Characterization of enteropathogenic and enteroaggregative Escherichia coli isolated from diarrheal outbreaks.

Virulence characteristics of diarrheal outbreak-associated Escherichia coli O55:NM, O126:NM, and O111:NM were examined. The E. coli O55:NM strains were atypical enteropathogenic E. coli (EPEC), while the E. coli O126:NM and O111:NM strains should be classified as enteroaggregative E. coli (EAggEC). The contributions of EPEC and EAggEC to the human disease burden in Japan might be significantly greater than is currently appreciated.     

Species specificity and lack of production of STb enterotoxin by Escherichia coli strains isolated from humans with diarrheal illness.

Escherichia coli strains produce at least two heat-stable enterotoxins, STa and STb. STa is well known to be important in the pathogenesis of human diarrheal disease; the role of STb has not been defined. Fifty-two E. coli strains recovered from human diarrheal illness in northeast Brazil or Bangladesh were examined in weaned porcine ligated intestinal segments for STb activity. A total of 113 E. coli strains from human diarrheal disease in northeast Brazil and 28 E. coli strains from Bangladesh were examined for DNA hybridization to a STb gene probe. None of these strains produced STb as detected by enterotoxic activity or by the gene probe. We also examined adult human ileal mucosa for responses to STb in the Ussing chamber in vitro. In contrast to piglet jejunum, which consistently responds electrogenically to crude STb, human ileal tissue showed no response to STb but responded electrogenically to theophylline (10 mM). These results suggest that STb-producing E. coli strains are not a major cause of diarrheal illness in humans.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Phylogenetic group,virulence factors and antimicrobial resistance of Escherichia coli associated with bovine mastitis

Escherichia coli is an important pathogen involved in the etiology of bovine mastitis. A total of 70 E. coli isolates recovered from clinical and subclinical mastitis samples were characterized with respect to their phylogenic group, virulence factors and antimicrobial susceptibility. Based on the presence of the specific genes chuA, yjaA and TspE4.C2, these isolates were found to belong to three different groups: group A(25), group B1(41) and group D(4). Twenty-five (35.7%) isolates harbored at least one virulence gene, and the most prevalent virulence genes were f17A, irp2, astA, iucD and colV. The irp2-coding gene was more often detected in group A than in group B1 isolates; in contrast, colV was identified more often in group B1 isolates. The majority of isolates (87.1%) were resistant to at least one antimicrobial compound. Forty-seven isolates (67.1%) were resistant to streptomycin, and those from group B1 were more resistant to streptomycin than isolates from group A. The latter feature was supported by the distribution of streptomycin resistance genes observed in group B1 compared to group A.     

Peyer's patch adherence of enteropathogenic Escherichia coli strains in rabbits.

RDEC-1 (serotype O15) is an attaching and effacing strain of rabbit enteropathogenic Escherichia coli (REPEC) that causes diarrhea in postweanling rabbits. It expresses AF/R1 pili that mediate Peyer's patch M-cell adherence. We investigated Peyer's patch adherence, the presence of virulence genes, ileal brush border aggregation, and pilus expression in 9 strains representing several serotypes of REPEC as well as in two commensal strains. Postweanling rabbits were inoculated with 10(6) organisms and sacrificed at 24 h, and tissues were prepared for examination by light microscopy. Strains B10 and RDEC-1 were also studied at 12 and 72 h postinoculation. All REPEC strains were eaeA positive, expressed pili, and adhered to ileal brush borders. Both commensal strains expressed pili, and one strain adhered to brush borders. All REPEC strains demonstrated some degree of Peyer's patch lymphoid follicle adherence, ranging from diffuse coverage to small patches covering two to three dome epithelial cells. Strains C102 and C110 had genes homologous with the structural subunit gene of the AF/R1 pilus (afrA) of RDEC-1, which correlated with greater degrees of lymphoid follicle adherence and lesser degrees of ileal villus adherence. The observation that all REPEC strains adhere to Peyer's patch epithelium suggests the possibility that human strains of enteropathogenic E. coli (EPEC) might do likewise. EPEC strains might thus serve as mucosal vaccine vectors in humans. Better understanding of the molecular mechanism of REPEC adherence should provide a model for the targeting of the Peyer's patch in humans.     

A novel cryohemagglutinin associated with adherence of enteroaggregative Escherichia coli.

