仔猪腹泻源大肠杆菌耐药性调查及耐药基因检测

对泰州地区分离的仔猪腹泻源大肠杆菌的耐药性及其携带的相关耐药基因情况进行检测,为养殖场今后合理用药提供科学依据。采用微量肉汤稀释法对分离的49株大肠杆菌进行8种抗菌药物的药敏试验;采用PCR方法对多黏菌素耐药基因mcr-1及超广谱β-内酰胺酶(ESBL)耐药基因blaCTX-M、blaSHV和blaTEM进行检测。药敏试验结果显示:大肠杆菌对氯霉素、氟苯尼考、氨苄西林、四环素、庆大霉素和恩诺沙星等的耐药率分别达到100%、97.96%、95.92%、89.80%、59.18%和51.02%;而对多黏菌素E和头孢噻呋的耐药率也达到44.90%和32.65%。进一步耐药基因检测结果显示:ESBL耐药基因中blaCTX-M的检出率最高,达到12.24%(6/49);其次为blaSHV,8.16%(4/49);有一株大肠杆菌携带blaTEM基因。其中一株同时携带blaCTX-M和blaSHV。多黏菌素耐药基因mcr-1的检出率达到30.61%(15/49),其中4株同时携带blaCTX-M基因。泰州地区分离大肠杆菌耐药情况和多重耐药现象较为严重,多黏菌素耐药基因mcr-1及ESBL耐药基因blaCTX-M、blaSHV和blaTEM的广泛分布表明这些耐药基因存在快速传播的风险。因此,有必要加强猪场细菌耐药性监测。     

仔猪腹泻致病性大肠杆菌分型鉴定及耐药性分析

为了解贵州省规模化养猪场腹泻仔猪致病性大肠杆菌流行情况及耐药性变化,本研究运用凝集试验、PCR和药敏纸片琼脂扩散法等方法对分离的78株致病性大肠杆菌进行血清型、毒力基因及耐药性分析。结果显示,78株致病性大肠杆菌以O138、O87血清型为主,占定型菌株的60.8%;其中62株致病性大肠杆菌检出毒力基因,检出率为79.5%,可分为8种毒力基因类型,分属肠致病性大肠杆菌（EPEC）、肠产毒性大肠杆菌（ETEC）和肠聚集性大肠杆菌（EAEC）,毒力基因eaeA、elt和escV检出率较高,分别为38.5%、28.2%和21.8%;分离到的致病性大肠杆菌对β-内酰胺类药物高度耐药,均为多重耐药株,耐药种类可达8种以上。结果表明,当前贵州省规模化养猪场腹泻仔猪致病性大肠杆菌的毒力基因检出率较高且基因型复杂,耐药性严重。本试验结果可为规模化养猪场防控仔猪腹泻提供基础资料及理论依据。     

四川宜宾地区规模猪场腹泻仔猪大肠杆菌耐药性监测及血清型鉴定

为有效监测四川宜宾地区规模化猪场仔猪大肠杆菌耐药性及优势血清型变化,对采自该地区腹泻仔猪的38株大肠杆菌进行药物敏感性检测和O抗原血清型鉴定。结果表明,菌株对14种药物存在不同程度的耐药性,其中对氨苄西林、林可霉素、四环素和磺胺嘧啶极度耐药,耐药率分别为100%、100%、92.11%、89.47%;敏感性最高的是庆大霉素,敏感率为68.42%,氧氟沙星、丁胺卡那、环丙沙星和氟苯尼考相对比较敏感;所有菌株至少对4种药物耐受(4耐),最高达14耐。通过血清型鉴定,确定了15种血清型,共27株大肠杆菌,其中以O60、O101和O8为优势血清型。     

羊源大肠杆菌血清型鉴定、毒力基因检测及耐药性分析

为分析内蒙古扎兰屯地区羊源大肠杆菌血清型分布、毒力基因及耐药性流行情况,采集患羊鼻腔拭子150份,分离大肠杆菌,并采用O血清型玻板凝集试验、PCR法和K-B药敏纸片法对分离的大肠杆菌致病菌株进行血清型、毒力基因及耐药性检测。结果显示, 45株大肠杆菌致病菌株分属10种血清型,以O111、O127、 O38为流行的优势血清型,分别占致病菌株的26.7%、22.2%和17.8%;分离菌株毒力基因irp2、fyuA、hlyA、hlyE、Ler、eaeA、Ehly检出率在33.3%以上,其他毒力基因检出率在4.4%～14.3%之间;分离菌株对氨苄西林、阿莫西林、新霉素9种药物耐药率在53.3%以上,对其他药物的耐药率在13.3%～26.7%之间。研究表明,内蒙古扎兰屯地区羊源大肠杆菌血清型复杂,携带多种毒力基因,耐药性严重。     

