广东屠猪肉样品中大肠杆菌耐药性与毒力特征的分析

为分析广东地区屠猪肉中大肠杆菌(E.coli)药物敏感菌株的血清型、毒力基因和系统进化背景,本研究从屠猪肉样品中分离出112株E.coli,采用玻片凝集法鉴定血清型,琼脂稀释法测定10种抗菌药的敏感性,PCR方法检测7种毒力相关基因,多重PCR方法进行系统进化背景判定。结果显示,112株E.coli中,定型菌株95株,分别属于15种血清型,其中O65、O131、O8和O158为优势血清型。几乎所有菌株对氟苯尼考、多西环素和四环素高度耐药,而对头孢曲松高度敏感,其中多重耐药菌株多数耐5种以上药物,常见的多重耐药表型是氟苯尼考/氯霉素/多西环素/四环素/氨苄西林。PCR鉴定结果表明,含有毒力基因的菌株中38%至少具有两个毒力基因,其中EAST1+Stx2e和hlyF+Stx2e比较常见。比较常见的毒力基因为Stx2e和EAST1,STb基因仅在一株菌中检测到。多重PCR鉴定结果显示,屠猪肉样品中E.coli主要分布为共生型的A组和B1组。本研究为大肠杆菌病的控制和合理使用抗生素提供实验依据。     

牛产肠毒素大肠杆菌毒力因子多重PCR检测方法的建立

通过多重PCR扩增产肠毒素大肠杆菌（enterotoxigentic E.coli，ETEC）的毒力因子F41菌毛、K99菌毛和STa肠毒素的编码基因来检测和鉴定ETEC。试验中对影响PCR扩增的dNTP、Mg^2＋、引物浓度以及退火温度等因素进行优化，在优化条件的基础上，确定多重PCR的特异性和灵敏性，以此建立同时检测ETEC多个毒力因子的多重PCR方法。用该方法对分离于犊牛腹泻和犊牛肠毒血症的7株大肠杆菌进行检测，结果2株为F41、K99和STa阳性，4株为F41、STa阳性，1株为K99STa阳性。这与玻片凝集试验检测菌毛的结果一致。试验表明，该方法特异性强、敏感性高、简便、快速，适用于临床鉴定和检测牛ETEC菌株。     

Antimicrobial resistance of Escherichia coli isolated from chickens

Faecal Escherichia coli isolated from healthy farm chickens, from farm chickens with avian influenza, and from chickens with diarrhoea were more resistant to antimicrobial agents (94-100%) than those isolated from healthy domestic chickens (20%). Transfer of drug resistance was readily achieved from strains isolated from both healthy and sick farm chickens, and from diarrhoeic chickens; it was more difficult to demonstrate in strains from domestic chickens. Resistant E. coli showing serotypes suspected to be enteropathogenic for man, i.e 0126:K71(B16), 044:K74 (L) and 0119:K69(B14), were isolated from faecal samples of healthy and sick farm chickens, but not from healthy domestic birds.     

Antimicrobial resistance of commensal Escherichia coli from dairy cattle associated with recent multi-resistant salmonellosis outbreaks

The use of antimicrobial drugs in livestock is suspected to contribute to bacterial antimicrobial resistance (AR) development. Dairy farms experiencing recent outbreaks of salmonellosis involving multi-resistant (MR) Salmonella strains were compared to control farms with respect to AR among bovine commensal E. coli isolates. For most antimicrobials tested, the percentage of AR E. coli isolated from salmonellosis-affected farms was significantly higher than that from control farms. Calf E. coli from both case and control farms had greater levels of AR than cow isolates. Commensal E. coli isolates from case farms and calves tended to more frequently be MR. These data are consistent with the existence of higher antimicrobial selection pressure on farms with recent salmonellosis outbreaks, however, the directionality of the relationship remains to be elucidated. An improved understanding of the epidemiology of AR bacteria in livestock production, both at the herd and molecular level, is essential to mitigate risk to public health and food safety.     

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.     

Serum resistance and virulence of Escherichia coli isolated from turkeys

Twenty-five strains of Escherichia coli isolated from turkeys were characterized for their serum resistance and virulence. An in vitro bactericidal assay was used to determine the serum resistance of E coli. Virulence was determined by survival time after IV inoculation of each strain into 3-week-old turkeys. Serum-resistant E coli strains were generally found to be virulent for turkeys, whereas serum-sensitive E coli strains were avirulent. Of the 25 strains, 18 strains were placed in the 2 categories of serum-resistant/virulent and serum-sensitive/avirulent. Five strains were serum-resistant and avirulent, and 2 strains were serum-sensitive and virulent. Serum resistance appears to be an important determinant of virulence for E coli in turkeys; however, the requirement for other virulence factors, in addition to serum resistance, was suggested by the finding that 5 serum-resistant strains were avirulent in turkeys.     

