Relative importance of enterotoxigenic and invasive enteropathogenic bacteria in infantile diarrhoea

Swedish children and adults (648 patients) with acute diarrhoea were investigated for enterotoxigenic strains in stool cultures. A total number 74 strains were isolated from 28 patients and assayed in the rabbit intestinal loop test and the adrenal cell test. Only three of the enterotoxigenic E. coli (ETEC) isolates belonged to classical enteropathogenic serotypes of E. coli (EPEC). Two enterotoxigenic strains of Proteus morganii, two of Enterobacter hafniae and one of Citrobacter freundii were isolated. None of 67 EPEC strains were found to produce a heat-labile enterotoxin (LT) in either of the two test systems. A number of Yersinia enterocolitica and Pseudomonas aeruginosa strains from stool cultures often produced toxic effects in the cell test but no enterotoxin activity was detected for any of the strains investigated either in the adrenal cell test for heat-labile enterotoxin (LT) or the suckling mouse test for heat-stable enterotoxin (ST). All EPEC isolates were also tested for ST and for invasive properties in the Sereny test; each isolate was negative in both test systems. It is concluded that production of LT and ST enterotoxin were common in stool isolates from Ethiopian children but a rare phenomenon among Swedish children with acute infantile diarrhoea. Isolation of aerobic stool bacteria with invasive properties seems to be uncommon both in Ethiopian and Swedish children. However, since both LT and ST as well as invasive properties seem to be very unstable genetic properties in many of these stool isolates improved sensitive methods for the last two properties will probably change this picture in the future.     

Evolutionary relationships among pathogenic and nonpathogenic Escherichia coli strains inferred from multilocus enzyme electrophoresis and mdh sequence studies

Within the species Escherichia coli, there are commensal strains and a variety of pathogenic strains, including enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and urinary tract infection (UTI) strains. The pathogenic strains are identified by serotype and by possession of specific virulence determinants (toxins and adhesions, etc.) encoded by either monocistronic genes, plasmids, or pathogenicity islands. Although there are studies on the relationships between selected pathogenic strains, the relatedness among the majority of the pathogenic forms to each other, to commensal E. coli, and to the genus Shigella (which has often been suggested to be part of E. coli) has not been determined. We used multilocus enzyme electrophoresis (MLEE) at 10 enzyme loci and the sequence of the mdh housekeeping gene to study the genetic relationships of pathogenic E. coli strains (including Shigella clones), namely, 5 EPEC strains (serotypes O111 and O55), 3 EHEC strains (serotype O157), 6 ETEC strains (serotypes O78, O159, and O148), 5 EIEC strains (serotypes O124, O28, and O112), and 13 Shigella strains representing clones Flexneri, Dysenteriae, Boydii, and Sonnei, to commensal E. coli strains. Both the MLEE and mdh sequence trees reveal that EPEC, EHEC, ETEC, EIEC, and UTI strains are distributed among the ECOR set groups, with no overall clustering of EPEC, ETEC, EIEC, or UTI strains. The genus Shigella is shown to comprise a group of closely related pathogenic E. coli strains. Six pathogenic strains, i.e., M502 (EIEC; O112ac:NM), M503 (EPEC; O111:H12), M526 (ETEC; O159:H4), M522 (EPEC; O111ac:H12), M524 (ETEC; O78:H11), and M506 (ETEC; O78:H11), were found to have mdh sequences identical to those of five ECOR group A strains (ECOR5, ECOR10, ECOR14, ECOR6, and K-12). All 11 strains are closely related by MLEE. The results indicate that pathogenic strains of E. coli do not have a single evolutionary origin within E. coli but have arisen many times. The results also suggest the possibility that any E. coli strain acquiring the appropriate virulence factors may give rise to a pathogenic form.     

Diffuse adherence, ST-I enterotoxin and CFA/IV colonization factor are encoded by the same plasmid in the Escherichia coli O29:H21 strain

Escherichia coli O29:H21 is a human enterotoxigenic serotype that produces heat-stable (ST-I) enterotoxin, adheres diffusely to HeLa cells, and presents colonization factor antigen IV (CFA/IV) composed of CS5CS6 surface antigens. In one strain studied the genes for diffuse adherence and CFA/IV (CS5CS6) production were found to be present in the same plasmid encoding ST-I. The virulence plasmid (Ent) presented two unrelated basic replicons homologous to repFIC and repW. Gene(s) encoding diffuse adherence did not share homology with the probe for F1845 fimbrial adhesin which is responsible for this phenotype in other E. coli strains. Ent plasmids containing genes for diffuse adherence have not been described previously.     

