Evaluation of repetitive extragenic palindromic-PCR for discrimination of fecal Escherichia coli from humans, and different domestic- and wild-animals

The objective of this study was to investigate the potential of repetitive extragenic palindromic anchored polymerase chain reaction (rep-PCR) in differentiating fecal Escherichia coli isolates of human, domestic- and wild-animal origin that might be used as a molecular tool to identify the possible source(s) of fecal pollution of source water. A total of 625 fecal E. coli isolates of human, 3 domestic- (cow, dog and horse) and 7 wild-animal (black bear, coyote, elk, marmot, mule deer, raccoon and wolf) species were characterized by rep-PCR DNA fingerprinting technique coupled with BOX A1R primer and discriminant analysis. Discriminant analysis of rep-PCR DNA fingerprints of fecal E. coli isolates from 11 host sources revealed an average rate of correct classification of 79.89%, and 84.6%, 83.8%, 83.3%, 82.5%, 81.6%, 80.8%, 79.8%, 79.3%, 77.4%, 73.2% and 63.6% of elk, human, marmot, mule deer, cow, coyote, raccoon, horse, dog, wolf and black bear fecal E. coli isolates were assigned to the correct host source. These results suggest that rep-PCR DNA fingerprinting procedures can be used as a source tracking tool for detection of human- as well as animal-derived fecal contamination of water.     

Fidelity of bacterial source tracking: Escherichia coli vs Enterococcus spp and minimizing assignment of isolates from nonlibrary sources

AIMS: The goal of the study was to improve the fidelity of library-dependent bacterial source tracking efforts in determining sources of faecal pollution. The first objective was to compare the fidelity of source assignments using Escherichia coli vs Enterococcus spp. The second objective was to determine the efficacy of using thresholds during source assignments to reduce the rate of misassignments when nonlibrary isolates (i.e. isolates from animals not used in building the identification library) are present. METHODS AND RESULTS: E. coli and Enterococcus isolates from 784 human, cow, deer, dog, chicken, and gull faecal samples were fingerprinted using BOX-PCR. Jack-knife analysis of the fingerprints showed that the overall rate of correct assignment (ORCA) of 867 E. coli isolates was 67% compared with 82% for 1020 Enterococcus isolates. In a separate blind test using similarity value and quality factor thresholds, the ORCA of 130 E. coli and 131 Enterococcus isolates were 70% and 98%, respectively. The use of these thresholds reduced misassignment of 262 nonlibrary enterococcal isolates from horses, goats, pigs, bats, squirrels, ducks, geese, and migratory song birds. Misassignment was reduced from 100% when thresholds were not used, to 47% using similarity threshold alone, and to 12% when both thresholds were used. CONCLUSIONS: The use of enterococci provides higher rates of correct source assignment compared with E. coli. The use of similarity thresholds to decide whether to accept source assignments made by computer programmes reduces the rate of misassignment of nonlibrary isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Although both E. coli and Enterococcus spp. are still used in microbial source tracking, the use of enterococci should be preferred over the use of E. coli in DNA fingerprint-based efforts. In addition, because environmental isolates are not limited to those from animals used to build source tracking libraries, similarity thresholds should be used during source assignments to reduce the rate of misassignments.     

Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio

A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.     

制备禽致病性大肠杆菌菌蜕的三种方法比较

菌蜕(Bacterial ghosts)是一种只含有细菌内、外膜结构的细菌空壳,可作为新型疫苗和传递载体。本研究通过3种方式制备禽致病性大肠杆菌(Avian pathogenicity Escherichia coli,APEC)分离株DE17的菌蜕,评价不同的菌蜕制备方法。结果表明,利用噬菌体Phi X174的裂解基因E构建的溶菌质粒pBV220-E制备DE17菌蜕,对DE17菌株裂解率可达99.9%,扫描电镜观测结果表明,在DE17两端或中部形成可见的跨膜孔道,呈现典型的菌蜕结构。利用合成的细胞穿透肽MAP(KLALKLALKALKAALKLA)作用于DE17制备菌蜕,结果表明,10μmol/L的MAP可实现对OD_(600)=0.1的DE17完全灭活,扫描电镜虽未看到明显的跨膜孔道,但细菌的膜结构呈现沟壑状,而构建的可表达MAP的溶菌质粒pBV220-MAP并不能实现对DE17的裂解作用。本研究通过比较分析不同APEC菌蜕的制备方式,为进一步研究菌蜕疫苗和提高菌蜕疫苗的生物安全性提供参考。     

