Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.     

Characterization of enteroaggregative Escherichia coli isolates

Forty enteraggregative Escherichia coli (EAggEC) previously characterized by their ability to adhere to HEp-2 cells or/and their hybridization with the 1-kb EAggEC DNA probe were investigated for the presence of adherence factors and heat-stable enterotoxin (EAST1)-encoding genes. Only 45% of the isolates harbored the EAST1-encoding genes as detected by polymerase chain reaction. None of them hybridized with an AAF/II-encoding gene specific DNA probe and 35% (14/40) were positive in a PCR assay using primers specific for aggC, an accessory gene of the AAF/I-encoding operon. Cloning and sequence analysis of the aggA variant from one isolate, EAggEC 457, revealed 68.9% identity between its deduced amino acid sequence and those of the aggA product from the AAF/I-producing reference strain, E. coli 17.2. No major protein subunit was detected at the surface of EAggEC 457 compared to the bacterial surface extract of E. coli 17.2.     

Enterovirulent <Emphasis Type="Italic">E. coli</Emphasis> in inflammatory and noninflammatory bowel diseases

We determined the incidence of enterovirulent E. coli (EVEC; which can to cause gastrointestinal infections) in strains isolated from patients with both of the major inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) and from patients with noninflammatory bowel diseases (nonIBD). Cell detachment E. coli (CDEC) were detected in 14 % of all strains. A significant difference in the presence of CDEC was found between the groups of strains isolated from UC (24.1 %), nonIBD (11.9 %) and CD (4.7 %). Enteroaggregative E. coli (EAggEC) were detected in 2.5 %, typical enteropathogenic strains (EPEC) in 1.3 % and enterotoxigenic ones (ETEC) in 1.5 %. Enteroinvasive (EIEC) and shigatoxin producing E. coli (STEC) were not detected. Some strains showed a high invasion level in gentamicin-protection assay. These strains could therefore belong to adherent-invasive E. coli (AIEC) because they are free of genes encoding invasins (ipaH, ial) and are equipped with fimA gene. However, complete characterization of these strains and their classification as AIEC will require further tests.     

Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin‐producing Escherichia coli isolated from raw products in Argentina

A total of 73 Shiga toxin‐producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin‐binding fimbriae), sfpA (sorbitol‐fermenting EHEC O157 fimbriae plasmid‐encoded) and of the toxigenic gene cdt‐V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt‐V toxin in LEE‐negative STEC strains that occur in foods, and this traits could increase their pathogenic potential.

Significance and Impact of the Study

Meat products are one of the main vehicles of Shiga toxin‐producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.     

Virulence characteristics of Shiga toxin‐producing Escherichia coli from raw meats and clinical samples

Shiga toxin producing Escherichia coli (STEC) are dangerous foodborne pathogens. Foods are considered as important sources for STEC infection in human. In this study, STEC contamination of raw meats was investigated and the virulence factors of 120 clinical STEC strains characterized. STEC was detected in 4.4% of tested samples. Among 25 STEC strains from meats, five strains (20%) were positive for the eae gene, which encodes intimin, an important binding protein of pathogenic STEC. The remaining strains (80%) were eae‐negative. However, 28% of them possessed the saa gene, which encodes STEC agglutinating adhesin. The ehxA gene encoding for enterohemolysin was found in 75% of the meat strains and the subAB gene, the product is of which subtilase cytotoxin, was found in 32% of these strains. The stx_2a gene, a subtype of Shiga toxin gene (stx), was the most prevalent subtype among the identified meat STEC bacteria. None of the meat STEC was O157:H7 serotype. Nevertheless, 92% of them produced Shiga toxin (Stx). Among 120 clinical STEC strains, 30% and 70% strains harbored single and multiple stx subtypes, respectively. Most clinical STEC bacteria possessed eae (90.8%) and ehxA (96.7%) genes and 92.5% of them showed Stx productivity. Our study shows that some raw meat samples contain non‐O157 STEC bacteria and some strains have virulence factors similar to those of clinical strains.     

Marine hydrocarbonoclastic bacteria as whole‐cell biosensors for n‐alkanes

Whole‐cell biosensors offer potentially useful, cost‐effective systems for the in‐situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole‐cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25‐fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3–4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 μM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis.     

