The incidence and the valuation of eaeA gene of enteropathogenic Escherichia coli (EPEC) or aggR gene of enteroaggregative E. coli (EAggEC) in the strains isolated from patients in sporadic diarrhea cases

To clarify the pathogenic role of enteropathogenic Escherichia coli (EPEC) or enteroaggregative E. coli (EAggEC), the possession of eaeA gene of EPEC or aggR gene of EAggEC in the strains isolated from 525 patients in sporadic diarrhea cases during 3 years (1998-2000) in Tama, Tokyo was investigated by a PCR method. The eaeA-positive E. coli strains were confirmed from 23 cases including 5 cases detected verotoxin-producing E. coli (VTEC), and those except VTEC strains (18 cases, 3.4%) were the 5th predominant enteropathogen following rotavirus, Campylobacter, adenovirus, and Salmonella. By age, 17 eaeA-positive cases were from children < 10 years of age, and noticeably, of which 9 were from infants < 24 months of age. On the other hand, although aggR-positive E. coli strains were detected from 11 cases (2.1%), of which 6 also were from infants < 24 months of age. Clinical symptoms of patients whom eaeA or aggR gene-positive E.coli was isolated as the only potential enteric pathogen were similar, showing a mild gastroenteritic features. Only one strain of eaeA-positive E. coli and 4 of aggR-strains were typed with the commercial O-antisera, which were O55, and O86, O111 or O126. In antibiotic sensitivity tests for 9 agents, 22% of eaeA-strains and 91% of aggR-strains showed resistant, especially 10 aggR-strains had resistant to ABPC. These findings suggest that these organisms are a significant causative agents of infantile diarrhea and the PCR method is a useful procedure for the diagnosis of EPEC or EAggEC infectious disease.     

The prevalence of virulence-related genes, eaeA, aggR and astA, of localized and aggregative-adherent Escherichia coli (EPEC and EAggEC) in healthy children and age-matched patients with diarrhea

The prevalence of virulence-related genes of localized- and aggregated-adherent Escherichia coli (EPEC and EAggEC), such as eaeA, aggR and astA was compared between E. coli isolated from 0 to 5 year old children with and without diarrhea in Saga Prefecture. In the case of eaeA, 233 cases in Aichi Prefecture were included. The subjects were 74 diarrheal patients from which no diarrheagenic bacteria were detected besides E. coli. The control subjects were 304 nursery school children without diarrhea, and E. coli was isolated from 278 children in which 105 strains were of 0-serotype. EaeA-positive E. coli was isolated from nine (12.2%) Saga cases, 19 (8.2%) Aichi cases and 6 (5.7%) control subjects; aggR-positive E. coli was isolated from 10 (13.5%) cases and 6 (5.7%) control subjects and astA-positive E. coli from 10 (13.5%) cases and 14 (13.3%) control subjects. No significant difference (p > 0.05) was observed in the prevalence of eaeA, aggR and astA between healthy and diarrheal children, even in age-matched and 0-serotypable E. coli limited comparisons. The pathogenicity of EPEC and EAggEC should be investigated, considering other known or unidentified factors.     

Atypical enteropathogenic Escherichia coli strains: phenotypic and genetic profiling reveals a strong association between enteroaggregative E. coli heat-stable enterotoxin and diarrhea

The virulence profiles of most atypical enteropathogenic Escherichia coli (EPEC) strains are unknown. A total of 118 typical and atypical strains of EPEC serotypes and non-EPEC serogroups isolated from children with or without acute diarrhea who were from different cities in Brazil were examined for virulence-associated markers and adherence to HEp-2 cells, and also had random amplified polymorphic DNA (RAPD) analysis performed. Atypical strains were identical to typical strains with regard to the virulence factors encoded on the locus of enterocyte effacement (LEE). In contrast with typical EPEC strains, none of the atypical strains reacted with the bfpA probe, and half of the strains hybridized with the perA probe. Most atypical strains presented Tir sequences that correlated with enteropathogenic or enterohemorrhagic E. coli (98%), had LEE inserted in either selC or pheU (88%), and presented a typeable intimin (52%). Eighteen new serotypes were found in the EPEC strains. Atypical and typical EPEC strains belonged to different RAPD clusters. Most atypical strains showed a localized-like adherence pattern (61.5%). Of the non-LEE-encoded virulence factors, enteroaggregative E. coli heat-stable enterotoxin was noted most frequently (45%) and was significantly associated with diarrhea (P=.01). Thus, this virulence marker may be used as an additional tool for the diagnosis of truly atypical pathogenic strains.     