Comparison of repetitive PCR and pulsed-field gel electrophoresis for the genotyping of Mannheimia haemolytica

Mannheimia haemolytica is an opportunistic pathogen that can cause fibrinonecrotic pneumonia in cattle and is the main bacterial agent implicated in bovine respiratory disease-complex (BRD). Despite its economic importance to the cattle industry, few studies have characterized the genetic nature of M. haemolytica and none have genotyped isolates from feedlots. Identifying and monitoring genetic variants of M. haemolytica is important to understanding the etiology of BRD in cattle. We investigated the capacity of three genotyping techniques (BOX-PCR, (GTG)₅-PCR and PFGE analysis of SalI-restricted DNA) to discriminate among 24 reference strains from the family Pasteurellaceae and 40 M. haemolytica isolates collected from feedlot cattle. From cluster analysis of the M. haemolytica isolates, PFGE was revealed as most discriminating, followed by BOX-PCR and then (GTG)₅-PCR (Simpson's diversity index > 0.98, 0.82, and 0.72, respectively). Of these methods, PFGE also had the greatest mean repeatability (0.96). The PFGE and BOX-PCR assays grouped all M. haemolytica in a single cluster but only BOX-PCR and (GTG)₅-PCR grouped the Mannheimia glucosida and Mannheimia ruminalis strains together. Refinement of genotyping procedures for M. haemolytica could offer new insight into the etiology of this pathogen in BRD.     

Purification and characterization of heat-labile enterotoxin isolated from chicken enterotoxigenic Escherichia coli

Abstract The heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli was purified to homogeneity and its molecular and antigenic properties were compared with those of purified LTs from porcine and human enterotoxigenic Escherichia coli (LTp, LTh). The A subunit of LTc was identical to that of LTp and the B subunit of LTc was identical to that of LTh but not that of LTp, in mobility on SDS-polyacrylamide gel electrophoresis. Ouchterlony tests demonstrated that LTc is antigenically identical to LTh but not with LTp. The p I point and amino acid composition of LTc were also compared and the results suggest that chicken enterotoxigenic E. coli produced an LT similar to LTh.     

Development of a 5'-nuclease PCR assay for the identification of Escherichia coli strains expressing the flagellar antigen H21 and their detection in food after enrichment

Aims: To develop a real‐time PCR assay targeting the Escherichia coli flagellar antigen H21 for identification and surveillance of clinically important Shiga toxin‐producing E. coli (STEC) serotypes classified in seropathotype C. Methods and Results: The fliC allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The nucleotide sequence obtained was compared with fliC genes of E. coli O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers and a TaqMan^® minor groove binder probe specific for fliC‐H21 were designed and used in a 5′‐nuclease PCR assay. This method was evaluated using a panel of 138 diverse bacterial strains and was shown to be 100% specific for H21. PCR amplification of fliC‐H21 from one cell per reaction mixture was possible, and an initial inoculum of 10 STEC H21 colony‐forming units per 25 g of ground beef was detected after overnight enrichment. Conclusions: The PCR assay developed was found to be highly sensitive and specific for the identification and detection of E. coli H21 strains in ground beef. Significance and Impact of the Study: The real‐time PCR assay targeting the H21 flagellar antigen described here offers a valuable method for the rapid detection and molecular typing of pathogenic STEC H21 strains in food.     

