡源大肠杆菌与猪源大肠杆菌在耐药种类及耐药率上均呈现相似规律,对头孢他啶、头孢喹肟、头孢噻肟、头孢西丁、阿米卡星、安普霉素、庆大霉素、新霉素、氟苯尼考、氯霉素、亚胺培南、环丙沙星、喹乙醇等较敏感(鸡0~21.8%,猪0~14.3%),对氨苄西林、链霉素呈现轻度耐药(鸡29.1%~38.2%,猪23.8%~27.0%),对复方新诺明、四环素、多西环素呈现较严重耐药(鸡45.5%、76.4%和72.7%,猪50.8%、81.0%和68.3%)。鸡源大肠杆菌中3耐及3耐以上的菌株占56.4%,猪源大肠杆菌占47.6%。结果表明有机养殖模式下鸡、猪源大肠杆菌耐药率相对较低,多重耐药现象存在但相对轻微,对个别临床常用抗菌药耐药率虽较严重但比常规养殖场低。由于目前国内大部分学者致力于研究常规养殖方式下细菌耐药情况,本研究很好地填补了有机养殖模式下畜禽大肠杆菌耐药性研究的空白。     

Comparison of Drug Resistance of E.coli from Pigs in Different Regions under the Same Breeding Mode

In order to compare the resistance of E.coli from pigs of different regions under same breeding mode in Xinjiang, we collected fecal samples from pig farms of Hutubi (207), Manasi (210) and Changji (210) in a certain scale, respectively, a total of 627 samples, and isolation rate of E.coli from fecal samples were all 100.0%.The broth dilution method was used to detect resistance of E.coli to antimicrobials, and we compared the differences of resistance rate of E.coli from pig farms in three regions by chi square test. E.coli from pig farms of Hutubi region to apramycin, amikacin, amoxicillin-clavulanic acid and ceftiofur existed extremely significant differences comparing with another two regions (P< 0.01).E.coli from pig farms of Manasi region to ciprofloxacin existed significant differences comparing with another two regions (P< 0.05), and existed extremely significant differences to norfloxacin (P< 0.01).E.coli from pig farms of Changji region to norfloxacin had extremely significant differences comparing with Hutubi region (P< 0.01), and existed significant differences to amoxicillin-clavulanic acid and ampicillin comparing with Manasi region (P< 0.05).3 to 8 resistant E.coli strains accounted for 94.7% in Hutubi region, 3 to 7 resistant E.coli strains accounted for 89.5% in Manasi region and 4 to 8 resistant E.coli strains accounted for 82.4% in Changji region, respectively.There was no significant difference among different regions on multi-drug resistance (P> 0.05).The results indicated that E.coli from pigs of different regions under the same breeding mode had different characteristics of resistance.In addition, the resistance of E.coli from pigs were very serious and mainly multi-drug resistant, drug resistance patterns were diversified.     

同一养殖模式下不同地区猪源大肠杆菌耐药性比较

本试验旨在比较新疆相同养殖模式下不同地区猪源大肠杆菌的耐药情况。分别在某规模化养猪场呼图壁(207份)、玛纳斯(210份)和昌吉(210份)地区采集粪样,共计627份样品,各地区猪源大肠杆菌的分离率均为100.0%。采用微量肉汤稀释法对分离出的大肠杆菌进行临床常用抗菌药物的药敏试验,并通过卡方检验比较3个地区猪源大肠杆菌耐药率的差异。结果显示,呼图壁地区猪源大肠杆菌对安普霉素、阿米卡星、阿莫西林—克拉维酸和头孢噻呋的耐药率均极显著高于另外两地区(P< 0.01);玛纳斯地区猪源大肠杆菌对环丙沙星的耐药率显著高于另外两地区(P< 0.05),对诺氟沙星的耐药率极显著高于另外两地区(P< 0.01);昌吉地区猪源大肠杆菌对诺氟沙星的耐药率极显著高于呼图壁地区(P< 0.01),而对阿莫西林—克拉维酸和氨苄西林的耐药率均显著高于玛纳斯地区(P< 0.05)。呼图壁地区3~8耐菌株占94.7%,玛纳斯地区3~7耐菌株占89.5%,昌吉地区4~8耐菌株占82.4%。不同地区之间3~7耐菌株数差异不显著(P> 0.05)。结果表明,在相同的养殖模式下,不同地区猪源大肠杆菌的耐药情况不同。此外,养殖场猪源大肠杆菌的耐药问题严重,以多药耐药为主,耐药谱型呈多样化。     

Screening of Sensitive Drugs for Bovine Pathogenic Escherichia coli and Bacteriostatic Test of Sodium Humate

In order to get more information about the distribution of virulence genes and drug resistance genes of Escherichia coli(E.coli) isolated from calf diarrhea in some large-scale cattle farms in Heilongjiang,sensitive drugs were screened to analyze the preventive and therapeutic effect of sodium humate on E.coli.In this study,40 samples were collected and identified by separation and purification,staining microscopy,biochemical identification and molecular biology,and the virulence and drug resistance genes were detected by PCR and the drug sensitivity was detected by K-B test.The inhibitory effect of different concentrations of sodium humate on E.coli was measured by serial dilution and plate counts.Moreover,the protective effect of sodium humate on mice were studied through gastric administration.The results showed that 30 strains of E.coli were isolated,of which 25 strains had virulence genes and 8 virulence genes were detected out of 10,with detection rate of 83.3%.There were many virulence gene combinations.The highest detection rates for bla_TEM and bla_SHV genes were 100% and 13.3% respectively,and the detection rate of other resistance genes was also high.The results of drug sensitive test showed that 30 isolated strains were most sensitive to meropenem,followed by amikacin.The results of serial dilution and plate counts showed that 5% sodium humate was the most bacteriostatically effective,and the results of toxicity test showed that sodium humate could improve the survival rate of mice.This study provided a basis for the rational use of antibiotics and the clinical application of sodium humate in this area.     