Virulence factors and markers in Escherichia coli from calves with bacteremia

Relative pathogenicity of 151 Escherichia coli isolates from 36 calves with bacteremia after necropsy was studied by measurement of the LD50 after mice were inoculated IP with E coli isolates. Study of virulence factors and markers revealed that the pathogenicity of E coli was associated with the production of hydroxamate siderophores and with resistance to serum bactericidal effects. Production of colicins, including colicin V, and of surface antigen 31A was correlated with virulence. The close association between phenotypic expression of virulence factors and markers was consistent with a hypothesis of a localization of genes coding for virulence factors and markers on the same plasmid.     

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。     

Longitudinal prevalence study of diarrheagenic Escherichia coli in dairy calves

A longitudinal study (cohort study) elaborating 1,224 rectal swabs from 221 calves aging between 1 and 12 weeks was conducted on 11 dairy farms (i) to ascertain associations between diarrhea and shedding of diarrheagenic E. coli and (ii) to facilitate the zoonotic potential assessment of E. coli strains shed by young calves. Calves were screened weekly by PCR of swab cultures for shedding of enterotoxigenic E coli [ETEC; by detection of heat stable (est) and heat labile enterotoxin genes (elt)], diffusely adhering E. coli [DAEC; diffuse adhesion (daa)], typical enteropathogenic E. coli [EPEC; bundle-forming pili (bfpA) and intimin (eae)] as well as enterohemorrhagic E. coli [EHEC, intimin (eae) and Shiga toxin (stx)]. In addition, EHEC-hemolysin- (Hly(EHEC)) and alpha-hemolysin- (alpha-Hly) producing E. coli were detected by inoculation of blood agar plates. Within the 221 calves, prevalences were 69.7% (25.2% of the 1,224 samples) for Hly(EHEC)-producing E. coli, 55.3% (19.3%) for eae, and 18.2% (4.5%) for stx. E. coli strains exhibiting an alpha-Hly phenotype were detected in 66.5% of the calves and 21.9% of fecal samples. The est gene was detectable in 31.7% of the calves from only 9 of 11 herds and in 7.8% of the samples. Calves shedding DAEC or typical EPEC were not identified. The detection frequency of virulence traits significantly depended on the calves' age and shedding dynamics differed between the traits. A significant correlation between calf diarrhea and shedding of EHEC virulence traits was determined for several postnatal periods (1 week: Hly(EHEC); 1st & 10th week: eae; 4th week stx). Shedding of ETEC (est) was associated with diarrhea in newborn calves (1st week) only. Hly(EHEC)- and alpha-Hly-producing E. coli were shed significantly more frequently by diarrheic calves in 1st and 8th week of life, respectively. The knowledge gained in this study highlights the high prevalence of zoonotic E. coli already in calves.The age-dependent shedding dynamic of the various E. coli pathovars has to be considered regarding prophylaxis as well as planning intervention studies, both for calves and humans.     

Relationship of complement resistance and selected virulence factors in pathogenic avian Escherichia coli.

Complement resistance, antibiotic resistance profiles, and virulence profiles of 80 Escherichia coli isolates from the intestines of normal chickens (40 isolates) and chickens diagnosed as having colisepticemia (40 isolates) were compared. Differences were observed between the two groups for antibiotic resistance, siderophore production, presence of type 1 pili, complement resistance, motility, and size of plasmids. The systemic isolates were more likely to have siderophores and type 1 pili, and to be complement-resistant and motile than were the intestinal isolates. No differences between the two groups were observed for colicin production. Further comparison of the 10 most complement-resistant isolates from the systemic group and 10 most complement-sensitive isolates from the intestinal group revealed a correlation between an isolate's resistance to complement and its ability to kill embryos, express type 1 pili, and be motile. Virulence of avian E. coli strains appears to be correlated with complement resistance and the interaction of this resistance with the ability to produce type 1 pili and be motile.     

Antibiotic resistance among enterotoxigenic Escherichia coli from piglets and calves with diarrhea

In vitro resistance to 8 antimicrobials among enterotoxigenic Escherichia coli from piglets and calves over a 13-year period was evaluated. Least resistance occurred against ceftiofur for all, followed by apramycin and gentamicin for porcine and florfenicol for bovine isolates. No significant differences were found between the first 8 and last 5 years.     