54株猪源肠外致病性大肠杆菌血清型、系统进化群和基因型

摘 要:[背景]近年来,我国规模猪场着重加强了对猪繁殖与呼吸综合征、猪圆环病毒病、猪瘟、猪伪狂犬病、猪链球菌病、副猪嗜血杆菌病等疫病的防控,却忽视了由肠外致病性大肠杆菌(Extraintestinal Pathogenic Escherichia coli,ExPEC)对猪群健康产生的潜在危害性,了解和掌握猪源ExPEC流行特征意义显著。[目的]探究临床分离的54株猪源ExPEC血清型、系统进化群和基因型的分布及流行特征。[方法]应用玻板凝集试验和试管凝集试验鉴定O抗原血清型,采用PCR技术检测系统进化群鉴定相关基因、28个ExPEC相关毒力基因以及多位点序列分型相关基因。[结果]受试菌中有52株确定了O抗原血清型,其中40株为O38 (74.1%),为优势血清型;8株为O127 (14.8%),O93和O11均2株(各占3.7%)。受试菌中44株为B2群(81.5%),是主要系统进化群,D群和B1群均5 株(各占 9.3%);28 个 ExPEC 相关毒力基因中ompA、ibeA、fimH、traT、focD、papA、iroN、iutA、iucD、cvaC、tsh、kpsMT Ⅱ、iss和ompT出现的频率超过50%,其中ompA和ibeA检出率分别达100%和96.3%,为高度流行的毒力基因,未检到cnf1,而bmaE、malX和iha更倾向分布于D群菌株中。受试菌共呈现31种ST型,其中ST10和ST648均5株(各占9.3%),ST410和ST101均4株(各占7.4%)。[结论]猪源ExPEC优势血清型及系统进化群在不同地区、不同时段上的流行分布均存在一定差异,呈现动态过程,O38作为优势血清型目前尚未见报道,具有高致病性的B2群和D群菌株有逐渐增多的趋势。ST型复杂多样,呈现遗传多样性,在一定程度上与人源和禽源ExPEC具有相同的遗传背景。     

Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

Molecular typing of Klebsiella species has become important for monitoring dissemination of β-lactamase-producers in hospital environments. The present study was designed to evaluate poly-trinucleotide (GTG)₅- and rDNA intergenic transcribed spacer (ITS)-PCR fingerprint analysis for typing of Klebsiella pneumoniae and Klebsiella oxytoca isolates. Multiple displacement amplified DNA derived from 19 K. pneumoniae (some with an ESBL-phenotype), 35 K. oxytoca isolates, five K. pneumoniae, two K. oxytoca, three Raoultella, and one Enterobacter aerogenes type and reference strains underwent (GTG)₅ and ITS-PCR analysis. Dendrograms were constructed using cosine coefficient and the Neighbour joining method. (GTG)₅ and ITS-PCR analysis revealed that K. pneumoniae and K. oxytoca isolates, reference and type strains formed distinct cluster groups, and tentative subclusters could be established. We conclude that (GTG)₅ and ITS-PCR analysis combined with automated capillary electrophoresis provides promising tools for molecular typing of Klebsiella isolates.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Escherichia coli and enterococci are sensitive and reliable indicators for human,livestock and wildlife faecal pollution in alpine mountainous water resources

Aims: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. Methods and Results: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7–7·0 CFU g^?1) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0–8·4 CFU g^?1). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3–7·7 CFU g^?1. To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi‐parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment ( Reischer et al. 2008 ; Environ Microbiol 10:2598–2608). Conclusion: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. Significance and Impact of the Study: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.     

