Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.     

RAPID detection and quantification of E. coli O157/O26/O111 in minced beef by real-time PCR

AIM: To develop a real-time PCR detection procedure for Escherichia coli O111, O26 and O157 from minced meat. METHODS AND RESULTS: Strains (n = 8) of each of E. coli O26, E. coli O111 and E. coli O157 were inoculated at ca 10-20 CFU g(-1) into minced retail meat and enriched for 6 h at 41.5 degrees C as follows: E. coli O26 in tryptone soya broth (TSB) supplemented with cefixime (50 microg l(-1)), vancomycin (40 mg l(-1)) and potassium tellurite (2.5 mg l(-1)); E. coli O111 in TSB supplemented with cefixime (50 microg l(-1)) and vancomycin (40 mg l(-1)); E. coli O157 in E. coli broth supplemented with novobiocin (20 mg l(-1)). DNA was extracted from the enriched cultures, and detected and quantified by real-time PCR using verotoxin (vt1 and vt2) and serogroup (O157 per gene; O26 fliC-fliA genes and O111 wzy gene) specific primers. CONCLUSIONS: The methods outlined were found to be sensitive and specific for the routine detection of E. coli O111, O26 and O157 in minced beef. SIGNIFICANCE AND IMPACT OF THE STUDY: The enrichment, isolation and detection procedures used in this study provide a rapid routine-based molecular method for the detection and differentiation of E. coli O26, O111 and O157 from minced meat.     

An amino acid substitution that blocks the deacylation step in the enzyme mechanism of penicillin-binding protein 5 of Escherichia coli

A mutant of Escherichia coli has been described that produces an altered form of penicillin-binding protein 5 which still binds penicillin but is unable to catalyse the release of the bound penicilloyl moiety. We show that the mutation is caused by a single nucleotide transition that results in a change from glycine at residue 105 of the wild-type sequence of penicillin-binding protein 5 to aspartate in the mutant.     

Alkaline phosphatase isozyme conversion by cell-free extract of Escherichia coli

Isozyme type 1 of alkaline phosphatase in Escherichia coli K-12 was converted to types 2 and 3 after incubation of type 1 isozyme with the supernatant of a sonicated cell-free extract prepared from the cells carrying the cloned iap+ gene on a multi-copy plasmid. By comparison, the lysate prepared from cells carrying the iap+ gene only on the chromosome showed much less isozyme-converting activity. The reaction was promoted by Mg2+ at concentrations of 10 to 50 mM. Protease inhibitors, antipain and leupeptin which inhibit the isozyme conversion in vivo, also inhibited the isozyme conversion in vitro. These results suggest that cells carrying the multiple copy iap+ plasmid overproduce a kind of proteolytic enzyme which removes the amino-terminal arginine residues from isozymes 1 and 2.     

一种用质粒DNA转化大肠杆菌感受态细胞的实用操作技巧

目的是建立一种简化、实用的用质粒DNA转化大肠杆菌的操作方法.采用氯化钙法制备大肠杆菌感受态细胞.以质粒pUC18,pCSN44,pAN52-1Not,pETts,pANth和植物双元表达栽体pCAMBIA1301分别转化用于质粒扩增与保存的常用大肠杆菌菌株Top10和DH5α以及用于原核表达的常用大肠杆菌菌株BL21(DE3)和TB1.质粒与感受态细胞的混合液置冰上作用一定时间后,直接涂布含有筛选抗生素的LB平板,于37℃培养12～16h.结果表明,用不同大小的质粒DNA转化不同的大肠杆菌菌株,都可以获得满足实验要求,转化效率可高达103～4阳性克隆/μg.该方法较标准的转化流程更加简便、省时、实用.     

Messenger RNA activities of four acute phase proteins during inflammation

Poly(A)+ RNA isolated from the livers of normal rats and of rats suffering from an acute inflammation was translated in a cell-free translation system from rabbit reticulocytes. The translation products were immunoprecipitated with specific antisera against alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin, alpha 1-proteinase inhibitor and albumin. 15 to 21 h after intramuscular injection of turpentine 73-, 66-, 2.8-, and 2-fold increases in translatable mRNAs for alpha 1-acid glycoprotein, alpha 2-macroglobulin, transferrin and alpha 1-proteinase inhibitor, respectively, were observed. For albumin a decrease in translatable mRNA to about 30% of controls was measured.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