Virulence factors in Escherichia coli isolated from calves with diarrhea in Vietnam

This study was conducted to determine the prevalence and characteristics of pathogenic Escherichia (E.) coli strains from diarrheic calves in Vietnam. A total of 345 E. coli isolates obtained from 322 diarrheic calves were subjected to PCR and multiplex PCR for detection of the f5, f41, f17, eae, sta, lt, stx1, and stx2 genes. Of the 345 isolates, 108 (31.3%) carried at least one fimbrial gene. Of these 108 isolates, 50 carried genes for Shiga toxin and one possessed genes for both enterotoxin and Shiga toxin. The eae gene was found in 34 isolates (9.8%), 23 of which also carried stx genes. The Shiga toxin genes were detected in 177 isolates (51.3%) and the number of strains that carried stx1, stx2 and stx1/stx2 were 46, 73 and 58, respectively. Among 177 Shiga toxin-producing E. coli isolates, 89 carried the ehxA gene and 87 possessed the saa gene. Further characterization of the stx subtypes showed that among 104 stx1-positive isolates, 58 were the stx1c variant and 46 were the stx1 variant. Of the 131 stx2-positive strains, 48 were stx2, 48 were stx2c, 11 were stx2d, 17 were stx2g, and seven were stx2c/stx2g subtypes. The serogroups most prevalent among the 345 isolates were O15, O20, O103 and O157.     

Animal and farm influences on the dynamics of antibiotic resistance in faecal Escherichia coli in young dairy calves

It is believed that the intensive use of antibiotics in the management of disease in pre-weaned calves contributes to high levels of antibiotic resistance in commensal and pathogenic bacteria. We described the temporal dynamics of antibiotic-susceptibility patterns seen in bovine enteric Escherichia coli in pre-weaned calves on dairy farms and dedicated calf-rearing facilities. Cohorts of 30 calves at each of six farms were sampled at 2-week intervals during the pre-weaning period. Faecal E. coli isolates were analyzed for antibiotic susceptibility to 12 antibiotics with the disk-diffusion method and grouped using cluster analysis of inhibition-zone patterns. The influences of calf age, farm-type, and individual-calf antibiotic therapy on the clusterings were assessed using stratified analyses and cumulative multinomial logistic regression using generalized estimating equation with antibiotic-resistance cluster as an ordinal-dependent variable. The model controlled for farm and cohort by a nested design and included a repeated measure on calf at each sampling occasion. E. coli from calves 2 weeks of age were more likely to be increasingly multiply resistant than E. coli from day-old calves (OR = 53.6), as were 4- and 6-week-old calves (OR = 29.8 and 16.4, respectively). E. coli from calves on dedicated calf-rearing facilities were more likely to be increasingly multiply resistant than E. coli from dairy-reared calves (OR = 2.4). E. coli from calves treated with antibiotics within 5 days prior to sampling were also more likely to be increasingly multiply resistant than E. coli from calves not exposed to individual antibiotic therapy (OR = 2.0).     

Virulence factors and antimicrobial susceptibility of Escherichia coli isolated from calves in Turkey

Escherichia coli isolates from calves were investigated by multiplex PCR assays for the presence of genes encoding K99, F41, F17-related fimbriae, heat-stabile enterotoxin a (STa), intimin (eae) and Shiga toxins (stx1 and stx2). A total of 120 E. coli isolates, 75 isolated from diarrhoeic or septicemic calves and 45 from clinically healthy calves aged between 1 day and 2 months were tested. Each isolate was obtained from different calves in different herds. Among the isolates from diseased animals, 12 (16%) isolates from 1- to 7-day-old diarrhoeic calves were detected as enterotoxigenic E. coli which possessed K99, F41 and STa in combination; F17-related fimbriae genes were detected in 33 (44%) isolates and they were found in combination with K99 + F41 + STa in two isolates. Of 120 isolates, 16 carried eae, eight stx1 and five stx2 genes alone or in combination. None of the eae- or stx-positive strains was identified as O157:H7. However, results indicate that calves may be carrier of Shiga toxin-producing E. coli which have potential as a human pathogen. Antimicrobial susceptibility of 75 isolates from diseased calves was determined by agar disk diffusion method for 14 antimicrobial agents. In 77.3% of the isolates, multiresistance was detected. Higher resistance rates were detected for cephalothin (72%), tetracycline (69.3%), kanamycin (69.3%), ampicillin (65.3%), nalidixic acid (53.3%), trimethoprim-sulphamethoxazole (52%) and enrofloxacin (41.3%), respectively. No resistance was found for ceftiofur and cefoxitin.     