广西大肠杆菌对多种抗菌药物的耐药率及部分耐药基因检测

为研究广西地区大肠杆菌对常用抗菌药物的耐药情况及耐药基因检出情况,采用2倍微量稀释法对实验室分离鉴定后甘油保藏的113株大肠杆菌进行头孢曲松钠、氟苯尼考等13种常见抗菌药物的药敏试验;筛选出42株对3种或3种以上药物最小抑菌浓度(MIC)≥3 200μg/mL的大肠杆菌,扩增bla_(TEM)基因、bla_(CTX-M-9)基因、sul 2基因。结果显示,细菌对头孢噻呋钠、氟苯尼考、磺胺间甲氧嘧啶、阿莫西林、环丙沙星、头孢噻肟钠、头孢曲松钠、左旋氧氟沙星、加替沙星的耐药率较高,分别为99.1%、99.1%、93.5%、92.9%、91.1%、89.9%、73.4%、72.5%、69.4%;对头孢他啶、磷霉素、阿米卡星、美罗培南的耐药率为56.6%、50.5%、32.7%、3.5%。在42株菌中,bla_(TEM)基因、bla_(CTX-M-9)基因、sul 2基因阳性率为19.0%、38.1%、45.2%。表明广西地区大肠杆菌对常见抗菌药物耐药情况趋于严重,耐药率呈升高趋势,以产超广谱β-内酰胺酶(ESBLs)大肠杆菌耐药基因及磺胺类sul 2耐药基因检出率偏高,为临床合理用药及兽医临床大肠杆菌耐药性研究提供理论依据。     

乌鲁木齐市宠物猫源大肠杆菌耐药性及耐药基因检测

为了解宠物猫源大肠杆菌对临床常用抗菌药物的耐药性及其耐药基因携带情况,在乌鲁木齐市多家宠物医院采集宠物猫肛拭子样品59份进行大肠杆菌的分离鉴定,通过琼脂稀释法测定10种抗菌药物的最小抑菌浓度,利用PCR方法检测13种耐药基因。结果:分离鉴定出大肠杆菌47株,分离率79.7%(47/59)。分离菌对四环素(TET)(83.0%)、阿莫西林-克拉维酸(A/C)(66.0%)和磺胺异噁唑(FIS)(66.0%)耐药情况严重;对头孢他啶(TAZ)、头孢曲松(CRO)、恩诺沙星(EN)、庆大霉素(GEN)耐药率在38.3%～48.9%;对环丙沙星(CIP)、阿米卡星(AMK)敏感性较好,未检出亚胺培南耐药菌株。菌株多药耐药分布在5～8耐,占比59.6%,耐药表型以1耐TET和6耐A/C+GEN+CRO+TAZ+TET+FIS为主。共检出ant(3″)-Ia、aac(6′)-Ib、bla_TEM、bla_CTX-M、qnrS、tetA、tetB、tetM、sulⅠ、sulⅡ10种耐药基因,检出率分布在8.5%～57.4%,未检出耐药基因oqxB、bla     

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。     

秦皇岛地区蛋鸡致病性大肠杆菌的血清型、毒力基因及耐药性检测分析

采用玻板凝集试验、PCR方法及K-B药敏纸片法分别对2021年秦皇岛地区分离鉴定的36株蛋鸡致病性大肠杆菌进行血清型、毒力基因、耐药基因及耐药性检测。结果显示,36株致病性大肠杆菌属于10种不同的血清型,以O78、O2、O1为主要流行的血清型,分别占比25.00%(9/36),19.44%(7/36),16.67%(6/36)。分离菌株携带14种不同的毒力基因,未检测到Ler、eaeA 2种毒力基因,其中iutB、tsh等5种毒力基因的检出率为66.67%～88.89%,而Vat、colv毒力基因检出率较低,分别为11.11%和16.67%。分离菌株对磺胺间甲氧嘧啶、阿莫西林等7种抗生素的耐药率在77.78%以上,对林可霉素、卡那霉素等4种抗生素的耐药率在27.78%以下。分离菌株均携带多重耐药基因,以Sul-1、dhpS等8种耐药基因为主,检出率为72.22%～94.44%,而SHV等4种耐药基因的检出率较低,为2.78%～25.00%,KPC、qnrA、qnrB基因未检出。本试验结果为河北省秦皇岛地区蛋鸡大肠杆菌的防制提供了用药指导。     

规模化猪场猪大肠杆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大肠杆菌分离株的药物敏感性和耐药基因的存在情况,以期为进一步研究细菌耐药的分子机制和临床用药提供资料,试验采用药物敏感试验、耐药基因PCR检测、基因测序等方法.结果表明:经K-B法检测20株猪源大肠杆菌对15种药物的敏感性,环丙沙星、氨苄西林、吡哌酸和青霉素等药物的耐药性强;利用PCR扩增技...     