重组人甲状旁腺激素肽（1-34）在大肠杆菌中的高效融合表达及纯化

人工合成人甲状旁腺激素1-34肽段 (PTH1-34)的cDNA序列,克隆到大肠杆菌蛋白表达载体pThioHis中,获得了高表达菌株。经发酵、破菌、金属鳌合层析、反相层析和凝胶层析后获得了纯度大于95%的hPTH1-34。hPTH1-34肽N端测序和质谱分子量测定结果与天然PTH1-34一致。生物学活性研究表明,hPTH1-34在体外具有刺激腺苷酸环化酶的作用。     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Identification of EaeA protein in the outer membrane of attaching and effacing Escherichia coli O45 from pigs

Abstract We have previously reported that the production of attaching and effacing lesions by Escherichia coli O45 isolates from pigs is associated with the eaeA ( E. coli attaching and effacing) gene. In the present study, expression of the EaeA protein, the eaeA gene product, among swine O45 E. coli isolates was examined. The majority (20/22) of attaching and effacing positive, eaeA⁺ E. coli O45 isolates, but none of ten attaching and effacing negative, eaeA⁻ or eaeA⁺ isolates, expressed a 97-kDa outer membrane protein as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. Amino-terminal amino acid sequencing demonstrated a high homology between this 97-kDa protein of swine E. coli O45 and the EaeA protein (intimin) of human enteropathogenic E. coli and enterohemorrhagic E. coli . In addition, a serological relationship between the EaeA proteins of swine O45, rabbit (RDEC-1) and human (E2348/69) attaching and effacing E. coli strains was observed. Our results indicate an association between expression of the EaeA protein and attaching and efficacing activity among O45 E. coli isolates. The data also suggest an antigenic relatedness of the EaeA proteins of swine, rabbit, and human attaching and effacing E. coli .     

产超广谱β-内酰胺酶大肠埃希菌Ⅰ类整合子检测与耐药性关系

目的了解I类整合子在产ESBLs和非产ESBLs大肠埃希菌中分布状况,分析I类整合子在细菌多重耐药中的作用。方法用PCR方法扩增I类整合酶基因,经电泳后检测扩增产物。用2χ检验进行统计学分析,P<0.05为差异有显著性。结果105株大肠埃希菌检出I类整合子46株,检出率为43.8%。I类整合子在产ESBLs菌的检出率为53.4%,明显高于非产ESBLs菌(31.9%),2χ检验,P<0.01。I类整合子阳性菌株多重耐药率为68.8%(33/48),明显高于阴性菌株(33.3%),P<0.05。I类整合子阳性菌株和产ESBLs菌均对青霉素类、喹诺酮类、磺胺类抗生素表现出较高的耐药率。所有菌株均对亚胺培南敏感。结论I类整合子携带与产ESBLs菌株耐药有关,I类整合子阳性菌株对多种抗生素的耐药率大于整合子阴性菌株。     

大肠杆菌44277(O2: K1) 中rmlB 基因功能

摘要: 【目的】确定rmlB 基因在大肠杆菌( O2: K1) L-型鼠李糖合成中的作用。【方法】将基因rmlB 进行原核表达并测定酶活; 用同源重组的方法将rmlB 基因敲除,分析表型变化,并运用质谱,以及核磁共振等手段分析脂多糖O 侧链的结构,以确定rmlB 在O 抗原合成中的作用。【结果】成功对rmlB 基因进行了表达并测定了重组蛋白的酶活,确定蛋白RmlB 具有dTDP-D-glucose 4,6-dehydratase 活性。成功构建了rmlB 基因缺失突变株,对突变株进行表型分析发现突变株的表型与野生株相比无变化。对突变株分析发现突变株中的O抗原仍含有L-型鼠李糖,说明在该菌株中可能存在RmlB 的同功能酶或者存在其它的L-型鼠李糖合成途径。【结论】rmlB 基因编码的蛋白具有dTDP-D-glucose 4,6-dehydratase 活性但此基因对于L-型鼠李糖的合成不是必需的。     

Effect of oligomannoside-type glycopeptides in diarrheal disease of rabbits induced by Escherichia coli strain RDEC-1

We examined the effects of a soluble receptor-like complex carbohydrate (an oligomannoside-type glycopeptide) on adherence of a type 1 fimbriated rabbit enteropathogen, Escherichia coli strain RDEC-1. Oligomannoside-type glycopeptide, but not a non-glycosylated peptide mixture used as control, inhibited adherence of type 1 fimbriated RDEC-1 to both rabbit ileal brush borders and guinea pig erythrocytes in vitro. In contrast, during RDEC-1 infection of rabbits the receptor-like oligomannoside did not affect the presence and severity of diarrhea, fecal shedding of organisms, luminal colonization by RDEC-1, or enteroadherence of organisms.     