产ESBLs禽源大肠杆菌毒力因子的调查与分析

为了获得产ESBLs和非产ESBLs菌株中毒力因子的携带情况,分析产ESBLs耐药菌株的毒力特性,为防治此类细菌性疾病提供依据。本试验采用CLSI标准方法和PCR方法分别检测青岛地区249株禽源大肠杆菌菌株的ESBLs和毒力基因。结果是83.13%的菌株产ESBLs;24种毒力基因均有检出;iutA、iroN、iss、iucA、sfa、fyuA、vat等7种毒力基因在产ESBLs菌株与非产ESBLs菌株中的检出率差异均有统计学意义。表明携带毒力因子的产ESBLs大肠杆菌菌株正在青岛地区流行和传播;毒力因子和耐药性之间具有一定相关关系,iutA、iroN、iss在产ESBLs禽源大肠杆菌中更加流行,而iucA、sfa、fyuA、vat在产ESBLs菌株中的流行率显著低于非产ESBLs菌株。     

Antimicrobial resistance and resistance gene determinants in clinical Escherichia coli from different animal species in Switzerland

Antimicrobial susceptibility testing was performed on a total of 581 clinical Escherichia coli isolates from diarrhea and edema disease in pigs, from acute mastitis in dairy cattle, from urinary tract infections in dogs and cats, and from septicemia in laying hens collected in Switzerland between 1999 and 2001. Among the 16 antimicrobial agents tested, resistance was most frequent for sulfonamides, tetracycline, and streptomycin. Isolates from swine presented significantly more resistance than those from the other animal species. The distribution of the resistance determinants for sulfonamides, tetracycline, and streptomycin was assessed by hybridization and PCR in resistant isolates. Significant differences in the distribution of resistance determinants for tetracycline (tetA, tetB) and sulfonamides (sulII) were observed between the isolates from swine and those from the other species. Resistance to sulfonamides could not be explained by known resistance mechanisms in more than a quarter of the sulfonamide-resistant and sulfonamide-intermediate isolates from swine, dogs and cats. This finding suggests that one or several new resistance mechanisms for sulfonamides may be widespread among E. coli isolates from these animal species. The integrase gene (intI) from class I integrons was detected in a large proportion of resistant isolates in association with the sulI and aadA genes, thus demonstrating the importance of integrons in the epidemiology of resistance in clinical E. coli isolates from animals.     

Secretion of virulence factors by Escherichia coli.

In order to interact with their host, pathogenic strains of E. coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease. Since E. coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes. E. coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems. In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes. It is a one-step mechanism. The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage. In E. coli, the best studied type I secreted protein is haemolysin. In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence. The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV). The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second. The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins. The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells. Such a system is found in attaching and effacing E. coli and in diffusely adhering E. coli.     

禽大肠杆菌毒力因子的研究进展

禽致病性大肠杆菌是毒力基因的贮存宿主,多种毒力因子的协同作用使其产生致病性,而耐药性的产生加大了对其防治的难度。本文主要对禽大肠杆菌毒力因子的种类、毒力特性及分子流行病学等研究进展进行了概述,为深入研究禽大肠杆菌病的致病机理和制定防控措施提供理论依据。     

Antimicrobial resistance in generic fecal Escherichia coli from 29 beef farms in Ontario

The occurrence of antimicrobial resistance in generic Escherichia coli can serve as an indicator of the pool of resistance genes potentially available for transfer to pathogenic organisms. This study was conducted on 29 volunteer beef farms in Ontario to describe the prevalence and patterns of antimicrobial resistance in E. coli, and to describe changes in the prevalence of resistance during the feedlot stage of production. From the pooled fecal samples on 28 of the 29 farms, 31% of isolates from feedlots (n = 993) and 12% of isolates from cow-calf farms (n = 807) were resistant to one or more of 16 antimicrobials tested. No isolates were resistant to ceftriaxone, ciprofloxacin, gentamicin, or nalidixic acid, and < 1% of the pooled isolates were resistant to ceftiofur. Two percent of both feedlot and cow-calf isolates were resistant to > or = 5 antimicrobials. Cow-calf farms were at significantly lower risk than feedlots for having E. coli isolates that were resistant to streptomycin, sulfamethoxazole, and tetracycline. On average, the prevalence of sulfamethoxazole resistant E. coli isolates was significantly higher in calves than in cows. No resistance was observed to ceftriaxone or ciprofloxacin among isolates (n = 1265) obtained from individually sampled feedlot animals on 2 farms. Less than 1% of these isolates were resistant to gentamicin, nalidixic acid, and ceftiofur. From the individually sampled feedlot animals, resistance to streptomycin (on 1 farm), sulfamethoxazole, and tetracycline increased significantly from arrival to mid-point during the feeding period, and these levels persisted until market-readiness.