Evaluation of three different STb assays and comparison of enterotoxin pattern over a five-year period in Swedish porcine Escherichia coli

The pig intestinal loop (PIL) assay, inhibition enzyme-linked immunosorbent assay (ELISA), and DNA hybridization assay were compared for analysis of Escherichia coli heat-stable enterotoxin b (STb) on 201 porcine E. coli strains. The DNA hybridization had a 95% correlation with the STb ELISA and was therefore chosen as the method for subsequent screening of enterotoxin genes: heat labile (LT), heat-stable a (STa), and/or STb. In contrast to the PIL assay, both the STb ELISA and DNA hybridization assays were more sensitive, reliable, reproducible, and showed good correlation with each other. Consequently, the STb ELISA is preferable for analysis of toxin preparations and screening of E. coli, whereas the DNA hybridization is better for large-scale epidemiologic screening. Escherichia coli strains (n = 437) associated with porcine diarrhea isolated in Sweden during 1989 were investigated. Of the strains, 135 (31%) were positive for at least one of these toxins and, therefore, designated enterotoxigenic E. coli (ETEC). Our results were compared with the enterotoxin pattern found in earlier studies of Swedish porcine strains. The only change in occurrence of toxins was found in strains isolated from piglets less than 1 week of age. LT- and STb-producing ETEC had decreased, and STa-producing ETEC had increased in prevalence. The occurrence of STb among ETEC of weaned pigs was 93%. This toxin was also found to be more common than STa when strains from all age groups were considered.     

Enteropathogenicity markers in Escherichia coli isolated from infants with acute diarrhoea and healthy controls in Rio de Janeiro, Brazil

Faeces from urban children < 2 years old with acute diarrhoeal illness and from non-diarrhoeal infants (controls) were examined for Escherichia coli and other enteropathogens. A total of 990 E. coli isolates from 100 patients and 50 controls was tested for enteropathogenic E. coli (EPEC) serotype (O:H), adherence to HEp-2 cells after incubation for 3 and 6 h, fluorescent actin staining (FAS), DNA hybridisation with EAF, eaeA, STh, STp and EAggEC probes and production of heat-labile enterotoxin (LT) and verocytotoxin (VT) with Y1 and Vero cells. EPEC were the most prevalent enteropathogens in patients (32.7%; and 14% in controls). Enteroinvasive E. coli (EIEC) and Vero cytotoxin-producing E. coli (VTEC) were not detected. The rate of isolation of enterotoxigenic E. coli (ETEC) was identical in both groups. Among the EPEC isolates the prevalent serotypes were O111:H2, O55:NM and O119:H6. Localised adherence (LA) was found significantly more frequently in isolates from patients (19.6%) than controls (2.1%). All LA-positive EPEC isolates were FAS+ and eaeA+, but only 75.2% of them hybridised with the EAF probe. Diffusely adhering E. coli (DAEC) and enteroaggregative E. coli (EAggEC) were found with equal frequency in patients and controls. Twenty-seven E. coli isolates were negative for EAF but positive for eaeA and FAS and produced LA in 6-h adherence tests. These EAF-/eaeA+ strains were the only putative enteropathogen identified in seven patients and were not found in controls. The ability of these strains to elicit ultrastructural cell alterations and cell-signalling events was evaluated in Caco-2 cells (human colon carcinoma cell line) by the gentamicin invasion assay and by transmission electron microscopy. The numbers of intracellular bacteria in cell invasion tests varied from 0.4% to 1.6% of the cell-associated bacteria after a 6-h incubation period. Tyrosine phosphorylation of host cell proteins was assessed in HEp-2 cells by immunofluorescence microscopy and all strains gave positive results. EAF-/eaeA+ E. coli strains express most of the virulence properties found among true EPEC strains and can be a relevant cause of infant diarrhoea in developing countries.     

Antibody responses in humans against coli surface antigen 6 of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) strains expressing only coli surface antigen 6 (CS6) have previously been isolated from patients with diarrhea, but the immunogenicity of CS6 has not been established in humans. We have detected CS6-specific immunoglobulin A responses in the feces and blood of patients convalescing from natural ETEC disease and of volunteers given an oral ETEC vaccine.     