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2%) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66%) were typical (escv+, bfp+) and 14 (34%) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90%) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.     

Prevalence of different virulence factors and biofilm production in enteroaggregative Escherichia coli isolates causing diarrhea in children in Ifakara (Tanzania)

This study investigated the prevalence of 19 virulence factors and biofilm production in 86 EAEC isolates causing diarrhea in children less than 5 years of age from Ifakara, Tanzania. Virulence factors were detected by PCR, whereas biofilm production was determined using a microtiter plate assay. No virulence factor, with the exception of the aat gene used to identify EAEC, was detected in 11/86 isolates (12.8%). The most frequently detected virulence factor was the aggR gene in 53 (61.6%) EAEC, followed by antigen 43 in 33.7%, dispersin in 26.7%, yersiniabactin in 22.1%; autrotransporter Sat in 20.9%; Shigella enterotoxin-1 in 16.3%, and heat-stable toxin-1 in 15.1%. Biofilm was produced in 66/86 (76%) isolates. AggR was the most prevalent virulence factor in the biofilm-forming group (65% versus 38%, P = 0.032). These results again show the high heterogeneity of virulence factors among EAEC isolates causing diarrhea in children, and that biofilm may be an important virulence factor, strongly associated with the presence of AggR.     

Rapid modulation of electrolyte transport in Caco-2 cell monolayers by enteropathogenic Escherichia coli (EPEC) infection

Background and aims—The pathophysiology ofenteropathogenic Escherichia coli (EPEC) diarrhoea remainsuncertain. EPEC adhere to enterocytes and transduce signals whichproduce a characteristic "attaching and effacing" (A/E) lesion inthe brush border membrane. The present in vitro study was designed todetermine whether signal transduction by EPEC also influenceselectrolyte transport.
Methods—Caco-2 cell monolayers were rapidlyinfected with wild type EPEC strain E2348/69, or the signaltransduction-defective mutant 14.2.1(1), and mounted in Ussing chambers.
Results—Strain E2348/69 stimulated a rapid buttransient increase in short circuit current (Isc) whichcoincided with A/E lesion formation; this Isc response wasabsent on infection with strain 14.2.1(1). While the initial rise inIsc induced by E2348/69 was partially (~35%) dependenton chloride, the remainder possibly represents an influx of sodium andamino acid(s) across the apical membrane.
Conclusions—The study directly shows that, afterinitial adhesion, EPEC induce major alterations in host cellelectrolyte transport. The observed Isc responses indicatea rapid modulation of electrolyte transport in Caco-2 cells by EPEC,including stimulation of chloride secretion, for which signaltransduction to host cells is a prerequisite.

Keywords:enteropathogenic Escherichia coli; Caco-2 cells; Ussing chambers; electrolyte transport; signaltransduction

     

Secretory immunoglobulin A (sIgA) in saliva versus virulence proteins of enteropathogenic Escherichia coli (EPEC) in ill and colonized children

IntroductionWe evaluated the presence of sIgA in saliva, versus Escherichia coli secreted proteins (Esp) related to the type III secretion system (T3SS), and its semi-quantitative concentration in children under 2 years-old (no longer breastfed) who were previously colonized or infected with enteropathogenic E. coli (EPEC).MethodsWe analyzed the presence of sIgA in 40 children, who previously had positive cultures for EPEC associated (n = 17) or not associated (n = 23) with diarrhea, using the Western Blot technique versus E. coli secreted proteins: EspABCD. A semi-quantitative measurement of the reaction for each protein was made by its density peaks (OD).ResultsWe found sIgA versus all or some EspABCD proteins in both groups. However, the ill patients had higher concentrations of these antibodies than colonized patients.DiscussionThe presence of sIgA in saliva could reflect an intestinal immune response and their levels could be related to a greater exposure and/or bacterial load.     