Serogroups of Escherichia coli strains producing cytotoxic necrotizing factors CNF1 and CNF2

The serogroups of 396 necrotizing Escherichia coli of human and bovine origin isolated in Spain between 1979 and 1991 have been determined. The 270 cytotoxic necrotizing factor strains belonged to 22 different O serogroups; however, 84% (226 of 270) were of one of seven serogroups (O2, O4, O6, O14, O22, O75 and O83). Although necrotizing E. coli producing cytotoxic necrotizing factor 2 belonged to 28 different serogroups, only six of them (O1, O3, O15, O55, O88 and O123) accounted for 60% (76 of 126) of cytotoxic necrotizing factor 2 strains. Furthermore, only 3% (4 of 126) of cytotoxic necrotizing factor 2 strains belonged to serogroups most common among strains producing cytotoxic necrotizing factor 1. The majority of necrotizing E. coli producing cytotoxic necrotizing factor 1 were obtained from human extraintestinal infections, whereas cytotoxic necrotizing factor 2 strains were isolated from stools of healthy and diarrhoeic calves.     

抗菌肽GK1在大肠杆菌中的融合表达

为高效表达抗菌肽GK1并避免GK1的高抗菌活性对大肠杆菌宿主菌的致命影响, 将经改造后的人胰岛素原(mhPI)与GK1的融合基因(mhPI-GK1)克隆到表达载体pET28a中, 构建出表达质粒pET28a-mhPI-GK1, 转化至大肠杆菌BL21(DE3)中进行表达。融合蛋白在大肠杆菌中以包涵体形式表达, 表达量占菌体总蛋白的20%。经CNBr裂解、阳离子交换层析和RP-HPLC纯化后, 每升发酵液可获得5.7 mg纯度大于97%的重组GK1。质谱检测显示重组GK1的分子量为2794.0 D, 抑菌活性实验表明纯化后的重组GK1和化学合成GK1具有相同的抗菌活性。为利用基因工程方法大规模生产GK1奠定了基础。     

Serological variety of flagellar antigen H1 in natural Escherichia coli population

Abstract Variation of the Escherichia coli flagellar antigen H1 was studied among 120 human isolates belonging to more than 25 O:K serovars. Factor-specific antisera were prepared and shown to be useful in the identification of serological subtypes of H1. The three subtypes found were defined as H1abc, H1acd and H1abe. The H1abc corresponded to the standard flagellar antigen H1 present in 84% of all strains. It was found in all O groups except O15, O17 and O83. Eight O15 and two O17 strains studied were of subtype H1abe, while the one O83 strain studied was H1acd. Both subtypes H1abc and H1acd were found among strains within O6:K5, O6:K13 or O-non-typeable:K5 serovars.     

嗜热藻BP-1中几个蓝细菌光敏色素结构域体内重组及光化学性质研究

通过蛋白质序列同源性比对分析,在嗜热藻（Thermosynechococcus elongatus BP-1）里面找到了与已知的Pb/Pg型蓝细菌光敏色素TePixJ和TeTlr0924同源的3个基因tlr0911、tlr1215和tlr1999。通过分子克隆技术把它们的GAF结构域分别构建在pET30a（＋）表达载体上,与可生成藻蓝胆素（PCB）的质粒pACYCDuet-ho1-pcyA在大肠杆菌BL21（DE3）体内重组,生成重组蛋白,利用亲和层析柱分离纯化,纯化后的蛋白质经过锌荧光和蛋白质酸性尿素变性以及荧光光谱和吸收光谱等实验分析鉴定,结果表明,Tlr0911-GAF存在蓝光吸收态Pb406 nm和绿光吸收态Pg527 nm之间的可逆光转换,它可共价结合两种藻胆色素,即藻紫胆素（PVB）和藻蓝胆素（PCB）,Tlr1999-GAF则存在蓝光吸收态Pb417 nm和青光吸收态Pt496 nm之间的可逆光转换,它同样共价结合PVB和PCB,而Tlr1215-GAF1和Tlr1215-GAF2不能自发结合藻胆色素,不具有光活性。