Simple Measurement of Blood NADP and Blood Levels of NAD and NADP in Humans

The tryptophan synthase genes, trpA and trpB, of Bacillus stearothermophilus IFO13737 were cloned by transformation of tryptophan auxotrophic mutations of the trp genes into Escherichia coli. The genes are located in the order of trpB and trp A, according to their coding orientation, in a 2.5 kb EcoRy-Hindlll DNA fragment. The complete nucleotide sequence of this DNA was determined. The trp A and trpB genes consist of 810bp (269 amino acid residues) and 1215bp (404 amino acid residues), respectively. The 5′-proximal portion of the trpB gene was found to overlap 20 nucleotides of the upstream coding region of the trpA gene. The homology of the amino acid sequences of the trp gene products of trp A and trpB of B. stearothermophilus is 35 and 50 %, respectively, to those of E. coli, and 55 and 70 %, respectively, to those of B. subtilis.     

Epidemiological investigation of <Emphasis Type="Italic">eaeA</Emphasis>-positive <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Escherichia albertii</Emphasis> strains isolated from healthy wild birds

Escherichia coli has commonly been associated with diarrheal illness in humans and animals. Recently, E. albertii has been reported to be a potential pathogen of humans and animals and to be carried by wild birds. In the present study, the prevalence and genetic characteristics of intimin-producing E. coli and E. albertii strains were evaluated in wild birds in Korea. Thirty one of 790 Enterobacteriaceae strains from healthy wild birds were positive for the intimin gene (eaeA) and twenty two of the 31 strains were identified as atypical enteropathogenic E. coli (aEPEC) that did not possess both EAF and bfpA genes. A total of nine lactose non-fermenting coliform bacterial strains were identified as E. albertii by PCR and sequence analysis of housekeeping genes. A total of 28 (90.3%) eaeA-positive strains were isolated from waterfowl. Fifteen aEPEC (68.2%) and two E. albertii (22.2%) strains had a β-intimin subtype and 14 aEPEC strains harboring β-intimin belonged to phylogenetic group B2. AU eaeA-positive E. albertii and 3 aEPEC strains possessed the cytolethal distending toxin gene (cdtB). The eaeA-positive E. coli and E. albertii strains isolated from healthy wild birds need to be recognized as a potential pathogroup that may pose a potential threat to human and animal health. These findings indicate that eaeA-positive E. coli as well as E. albertii can be carried by wild birds, posing a potential threat to human and animal health.     

和厚朴酚对大肠埃希氏菌生物被膜形成的抑制机制

【目的】研究不同浓度的和厚朴酚(honokiol)抑制大肠埃希菌(Escherichia coli)的供试菌株10389生物被膜(biofilm,BF)形成的作用机制。【方法】用氯化三苯基四氮唑比色法(TTC)和四唑盐减低法(XTT)测定honokiol抑制E.coli10389生物被膜形成的药物最低抑菌浓度(MIC)和最低杀菌浓度(MBC)及其抑制作用与时间的关系;通过qRT-PCR法检测不同浓度的honokiol对E. coli 10389生物被膜形成基因和群体感应系统相关基因表达量的影响;通过生物发光法和qRT-PCR法检测亚-MIC honokiol对E. coli 10389呋喃糖基硼酸二酯(AI-2)及其调控的与生物被膜形成相关的下游基因表达量的影响。【结果】Honokiol能抑制E.coli10389生物被膜的形成,但不同浓度的honokiol抑制E. coli 10389 BF形成的作用机制不同。其中,与对照组相比,MIC的honokiol能使E. coli 10389 BF形成相关基因编码毒素(hha)和细菌酸性调节因子(ari R) mRNA的表达量显著提高,抗毒素...     

Antimicrobial‐resistant faecal Escherichia coli in wild mammals in central Europe: multiresistant Escherichia coli producing extended‐spectrum beta‐lactamases in wild boars

Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli in populations of wild mammals in the Czech Republic and Slovakia. Methods and Results: Rectal swabs or faeces collected during 2006–2008 from wild mammals were spread on MacConkey agar and MacConkey agar containing 2 mg l^?1 of cefotaxime. From plates with positive growth, one isolate was recovered and identified as E. coli. Susceptibility to 12 antibiotics was tested using the disk diffusion method. Resistance genes, class 1 and 2 integrons and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). Extended‐spectrum beta‐lactamases (ESBL) were further characterized by DNA sequencing, macrorestriction profiling and determination of plasmid sizes. Plasmid DNA was subjected to EcoRV digestion, transferability by conjugation and incompatibility grouping by multiplex PCR. The prevalence of resistant isolates was 2% in small terrestrial mammals (rodents and insectivores, n_E. coli = 242), 12% in wild ruminants and foxes (n_E. coli = 42), while no resistant isolates were detected in brown bears (n_E. coli = 16). In wild boars (Sus scrofa) (n_E. coli = 290), the prevalence of resistant isolates was 6%. Class 1 and 2 integrons with various gene cassettes were recorded in resistant isolates. From wild boars, five (2%, n_{rectal smears} = 293) multiresistant isolates producing ESBL were recovered: one isolate with bla_CTX‐M‐1 + bla_TEM‐1, three with bla_CTX‐M‐1 and one with bla_TEM‐52b. The bla_CTX‐M‐1 genes were carried on approx. 90 kb IncI1 conjugative plasmids. Conclusions: Antibiotic‐resistant E. coli occured in populations of wild mammals in various prevalences. Significance and Impact of the Study: Wild mammals are reservoirs of antibiotic‐resistant E. coli including ESBL‐producing strains which were found in wild boars.     

Frequency of indicator bacteria,Salmonella and diarrhoeagenic Escherichia coli pathotypes on ready‐to‐eat cooked vegetable salads from Mexican restaurants

The presence of coliform bacteria, faecal coliforms, Escherichia coli, diarrhoeagenic E. coli pathotypes (DEP) and Salmonella were determined in ready‐to‐eat cooked vegetable salads (RECS) from restaurants in Pachuca city, Mexico. The RECS were purchased from three types of restaurants: national chain restaurants (A), local restaurants (B) and small restaurants (C). Two restaurants for each A and B, and three for C, were included. Forty RECS samples were purchased at each A and B restaurant and 20 at each C restaurant. Of the overall total of 220 analysed samples, 100, 98·2, 72·3, 4·1 and 4·1% had coliform bacteria, faecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC). The EPEC, ETEC and STEC were isolated each from 1·4% of samples. No E. coli O157:H7 were detected in any STEC‐positive samples. The analysis of Kruskal–Wallis anova and median test of microbiological data showed that the microbiological quality of RECS did not differ between the different restaurants (P > 0·05).

Significance and Impact of the Study

This is the first report regarding microbiological quality and Salmonella, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC) isolation from ready‐to‐eat cooked vegetable salads from Mexican restaurants. Ready‐to‐eat cooked vegetable salads could be an important factor contributing to the endemicity of EPEC, ETEC and STEC, and Salmonella caused gastroenteritis in Mexico.     

Cloning a Truncated Fragment (stx2a<Subscript>1</Subscript>) of the Shiga-Like Toxin 2A<Subscript>1</Subscript> Subunit of EHEC O157:H7: Candidate Immunogen for a Subunit Vaccine

Shiga toxins consist of enzymatically active A and B subunit multimers. The A subunit of shiga-like toxins can be proteolytically cleaved into two parts, A₁ and A₂, with A₁ being responsible for toxic activity. Antibody neutralizing the A₁ subunit of shiga toxin may protect against infection of the enterohemorrhagic Escherichia coli (EHEC O157:H7). It was difficult to express the full-length A₁ subunit of shiga toxin 2 (stx2A₁) in a previous study. We have now analyzed the full-length of stx2A₁ using bioinformatics software. The data show that the carboxyl terminal (of ~15 amino-acid residues) has strong hydrophobicity and low antigenicity. We cloned and expressed a truncated fragment of stx2A₁ (15 amino-acid residues of the carboxyl terminal being removed), designated stx2a₁, which can evoke a humoral immune response. Anti-Stx2a₁ antibodies can neutralize the native shiga toxin 2 both in vivo and in vitro, which suggests that Stx2a₁ serves as a candidate immunogen for a subunit vaccine that can also be used as the antigen to screen phage anti-shiga toxin antibody libraries. L. Liu and H. Zeng contributed equally to this study.     