Phenotypic and genotypic characteristics of Escherichia coli strains of non-enteropathogenic E. coli (EPEC) serogroups that carry EAE and lack the EPEC adherence factor and Shiga toxin DNA probe sequences

This study was conducted to characterize the virulence potential of 59 Escherichia coli strains carrying EAE and lacking the enteropathogenic E. coli adherence factor and Shiga toxin probe sequences. In hybridization studies, all strains carried the locus of enterocyte effacement (LEE)-associated DNA sequences. Of the other 15 virulence DNA sequences tested, HLY was the most frequent (44.1%); 17 combinations of these sequences were found, but strains carrying EAE only (EAE profile) were the most frequent (35.6%). Except for 1 cytodetaching strain, all others adhered to HeLa and Caco-2 cells, most of which (approximately 75.0%) showed variations of the localized adherence pattern. Actin accumulation was detected in 75.9% of the nondetaching strains. Most strains had LEE, probably inserted in pheU (49.2%), and presented a nontypeable intimin (83.1%). Translocated intimin receptor-derived DNA sequences correlated with enteropathogenic and enterohemorrhagic E. coli in 61.0% and 32.0% of the strains, respectively. Thirty-five different serotypes were found. Only strains with the EAE profile were associated with diarrhea (P=.039).     

Rapid Identification of Colonization Factor Antigens (CFA/I and CFA/II) of Enterotoxigenic E. coli (ETEC) and O-Antigens of Enteropathogenic E. coli (EPEC) by Coagglutination Test

Specific antisera for CFA/I (O78), CFA/II (O6) or enteropathogenic (EPEC) somatic Oantigens (serogroups O26, O86a and O111) were adsorbed to Staphylococcus aureus (strain Cowan I) to produce coagglutination reagents. Conventional agglutination tests with bacterial cells showed that antiserum against strain C922a-1 reacted with strain C91f, whereas anti-C91f did not agglutinate cells of strain C922a-1. Antisera raised against strains 4334f and C91f agglutinated their bacterial cells. Anti-303d did not react with strains C922a-1, 91f, nor with 4334f. Similar results were obtained with tests performed with COA-reagents of the above strains. Homologous antisera diluted 1: 1000 gave positive COA reaction, after being adsorbed to Cowan I. Such diluted antisera gave negative agglutination reaction in the conventional agglutination tests. The COA test is at least 100 times more sensitive than the conventional agglutination test. Hence, preparation of COA reagents implies practically economical use of antisera. Furthermore, the COA technique is much more sensitive and rapid (< 5 min) than immundiffusion test which takes ≥ 24 h to read and in which large molecular weight antigens fail to diffuse in to agarose. 30 CFA/I strains (100%) gave COA positive reaction and only 25 CFA/II strains (71,4%) were positive with CFA/II COA reagents. The coagglutination technique was also applicable to identification of enteropathogenic E. coli by using somatic O-antigens (026, 086a and Olli) antisera. Sensitized Staphylococcus aureus (Cowan I) by EPEC O-antigen sera diluted to ≥ 1/1500 could specifically aggregate strains belonging to corresponding O-serog-roup. In conclusion the COA technique is an appropriate, rapid diagnostic tool which can easily be used for epidemiological studies in developing and industrialized countries.     

Phenotypic diversity of enterotoxigenic Escherichia coli (ETEC) isolated from cases of travelers' diarrhea in Kenya.

BACKGROUND: The aim of this study was to characterize phenotypically enterotoxins, colonization factors (CFs) and the antibiotic susceptibility of enterotoxigenic Escherichia coli (ETEC) strains isolated from cases of acute diarrhea that occurred in Europeans traveling to resorts in Mombasa, Kenya; this information is critical for the development of vaccines and empirical treatment. METHODS: Over a 1-year period from 1996 to 1997, five E. coli-like colonies were obtained from each of 463 cases with acute diarrhea. These strains were characterized for enterotoxins using GM-1 ELISA, for CFs using a dot-blot assay, and for antibiotic susceptibility using antibiotic disks. RESULTS: Of 164 strains characterized for ETEC phenotype, 30 (18%) expressed heat-labile toxin (LT) only, 83 (51%) heat-stable toxin (ST) only, and 51 (31%) both LT and ST. Analysis for CF expression demonstrated that 107 (65%) of the strains were positive for CFs, including CFA/IV (46%), CFA/II (35%), and CFA/I (5%), while less than 4% expressed less common CFs. All ETEC strains tested were resistant to erythromycin and sensitive to ceftriaxone. Over one-third of the strains were resistant to sulfamethoxazole-trimethoprim or tetracycline. Six strains were resistant to nalidixic acid; none of these were resistant to ciprofloxacin. CONCLUSIONS: Cumulatively, our findings indicate that ETEC in this region comprises a highly diverse group of bacterial enteropathogens, and that the development of prophylactic agents against ETEC faces major challenges because of this diversity.     