Experimental investigation of the distribution of residual strains in the artery wall

Diarrheal diseases often result from ingestion of contaminated water or food. The population of La Paz, Bolivia is directly or indirectly exposed to the sewage-contaminated La Paz River. We conducted a bacteriologic survey of the La Paz River to quantify the level of bacterial contamination, with particular reference to enteropathogens. A total bacterial count exceeding 10(6) colony-forming units (CFU)/ml, including lactose fermenting and nonfermenting, gram-negative bacilli of approximately 10(5) CFU/ml, respectively, were detected in river water samples collected near two densely populated areas. A total bacterial count of 10(5) CFU/ml was also detected at the most downstream area of the river near a sparsely populated area. At four sampling locations, several enteropathogens were detected, including five enterotoxigenic Escherichia coli (ETEC) (serotype O6, O15, and O159), two enteropathogenic E. coli (EPEC) (serotype O44), two enteroinvasive E. coli (EIEC) (serotype O29), and three Salmonella O4 group isolates. The heat-labile enterotoxin gene and the invasive toxin gene were detected in all ETEC and EIEC isolates by polymerase chain reaction analysis. Nine isolates of E. coli were found by the agar dilution method to be susceptible to ampicillin, kanamycin, nalidixic acid, tetracycline, and chloramphenicol, and ampicillin resistance was found in only two isolates of EIEC 7-4 (serotype O29) and EPEC 7-5 (serotype O44). Ampicillin resistance was coded on plasmids and transferred conjugatively to E. coli chi1037 at a frequency of 10(-5) CFU/donor by the broth mating method. Strains of Aeromonas caviae, which can cause diarrheal disease in infants, were detected in vegetables grown in fields irrigated by water from the La Paz River. The survival of nine isolates of E. coli in filtered river water was compared with that of laboratory strains (E. coli chi1037, W3110, and ATCC29577). The survival time of seven isolates, excluding two ampicillin-resistant isolates, was markedly longer than that of the laboratory strains. Our results show a high bacterial contamination of the La Paz river and suggest that such levels may contribute to the high incidence of diarrheal disease in the city of La Paz.     

Binding of enterotoxigenic Escherichia coli to isolated enterocytes and intestinal mucus

Binding of human enterotoxigenic Escherichia coli (ETEC) to the small intestine is a prerequisite for colonization and is mediated by colonization factor (CF) antigens. Coli surface antigen 6 (CS6) is considered a CF but binding to isolated enterocytes has not been established. In this study bacteria expressing CS6 were analysed for binding to enterocytes from human and rabbit small intestine, isolated using either an EDTA-containing buffer or a buffer devoid of EDTA. We found that the bacteria bound to enterocytes from rabbit ileum and human duodenum, but only when the cells had been isolated in the absence of EDTA. Pretreatment of rabbit enterocytes with meta-periodate resulted in a decreased proportion of cells with bound bacteria. Purified CS6, and for comparison other ETEC CFs, were also tested for binding to different human and rabbit mucus fractions. These analyses showed that purified CS6 bound to mucus from rabbit duodenum and ileum as well as from human duodenum, jejunum and ileum and that this binding was abolished by pretreatment of the mucus material with meta-periodate or Proteinase K. CFA/I, CS1 to CS5, CS7, CS17, putative CF (PCF) O159 (CS12), PCFO166 (CS14), and CFA/III (CS8) also bound to the rabbit mucus material although with different patterns; the binding of CS2 and CS5 was abolished by meta-periodate treatment. Thus, ETEC bacteria expressing CS6 might bind to carbohydrate-containing structure(s) in the apical membrane of isolated rabbit ileal and human duodenal enterocytes that could probably be released by EDTA treatment. In addition, CS6 and other ETEC CFs bind to component(s), in some instances protein-associated carbohydrate structures, in mucus fractions from small intestine.     

Epidemiology of enterotoxigenic Escherichia coli diarrhea in a pediatric cohort in a periurban area of lower Egypt

Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype. In total, 242 Egyptian children aged <3 years were prospectively followed in 1993-1995. ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identified from monthly cross-sectional surveys. ETEC episodes were 0.6 per child-year. ST-only ETEC was 2.6 times (P<.001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation. Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes. Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea. These findings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation.     

eaeA genes in Escherichia coli derived from Japanese patients with sporadic diarrhea

To investigate the prevalence of attaching and effacing Escherichia coli, we examined 364 strains isolated from the feces of 9,684 patients with diarrhea at the Anjo Kosei Hospital in Japan for the presence of eaeA. Twenty-nine (8%) of the strains were eaeA positive. Of enteropathogenic E. coli (EPEC), 11 of the 87 (13%) strains were for the positive eaeA gene. The serotypes and the numbers of eaeA-positive strains among the strains tested were as follows: O26:H-(2/3), O55:H7 (4/4), O55:H-(2/ 2) and O128:H2(3/3). Two enterohemorrhagic E. coli (EHEC) strains (Verotoxin positive O157:H7) were also eaeA positive. Among 260 non-EPEC strains that were not categorized as diarrheagenic E. coli, 16 (6%) were eaeA positive. Those serotypes were as follows: O15:H2, O20:H6, O28:H28, O63:H6. O153:H7, O28:H6, O153:H19 and O157:H45. EPEC strains including O18:H7 and six other serotypes, enteroinvasive E. coli (EIEC), and enterotoxigenic E. coli (ETEC) were all eaeA negative.     