广东鹅场大肠杆菌耐药状况及blaCTX-M基因的传播特征

为揭示广东地区鹅场动物和环境源大肠杆菌的耐药情况及超广谱β-内酰胺酶CTX-M的流行与传播特征,本研究从广东省江门及阳江市共10处鹅场采集鹅及环境样品199份,采用MALDI-TOF-MS法分离鉴定大肠杆菌。采用琼脂稀释法对菌株进行耐药性分析,采用PCR法检测头孢噻肟耐药菌中bla_CTX-M基因及其基因环境,采用脉冲场凝胶电泳（PFGE）、接合转移和质粒复制子分型等方法探究bla_CTX-M基因的传播特征。结果显示,共获得196株大肠杆菌,对氨苄西林、多西环素、氟苯尼考和链霉素耐药率均超过50%,第三代头孢菌素耐药率为10%~25%,其中头孢噻肟耐药菌有49株（24.6%）。阳江地区大肠杆菌对受试药物的耐药率高于江门,且动物源高于环境源,尤其是头孢噻肟和头孢噻呋均存在显著差异（P<0.05）。头孢噻肟耐药菌中共检出19株携带bla_CTX-M基因,包括bla_CTX-M-55（n=17）、bla_CTX-M-27（n=1）和bla_CTX-M-65（n=1）,且bla_CTX-M基因阳性菌均可对5~11种药物耐药,呈现多重耐药的表型。bla_CTX-M-55基因环境均为ISEcp1-bla_CTX-M-55-orf477,且在ISEcp1与bla_CTX-M基因之间有3种长度的间隔序列;而bla_CTX-M-27和bla_CTX-M-65的基因环境均为ISEcp1-bla_CTX-M-27/65-IS903。19株bla_CTX-M基因阳性菌呈现10种PFGE谱型,存在一种主要流行的谱型（47.4%）,其包括多种来源菌株,暗示存在克隆传播现象。12株（63.2%）bla_CTX-M基因阳性大肠杆菌中bla_CTX-M基因转移成功,bla_CTX-M基因阳性接合子携带的复制子型为IncFⅡ（n=10）和IncHⅠ2（n=2）,且存在多西环素和氟苯尼考耐药表型与bla_CTX-M基因共转移现象。研究发现,阳江鹅场大肠杆菌耐药情况较为严重,bla_CTX-M基因存在一定的流行性且以bla_CTX-M-55亚型为主,bla_CTX-M基因阳性菌的克隆传播和质粒及插入序列ISEcp1介导的水平传播是导致该基因在鹅场大肠杆菌中扩散的主要原因,应引起高度重视。     

京津冀部分地区致犊牛腹泻大肠杆菌毒力基因、耐药基因检测及药物敏感性分析

【目的】探明京津冀地区犊牛腹泻大肠杆菌毒力基因与耐药基因流行情况,筛选敏感药物。【方法】于2020年12月至2021年7月从京津冀地区部分牛场采集146份犊牛腹泻样本,通过细菌分离纯化、革兰氏染色镜检及16S rRNA测序进行大肠杆菌分离鉴定;采用PCR方法对分离菌进行毒力基因（F17、K99、F41、STa、stx1、irp2和fyuA基因）和耐药基因（aac（6'）-Ⅰb、bla_CTX-M、bla_TEM、OqxB、tetA和sul1基因）检测;采用K-B纸片法进行药物敏感性试验。【结果】分离菌在鉴别培养基上的生长形态及革兰氏染色镜检结果均符合大肠杆菌生理生化特性,分离菌16S rRNA测序结果呈单一峰值,对拼接序列在NCBI中进行BLAST比对后发现,与大肠杆菌相似性均>96%,确定分离菌为大肠杆菌。试验共分离鉴定大肠杆菌142株,其中有88株携带毒力基因,占61.97%（88/142）,毒力基因F17、K99、F41、STa、stx1、irp2、fyuA阳性率分别为24.65%、0.70%、0、2.11%、1.41%、45.07%和21.83%,其中F17、irp2、fyuA为优势毒力因子,同时携带多重毒力因子的大肠杆菌检出率较低。aac（6'）-Ⅰb、bla_CTX-M、bla_TEM、OqxB、tetA和sul1 6种耐药基因皆被检出,bla_TEM基因检出率最高,为45.77%,aac（6'）-Ⅰb和OqxB基因检出率最低,均为9.15%,分离菌株主要携带1~3种耐药基因。药物敏感性试验结果显示,142株分离菌对诺氟沙星敏感率最高,其次为环丙沙星,对青霉素敏感率为0,耐药现象严重,耐2种以上抗菌药物的菌株达86.62%。【结论】京津冀地区犊牛腹泻大肠杆菌毒力基因与耐药基因流行广泛,耐药普遍,多重耐药现象严重。本研究可为京津冀地区犊牛腹泻的防治提供理论依据。