腹泻犊牛大肠杆菌的分离鉴定和药敏试验

犊牛腹泻是犊牛常见病之一.犊牛腹泻大肠杆菌有多种血清型,主要有导致初生犊牛腹泻的O8、O9、O20、O101大肠杆菌,感染犊牛发生败血症的O78、O15、O35、O115、O117、O137等血清型大肠杆菌[1].2005～2007年我们从南京地区部分奶牛场犊牛腹泻和犊牛败血症病例的犊牛,无菌采集直肠棉拭及病死犊牛的脾脏和关节液作病料,同时无菌采集部分临床健康的犊牛和成年奶牛直肠棉拭,划线于麦康凯平板,37℃培养24 h;     

Characterization of eae+ Escherichia coli isolated from healthy and diarrheic calves.

Strains of Escherichia coli from 101 healthy and 114 diarrheic calves were screened by PCR for the eae (intimin) gene and Shiga toxin genes (stx). Each eae+ and eae/stx+ strain was examined for antimicrobial susceptibility, enterohemolysin activity, and the somatic O antigen was determined. An immunoassay was used to detect Shiga toxin antigens for the eae/stx+ E. coli. Significantly more (p = 0.005) of the healthy calves carried eae+ and eae/stx+ E. coli in their feces when compared to strains from diarrheic calves. Moreover, Shiga toxin antigens were detected significantly more (p = 0.001) often among the eae/stx+ strains from healthy calves when compared to eae/stx+ strains from diarrheic calves. However, significantly more (p = 0.001) of the eae+ and eae/stx+ strains from diarrheic calves were resistant to at least one of the antimicrobials tested, and the strains from diarrheic calves had a significantly (p = 0.05) higher rate of antimicrobial resistance to at least two different antimicrobial classes. No significant difference (p> or =0.05) was detected among the eae+ and eae/stx+ strains from healthy and diarrheic calves for enterohemolysin production. Serogroups O-negative, O5, O26, and O111 were predominate among both healthy and diarrheic calves.     

Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves

We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E. coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves. Three types of cytotoxic responses were observed. Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E. coli from infant and piglet enteritis. Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated. Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains. Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures. Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts. Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically. In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.     

Subtypes of intimin among non-toxigenic Escherichia coli from diarrheic calves in Brazil.

One hundred and five strains of Escherichia coli that were isolated from calves with diarrhea in the state of São Paulo, Brazil, and were negative for enterotoxins and cytotoxins, were examined for the eae gene. Four (3.8%) strains were positive by polymerase chain reaction (PCR) and were shown to produce intimin by using Western blot with specific antiserum against the conserved N-terminal region of intimin. Subtyping of the intimins was done by PCR with specific primers and by Western blot with specific antisera against the C-terminal variable region of the protein. Three of these isolates (O?:H11, O26:H-, O123:H1) produced the beta subtype of intimin, and the 4th (0103:H2) produced intimin that was not typable. The 0103:H2 and the O26:H-isolates adhered to HEp-2 cells with diffuse adherence and localized-like adherence patterns, respectively. The other strains did not adhere to HEp-2 cells. To our knowledge, this is the first report of the occurrence of a subtype of intimin described for human enteropathogenic E. coli among bovine diarrheogenic E. coli. It is also the first report from Brazil demonstrating the presence of bovine E. coli harboring the eae gene.     

Characteristics of necrotoxigenic Escherichia coli isolated from septicemic and diarrheic calves between 1958 and 1970

A total of 434 Escherichia coli isolated from septicemic calves between 1958 and 1965 and 430 E. coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes. They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin. Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation. Twenty-six were confirmed as necrotoxigenic E. coli type 2 (NTEC2) and four as NTEC1 by PCR. Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation. Five of them were studied by PCR and confirmed as NTEC1. The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III. All adhesins studied were present in NTEC as well as in non-NTEC. NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive. This study shows that necrotoxigenic E. coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time.     

Molecular characterization of pathogenic Escherichia coli isolated from diarrheic and in-contact cattle and buffalo calves

Tropical Animal Health and Production - Escherichia coli field isolates from calves were characterized and categorized into the most significant diarrheagenic pathotypes using polymerase chain...     