An outbreak of foodborne illness caused by Escherichia coli O39:NM, an agent not fitting into the existing scheme for classifying diarrheogenic E. coli

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.     

The novel heat-stable enterotoxin subtype gene (ystB) of Yersinia enterocolitica: nucleotide sequence and distribution of the yst genes

The gene (ystB) encoding the novel subtype of the heat-stable enterotoxin (Y-STb) was cloned from the chromosome of a clinical isolate of Yersinia enterocolitica 84-50 (serotype O:5, biotype 1A) and the nucleotide sequence was determined. The ystB contained 216 base pairs that encoded a protein of 71 amino acid residues. The C-terminal 30 residues of the precursor protein exactly corresponded to the amino acid sequence of the Y-STb toxin, purified from the culture supernatant of the wild strain. Homology search revealed that there are 76.9% nucleotide sequence similarity between ystB and the Yersinia kristensenii ST gene, and 73.5% with the Y. enterocolitica prototype sequence of yst (ystA). When tested with the PCR generated ystB specific probe, 36 of 304 Y. enterocolitica strains from 18 countries hybridized with the probe. All the ystB probe positive strains belonged to biotype 1A and mostly to the so-called non-pathogenic serotype O:5, O:6, O:7,8 O:7,13 and O:10, while ystA was predominantly found among the pathogenic serotypes (78.5%). Out of 36 ystB gene positive strains, 18 were clinical origin from six countries, which were also positive in the suckling mice assay suggesting that ystB may play an important role in the pathogenesis, and the so-called non-pathogenic serotypes could be virulent for human.     

Studies on the bacterial flora of fish which are potential pathogens for human. Virulence factors of potential human pathogen isolated

Tests for various virulence factors, such as production of haemolysin on sheep blood agar plate, cytotoxin on HeLa cell line and enterotoxin in GM-1 ELISA and suckling mouse assay model, were done among the various strains of Aeromonas spp., Vibrio spp., Plesiomonas shigelloides and Esch. coli isolated from fresh water fish samples. Invasive properties of the isolates were also seen by using Sereny test. Haemolysin production was observed in 85.7% of Aeromonas, all (100%) of Vibrios, 13.3% of Esch. coli and none (0%) of P. shigelloides strains. Cytotoxin production was demonstrated in 60.8% of Aeromonas, 38.4% of Vibrios and none (0%) of P. shigelloides and Esch. coli strains. About 8% of Vibrio spp., were found positive for LT in GM-1 ELISA method whereas, none of the Aeromonas spp., Plesiomonas and Esch. coli. strains were found positive for LT and ST in GM-1 ELISA. By suckling mouse assay model 43.4% strains of Aeromonas were found positive for enterotoxin production whereas, strains of Vibrio spp., Plesiomonas and Esch. coli yielded negative results. Sereny test for invasive property was found negative in all the strains tested. The isolates from fish possess various virulence factors which contributes for pathogenicity in order to cause various diseases to susceptible individual.     

Ascaris suum: a revision of its early migratory path and implications for human ascariasis

The genetic diversity in a group of Escherichia coli strains belonging to serogroup O6 but expressing different H antigens was investigated by random amplification of polymorphic DNA (RAPD). Isolates of serotypes H16, H1, H31, and non-motile (NM) strains were typed using a set of 3 primers with different G + C contents. The amplified band arrays allowed the identification of 3 main clonal clusters corresponding to each O:H serotype analyzed. Based on their RAPD profiles NM strains could be assigned to either H1 or H31 serotypes. The results indicate that the flagellar antigen and the RAPD fingerprint represent reliable clonal markers in this E. coli group.     