Specific detection of Escherichia coli isolated from water samples using polymerase chain reaction targeting four genes: cytochrome bd complex, lactose permease, beta-D-glucuronidase, and beta-D-galactosidase

Aims: To develop a PCR-based method for reliable detection of Escherichia coli that enables its differentiation from biochemically and phylogenetically related bacteria. Methods and Results: Using multiplex PCR targeting four genes (cytochrome bd complex, lactose permease, β-d -glucuronidase, and β-d -galactosidase) the possibility of specific detection of various control E. coli strains was tested. It was found that four PCR fragments of the predicted size were observed only for E. coli strains, but not for relatives as close as Shigella sp. or other enterobacteria. Not surprisingly, this method enabled us to identify also E. coli strains which did not exhibit the β-d -glucuronidase activity. Our multiplex PCR was also successfully used for identification of 95 environmental isolates of E. coli. Conclusions: The developed PCR-based method, in which four genes coding for lactose permease, cytochrome bd complex, β-d -glucuronidase, and β-d -galactosidase, serve as target DNA sequences, allows precise and reliable detection of E. coli strains. Significance and Impact of the study: The suggested approach increases the specificity of detection of E. coli since it enables to distinguish E. coli from Shigella sp. and other relative enterobacteria.     

Detection of Campylobacter and Escherichia coli O157:H7 from filth flies by polymerase chain reaction

Flies (Diptera: Muscidae) that breed in faeces and other organic refuse (filth flies) have been implicated as vectors of pathogenic bacteria including Escherichia coli O157:H7, which cause haemorrhagic colitis in humans, and Campylobacter, which is the principal causative agent of human enteritis. The potential role of filth flies in the epidemiology of these pathogens in the United States was investigated by examining the prevalence of Campylobacter spp. and E. coli O157:H7 from two Arkansas turkey facilities. Polymerase chain reaction was conducted on DNA extractions of individual Musca domestica Linnaeus, Stomoxys calcitrans (Linnaeus), Hydrotaea aenescens (Wiedemann), Adia cinerella Fallen and turkey faecal samples using primers specific for E. coli H7, O157 and Campylobacter spp. Culturing verified that the flies were carrying viable Campylobacter spp. and E. coli O157:H7. Results from this study indicated that M. domestica, S. calcitrans, H. aenescens and Anthomyids are capable of carrying Campylobacter in North American poultry facilities and that the E. coli O157:H7 is carried by house flies and black dump flies associated with poultry. This PCR method provided a rapid and effective method to identify Campylobacter spp. and E. coli O157:H7 directly from individual filth flies.     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

Comparison of recombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changes

A stable high-copy-number plasmid pSYL105 containing the Alcaligenes eutrophus polyhydroxyalkanoic acid (PHA) biosynthesis genes was constructed. This plasmid was transferred to seven Escherichia coli strains (K12, B, W, XL1-Blue, JM109, DH5alpha, and HB101), which were subsequently compared for their ability to synthesize and accumulate ploy- (3-hydroxybutyric acid) (PHB). Growth of recombinant cells and PHB synthesis were investigated in detail in Luria-Bertani (LB) medium containing 20 g/L glucose. Cell growth, the rate of PHB synthesis, the extent of PHB accumulation, the amount of glucose utilized, and the amount of acetate formed varied from one strain to another. XL1-Blue (pSYL105) and B (pSYL105) synthesized PHB at the fastest rate, which was ca. 0.2 g PHB/g true cell mass-h, and produced PHB up to 6-7 g/L. The yields of cell mass, true cell mass, and PHB varied considerably among the strains. The PHB yield of XL1-Blue (pSYL105) in LB plus 20 g/L glucose was as high as 0.369 g PHB/g glucose. Strains W (pSYL105) and K12 (pSYL105) accumulated the least amount of PHB with the lowest PHB yield at the lowest synthesis rate. JM109 (pSYL105) accumulated PHB to the highest extent (85.6%) with relatively low true cell mass (0.77 g/L). Considerable filamentation of cells accumulating PHB was observed for all strains except for K12 and W, which seemed to be due either to the overexpression of the foreign PHA biosynthesis enzymes or to the accumulation of PHB. (c) 1994 John Wiley & Sons, Inc.