Prevalence of antimicrobial resistance and resistance genes in faecal Escherichia coli isolates recovered from healthy pets

Faecal samples of healthy dogs (n=39) and cats (n=36) obtained in Northern Portugal were seeded on Levine agar plates, and two Escherichia coli isolates per sample were recovered (78 of dogs and 66 of cats). The susceptibility to 16 antimicrobial agents was tested in this series of 144 E. coli isolates. Almost 20% of them showed tetracycline resistance and 12 and 15% presented ampicillin or streptomycin resistance, respectively. The percentage of resistance to the other antimicrobial agents was in all cases below 4%, and no resistant isolates were detected for ceftazidime, imipenem, cefoxitin or amikacin. Two isolates (from one dog) showed cefotaxime-resistance and harboured both the CTX-M-1 and OXA-30 beta-lactamases. A bla(TEM) gene was detected in 12 of 17 ampicillin-resistant isolates, the aac(3)-II gene in the three gentamicin-resistant isolates, aadA in 7 of 22 streptomycin-resistant isolates, and tet(A) and/or tet(B) gene in all 28 tetracycline-resistant isolates. The gene encoding class 1 integrase was detected in six E. coli isolates, including the four trimethoprim-sulfamethoxazole-resistant isolates and those two harbouring CTX-M-1 and OXA-30 beta-lactamases; different gene cassette arrangements were identified: dfrA1+aadA1 (two isolates), dfrA12+orfF+aadA2 (two isolates) and bla(OXA30)+aadA1 (two isolates). One amino acid change in GyrA protein (Ser83Leu or Asp87Tyr) was detected in four nalidixic acid-resistant and ciprofloxacin-susceptible isolates and two amino acid changes in GyrA (Ser83Leu+Asp87Asn) and one in ParC (Ser80Ile) were identified in one nalidixic acid- and ciprofloxacin-resistant isolate. Faecal E. coli isolates of healthy pets could be a reservoir of antimicrobial resistance genes.     

Antibiogram,virulotyping and genetic diversity of Escherichia coli and Salmonella serovars isolated from diarrheic calves and calf handlers

Antimicrobial resistance profile of E. coli and Salmonella serovars isolated from diarrheic calves and handlers in Egypt is unknown due to the absence of monitoring. Therefore, this study aimed to determine the virulence, genetic and antimicrobial resistance profiles of E. coli and Salmonella serovars associated with diarrhea in calves and handlers in intensive dairy farms in Egypt. A total of 36 bacterial strains (20 E. coli and 16 Salmonella) were isolated from fecal samples of 80 diarrheic Holstein dairy calves (10 E. coli and 13 Salmonella) and hand swabs of 35 handlers (10 E. coli and 3 Salmonella) in two intensive dairy farms in Sharkia Governate in Egypt. E. coli strains belonged to six different serogroups and O114:K90 was the most prevalent serogroup (30%). However, Salmonella strains were serotyped into four different serogroups and S. Kiel was the most prevalent serotype (50%). Thirteen (65%) E. coli isolates were harbouring either stx2, eaeA and/or astA virulence-associated genes. However, stn and spvC virulence genes were detected in 2 (12.5%) and 4 (25%) of Salmonella isolates, respectively. E. coli isolates showed marked resistance to ampicillin (75%), while Salmonella strains exhibited high resistance to amikacin (100%), gentamicin (93.75%) and tobramycin (87.5%). Results of the present study showed that E. coli and Salmonella serovars isolated from diarrheic calves and handlers in intensive dairy farms in Egypt exhibited resistance to multiple classes of antimicrobials, which may pose a public health hazard. Thus, the continuous monitoring of antimicrobial resistance is necessary for both humans and veterinary medicine to decrease the economic losses caused by antimicrobial-resistant strains in animals as well as the zoonotic risk.     

Proteomic analysis of multidrug resistant Escherichia coli strains from scouring calves

A number of researchers have used chemical inhibitors that target membrane efflux pumps as an experimental treatment strategy for multidrug resistant (MDR) bacterial infections. However, most of these compounds are toxic in vertebrate animals. The present research was therefore done to describe expression dynamics of drug resistance-associated Escherichia coli proteins that could serve as novel drug targets. Proteomes of MDR and antimicrobial susceptible (AS) E. coli were studied in two dimensional (2-D) polyacrylamide gels and liquid chromatography-mass spectrometry (LC-MS) was performed on proteins of interest. The number of recovered peptides per protein was used to elucidate the amounts of target proteins expressed in MDR and AS E. coli strains. Eight proteins that may be potentially involved in mechanisms of drug resistance were analyzed and identified by LC-MS. These were grouped into membrane porins (TolC, OmpA, OmpC, Nmpc Precursor), proteins involved in microbial protein synthesis (EF-Ts, EF-Tu, RpsA) and Dps, a protein of unknown location and function. Experimental data demonstrated variability in the expression patterns and quantities of the four porins (TolC, OmpA, OmpC, Nmpc precursor), the three microbial protein synthesis associated proteins (EF-Ts, EF-Tu and RpsA), and Dps which has been previously associated with drug resistance. While variability was seen in quantities and expression patterns of some of the proteins of interest, the present data falls short of determining the suitability of these proteins as novel drug targets. Further studies are required to explore how these proteins could be targeted for drug development.