Nucleotide sequence of the Escherichia coli prolipoprotein signal peptidase (lsp) gene.

The nucleotide sequence of the prolipoprotein signal peptidase (lsp) gene has been determined. The lsp gene was found to be adjacent to the isoleucyl-tRNA synthetase ( ileS ) gene, such that the termination codon of the ileS gene overlaps with the initiation codon of lsp. These two genes are transcribed in the same direction and the major promotor for the lsp gene appears to be upstream of ileS . Identification of the lsp gene was established by amplification of prolipoprotein signal peptidase activity in strains carrying a subcloned 1.1-kilobase Stu I-Acc I fragment and was further confirmed by introducing mutational alterations in the COOH terminus of the protein that caused a decrease in prolipoprotein signal peptidase activity. The deduced amino acid sequence indicates that prolipoprotein signal peptidase contains 164 residues. Unlike most exported proteins, there is no apparent signal peptide sequence for the lsp protein. Computer-assisted secondary structure analysis of the deduced amino acid sequence identified four hydrophobic regions that share features common to transmembrane segments in integral membrane proteins.     

No evidence of the Shiga toxin-producing E. coli O104:H4 outbreak strain or enteroaggregative E. coli (EAEC) found in cattle faeces in northern Germany, the hotspot of the 2011 HUS outbreak area

Background

Ruminants, in particular bovines, are the primary reservoir of Shiga toxin-producing E. coli (STEC), but whole genome analyses of the current German ESBL-producing O104:H4 outbreak strain of sequence type (ST) 678 showed this strain to be highly similar to enteroaggregative E. coli (EAEC). Strains of the EAEC pathotype are basically adapted to the human host. To clarify whether in contrast to this paradigm, the O104:H4 outbreak strain and/or EAEC may also be able to colonize ruminants, we screened a total of 2.000 colonies from faecal samples of 100 cattle from 34 different farms - all located in the HUS outbreak region of Northern Germany - for genes associated with the O104:H4 HUS outbreak strain (stx2, terD, rfb _O104, fliC _H4), STEC (stx1, stx2, escV), EAEC (pAA, aggR, astA), and ESBL-production (bla _CTX-M, bla _TEM, bla _SHV).

Results

The faecal samples contained neither the HUS outbreak strain nor any EAEC. As the current outbreak strain belongs to ST678 and displays an en-teroaggregative and ESBL-producing phenotype, we additionally screened selected strains for ST678 as well as the aggregative adhesion pattern in HEp-2 cells. However, we were unable to find any strains belonging to ST678 or showing an aggregative adhesion pattern. A high percentage of animals (28%) shed STEC, corroborating previous knowl-edge and thereby proving the validity of our study. One of the STEC also harboured the LEE pathogenicity island. In addition, eleven animals shed ESBL-producing E. coli.

Conclusions

While we are aware of the limitations of our survey, our data support the theory, that, in contrast to other Shiga-toxin producing E. coli, cattle are not the reservoir for the O104:H4 outbreak strain or other EAEC, but that the outbreak strain seems to be adapted to humans or might have yet another reservoir, raising new questions about the epidemiology of STEC O104:H4.     

The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli.

The in vitro and in vivo analysis of the ribonuclease E-deficient (rne-) and the altered mRNA stability protein-deficient (ams-) strains of Escherichia coli has demonstrated that they carry mutations in the same structural gene. Strains encoding either thermolabile RNase E (rne-3071) or Ams protein (ams-1) are defective in both rRNA processing and mRNA turnover. Immediately after a shift to the nonpermissive temperature, the chemical decay rate of bulk mRNA is slowed 2- to 3-fold, and within 70 min, precursors to 5S rRNA begin to accumulate. In addition, all of the phenotypes associated with either the rne-3071 or the ams-1 alleles were complemented by a recombinant plasmid carrying ams+. When taken together with previous genetic studies, these results suggest that the role of ribonuclease E in mRNA turnover involves endonucleolytic cleavages at the proposed ACAG(A/U)AUUUG consensus sequence.     