白花丹素对E.coli藻酸盐合成的抑制作用

细菌分泌的胞外多糖在生物被膜的形成和发展过程中发挥着重要作用。通过测定白花丹素对大肠埃希菌10389菌株(E.coli 10389)藻酸盐合成的影响及其对rse A和rpo E基因表达量的影响,探讨白花丹素对大肠埃希菌生物被膜(biofilm,BF)形成的抑制作用及机制。研究结果显示,白花丹素能抑制E.coli 10389生物被膜的形成,其抑杀E.coli 10389的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimal bactericidal concentration,MBC)分别为16和64μg/mL。白花丹素对成熟BF内的细菌也有抑制和杀灭作用,其抑杀E.coli 10389成熟BF内细菌的MIC和MBC分别为64和128μg/mL。白花丹素能够抑制E.coli 10389藻酸盐的合成,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组比,藻酸盐的合成量降低了34.83%(P0.01)。白花丹素可显著影响E.coli 10389 rse A和rpo E基因的相对表达量,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组相比,rse A的表达量上调了17.43%,rpo E的表达量降低了12.8%(P0.05)。结果表明,白花丹素能够抑制E.coli 10389 BF的形成,其作用机制可通过影响rse A和rpo E的基因表达量,进而抑制藻酸盐的合成来抑制大肠埃希菌生物被膜的形成。     

The prevalence of plasmid‐mediated quinolone resistance determinants among clinical isolates of ESBL or AmpC‐producing Escherichia coli from Chinese pediatric patients

Three kinds of PMQR determinants (qnr genes, aac(6’)‐Ib‐cr, and qepA) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants in ESBL or AmpC‐producing E. coli clinical isolates in Chinese children, a total of 292 ESBL or AmpC‐producing E. coli clinical isolates collected from five children's hospitals in China from 2005 to 2006 were screened for PMQR determinants by PCR. Twenty (6.8%) of the 292 isolates were positive for PMQR determinants. A total of 12 (4.1%) isolates were positive for qnr genes, comprising three positive for qnrA (1.0%), three for qnrB (1.0%), and six for qnrS (2.1%). Twenty‐four (8.2%) isolates were positive for aac(6’)‐Ib, of which 10 (3.4% of 292) had the –cr variant. There was no qepA gene detected in the isolates. Conjugation revealed that qnr, aac(6’)‐Ib‐cr, and ESBL‐encoding genes were transferred together.     

Effective antimicrobial activity of Cbf‐K16 and Cbf‐A7A13 against NDM‐1‐carrying Escherichia coli by DNA binding after penetrating the cytoplasmic membrane in vitro

New Delhi metallo‐beta‐lactamase‐1(NDM‐1)‐carrying isolates, which are resistant to most clinical used antibiotics except for tigecycline and colistin, have been found worldwide. Cathelicidin‐BF (BF‐30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity. Cbf‐K₁₆ and Cbf‐A₇A₁₃ were obtained by mutating Lys¹⁶, Ala⁷, and Ala¹³ of BF‐30, respectively. To investigate their antimicrobial activities against NDM‐1 carrying bacteria, recombinant Escherichia coli BL21 (DE3)‐NDM‐1 with high NDM‐1 activity was constructed by inserting the Klebsiella pneumoniae NDM‐1 gene (GenBank accession no. HQ328085) into a pET28a vector and transforming it into E. coli BL21 (DE3). The peptides showed effective antimicrobial activities against NDM‐1‐carrying E. coli, and the minimum inhibitory concentrations of Cbf‐K₁₆ and Cbf‐A₇A₁₃ were only 4 and 8 µg/ml, whereas those of minimum bactericidal concentrations were 8 and 16 µg/ml, respectively. A time course experiment showed that colony forming unit counts rapidly decreased, and bacteria were thoroughly eliminated within 3 and 6 h by the Cbf‐K₁₆ and Cbf‐A₇A₁₃ treatments, respectively. The peptides penetrated the bacterial cell membrane and enabled β‐galactosidase leakage, and caused the cytoplasmic membrane to become permeable, and finally bound to the DNA. The genomic DNA of E. coli was completely unable to migrate on an agarose gel after Cbf‐K₁₆ treatment (8 µg/ml). These data demonstrated that Cbf‐K₁₆ and Cbf‐A₇A₁₃ possess effective antimicrobial activity against drug‐resistant strains, including NDM‐1 carrying E. coli BL21 (DE3)‐NDM‐1, by binding to DNA after penetrating the cytoplasmic membrane in vitro, which may have potential therapeutic value for the treatment of NDM‐1‐carrying bacterial infections. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.