Epitope analysis of the CS3 fimbrial subunit of human enterotoxigenic Escherichia coli and the construction of novel CS3::ST and CS3::LT-B immunogens

Enterotoxigenic E. coli (ETEC) are the major cause of traveler's diarrhoea and the CS3 fimbriae/fibrillae are expressed by most strains bearing the colonization factor CFA/II. The cstAH gene cluster determining CS3 biosynthesis has been previously cloned and sequenced and it has been shown that cstH encodes the major fimbrial subunit and cstA-G encode an assembly cassette. In the work described here we have sought to define the surface exposed domains on CS3 and to manipulate them so that CS3 can be used as a means of expressing foreign antigenic determinants on the bacterial surface. Using a panel of 21 monoclonal antibodies, which we have used in western blotting, immunofluorescence microscopy and colony blotting, together with computer predictions, we have identified three domains within CstH. Two of these sites were permissive for insertion and we have introduced, in-frame, either an epitope from the B subunit of LT (heat labile toxin) or the entire coding sequence of mature ST (heat stable toxin) to construct hybrid proteins. These proteins could be assembled into hybrid fimbriae which could be recognized by antibodies to both CS3 and the foreign epitope as shown by immunofluorescence microscopy and colony blotting. The immunogenicity of the constructs has been evaluated following both oral and intraperitoneal immunization of mice with the attenuated Salmonella typhimurium strain G30 harbouring the hybrid cst operons. Although plasmid stability is currently a problem, these experiments showed that antibodies to both the carrier and the foreign epitope were generated.     

Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: relationship with toxic phenotypes

Seventy-four E. coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v = VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods. In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays. DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions. The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain). Apparently, in Spain three seropathotypes are predominant: (i) O149:K91:H10 K88+ LT-I+ STb+, (ii) O141:K85ab:H- P987+ STaP+, and (iii) O138:K81:H14 or H- STaP+ VT2v+. We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.     

Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohemorrhagic Escherichia coli strains

PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred with serotype O?:H7, O19:H7, O121:H7, O88:H7, and O156:H7 strains. Despite these differences, the nucleotide sequences of the E. coli E32511 (O157:NM) and U5-41 (O1:H7) fliC genes were 97% homologous. Two PCR primer pairs synthesized on the basis of the E32511 H7 fliC sequence amplified specific DNA fragments from all E. coli H7 strains, but did not amplify DNA fragments from the other bacterial strains. The H7-specific primers were used in combination with other primers which target the Verotoxin 1(VT1) and VT2 genes and the E. coli O157:H7 eaeA gene in multiplex PCR assays. In these assays, vt and eaeA PCR products were observed with DNAs from the majority of EHEC strains and vt, eaeA, and fliC PCR products were observed with DNAs from E. coli O157:H7 or NM strains. Only eaeA PCR products were present with DNA from enteropathogenic E. coli, and only vt PCR products occurred with VT-producing E. coli which are not EHEC. The multiplex PCR assays described allow for the specific identification of E. coli O157:H7 or NM and other EHEC strains.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non-E. coli bacteria. Its detection limit was 10(2)-10(3) cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10(0) cells 100 ml-1 of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Proteinaceous factor(s) in culture supernatant fluids of bifidobacteria which prevents the binding of enterotoxigenic Escherichia coli to gangliotetraosylceramide

We have examined the competitive binding of several species of Bifidobacterium and Escherichia coli Pb176, an enterotoxigenic E. coli (ETEC) strain, to gangliotetraosylceramide (asialo GM1 or GA1), a common bacterium-binding structure, and identified a factor(s) in the Bifidobacterium culture supernatant fluid that inhibits the binding of E. coli Pb176 to GA1. The ETEC strain we used expresses colonization factor antigen (CFA) II, which consists of coli surface-associated antigens CS1 and CS3. Competitive exclusion of ETEC from GA1 molecules by Bifidobacterium cells was found by an in vitro thin-layer chromatography overlay binding suppression assay. However, the ETEC cells were less effective in blocking the adherence of Bifidobacterium cells to GA1. These findings suggest that the two bacterial species recognize different binding sites on the GA1 molecule and that the mechanism of competitive exclusion is not due to specific blockage of a common binding site on the molecule. The neutralized culture supernatant fluids of Bifidobacterium species, including that of Bifidobacterium longum SBT 2928 (BL2928), showed remarkable inhibition of the ETEC binding to GA1. Our results suggest that the binding inhibitor produced by BL2928 is a proteinaceous molecule(s) with a molecular weight around or over 100,000 and a neutral isoelectric point. The binding inhibitor produced by BL2928 and other Bifidobacterium species is estimated to contribute to their normal anti-infectious activities by preventing the binding of pathogenic strains of E. coli to GA1 on the surface of the human intestinal mucosa.     

Detection and characterization of the fimA gene of Escherichia coli O157:H7

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.