Interaction of Escherichia coli dnaB and dnaC(D) gene products in vitro.

Purified E. coli dnaB and dnaC(D) gene products interact physically and functionally in vitro. This interaction was demonstrated as follows: (a) A complex of dnaB and dnaC(D) gene products was isolated by gel filtration; ATP specifically was required for isolation of the complex. (b) The DNA-independent ribonucleoside triphosphatase activity associated with dnaB gene product was inhibited by dnaC(D) gene product. (c) The dnaC(D) gene product was protected from inactivation by N-ethyl-maleimide by the combination of dnaB gene product and ATP; this protection required ATP specifically.     

Cloning of the structural gene (ompA) for an integral outer membrane protein of Escherichia coli K-12.

The gene (ompA) for the major outer membrane protein II* from Escherichia coli K-12 has been cloned on a 5-megadalton EcoRI fragment by using phage lambda as vector. The gene is expressed during the lytic cycle of the recombinant phage and the insoluble membrane-bound protein was detected in phage plaques with a simple radioimmunoassay. Transfer of the EcoRI fragment into plasmid pSC101 and expression in a host lacking protein II* led to overproduction of protein II* and decreased production of two other major outer membrane proteins. Expression of the plasmid pSC101-ompA+ in minicells derived from an ompA minicell-producing strain led to synthesis, at high rates, of this protein and massive accumulation of a second cell envelope protein most likely representing the biosynthetic precursor of protein II*.     

Reactivity of primary biliary cirrhosis sera with Escherichia coli dihydrolipoamide acetyltransferase (E2p): characterization of the main immunogenic region.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial autoantibodies in the serum. The major antigens recognized by the antibodies are the E2 components of the 2-oxo acid dehydrogenase complexes, all of which possess covalently attached lipoic acid cofactors. A bacterial etiology has been proposed for the disease, and patients' antibodies are known to recognize the E2 subunits (E2p) of both mammalian and bacterial pyruvate dehydrogenase complexes. Immunoblotting and ELISA inhibition techniques using extracts of Escherichia coli deletion strains, genetically restructured E2 polypeptides, and isolated lipoyl domains demonstrate that (i) the E2o subunit of the E. coli 2-oxoglutarate dehydrogenase complex is recognized by patients' antibodies; (ii) the main immunogenic region of E2p lies within the lipoly domains; (iii) the presence of a lipoly residue within the domain is crucial for effective recognition by the antibodies; and (iv) octanoylated E2p, octanoylated E2o, and octanoylated lipoyl domain, produced by a mutant deficient in lipoate biosynthesis, are recognized by patients' antibodies but not as effectively as their lipoylated counterparts. These findings indicate that antibodies in PBC patients' sera bind to a unique peptide-cofactor conformation within the lipoyl domains of the E2 polypeptides and that this epitope is partially mimicked by substituting the lipoyl cofactor with an octanoyl group.     

Isolation and characterization of a second RNase H (RNase HII) of Escherichia coli K-12 encoded by the rnhB gene.

An additional RNase H (EC 3.1.26.4), RNase HII, has been isolated from Escherichia coli K-12. By screening a library of E. coli DNA for clones that suppressed RNase H deficiency of an E. coli rnh mutant, a clone was obtained that produced a protein with RNase H activity. The overexpressed RNase HIII protein in E. coli was purified to near homogeneity and exhibited a strong preference for the ribonucleotide moiety of RNA-DNA hybrid as substrate. The terminal 11 amino acids were determined and were identical to those predicted from the nucleotide sequence. The rnhB gene, which encodes RNase HII, was distinct from rnhA by its map position (4.5 min on E. coli genetic map, between lpxB and dnaE) and by the lack of significant amino acid sequence similarity. The presence of a second RNase H in E. coli indicates that multiple RNase H genes per genome is a general feature of a general feature of a wide variety of organisms.