Strain O42 (serotype O44:H18) of enteroaggregative Escherichia coli (EAggEC) has been shown to be pathogenic in volunteer experiments. This strain exhibited plasmid (pO42)-encoded D-mannose-resistant hemagglutinating activity (MRHA) that was detected only at low temperatures (e.g., 0 degrees C) and only with human erythrocytes. The production of this cryogenic MRHA (cryo-MRHA) was observed when the bacteria were grown in liquid media and was strictly regulated by bacterial growth temperatures. Transposon-insertion mutagenesis revealed that this MRHA is associated with (i) bacterial clump formation in liquid cultures, (ii) bacterial adherence to HEp-2 cells as well as (Formalin-fixed) human colonic mucosa, and (iii) production of a 16-kDa outer membrane protein. The PCR designed on the basis of the determined cryo-MRHA-associated DNA sequence sharply distinguished strain O42 from eight other EAggEC strains whose MRHAs were detected at both cold and room temperatures to the same (or similar) extent. Strain O42 possessed a surface layer that may enhance the pO42-mediated adherence. The data suggest that a plasmid-encoded cryo-MRHA is a candidate for a major adhesin of EAggEC strain O42.     

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.     

Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype.

Two outbreaks of hemorrhagic colitis, a newly recognized syndrome characterized by bloody diarrhea, severe abdominal pain, and little or no fever, occurred in 1982. No previously recognized pathogens were recovered from stool specimens from persons in either outbreak. However, a rare E. coli serotype, O157:H7, was isolated from 9 of 20 cases and from no controls. It was also recovered from a meat patty from the implicated lot eaten by persons in one outbreak. No recovery of this organism was made from stools collected 7 or more days after onset of illness; whereas 9 of 12 culture-positive stools had been collected within 4 days of onset of illness. The isolate was not invasive or toxigenic by standard tests, and all strains has a unique biotype. Plasmid profile analysis indicates that all outbreak-associated E. coli O157:H7 isolates are closely related. These results suggest that E. coli O157:H7 was the causative agent of illness in the two outbreaks.     

Virulence and antimicrobial resistance profiles among Escherichia coli strains isolated from human and animal wastewater

To gain insight into whether Escherichia coli isolated from humans and resistant to some common antimicrobial agents are derived from animals, 85 E. coli strains were selected by ERIC-PCR from human and animal wastewater samples. Phylogroup, pathogenicity islands (PAIs), resistance to quinolones, fluoroquinolones and presence of extended-spectrum beta-lactamases (ESBLs) were analyzed. Among the total, 55% were resistant to nalidixic acid and 38% to ciprofloxacin; 12% produced ESBLs. Chicken-derived strains were associated with quinolone and fluoroquinolone resistance and presence of ESBLs, while human strains were associated with susceptibility. Group B2 E. coli strains were associated with human origin, susceptibility to fluoroquinolones and presence of PAIs, whereas groups A, B1 and D showed a low virulence profile and a high level of antimicrobial resistance. In both human and animal wastewater, E. coli A, B1 and D were prevalent, and strains from both origins showed a similar virulence profile in each phylogroup. These findings led us to hypothesize that abusive antibiotic use in food animal production may promote the development of resistance among these intestinal E. coli phylogroups, which could later be transmitted to humans through the food supply. The low prevalence of E. coli group B2 in the animal gut may explain, at least in part, the absence of emergence of resistant B2 isolates.     

Isolation,genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.     

A protein factor associated with serum resistance in Escherichia coli.

Immunogel-diffusion studies showed that 60 degree C LiCl extracts of the smooth serum-resistant mutant Escherichia coli strain 17 contained greater amounts of a protein antigen than did extracts of the parent strain LP729. An extract of strain 17 was fractionated on Sepharose 4B and the protein antigen was found as the only detectable antigen in a number of fractions; sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that these fractions contained one major polypeptide band with a molecular weight of 46 000 daltons. We suggest that this protein antigen may be partly responsible for the serum resistance of strain 17 though its presence in other serum-sensitive strains suggests that additional factors are essential for full serum resistance.     

Serotypes of Enterotoxigenic Escherichia coli Isolated in the United States

Strains of enterotoxigenic Escherichia coli isolated from humans in the United States were found in 11 of 16 serotypes that previously were documented in the international literature as associated with enterotoxin production. Of 68 strains belonging to these 11 serotypes, 28 (41%) were enterotoxigenic; none of 46 strains belonging to 5 other previously implicated serotypes was enterotoxigenic. Control cultures of various serotypes were selected for comparison and found to contain 0 to 7% enterotoxigenic E. coli. E. coli belonging to documented enterotoxin-associated serotypes, characterized by both O and H antigens, were selected for toxin testing to determine their prevalence and potential pathogenicity in this country. In this study, a strain possessing any combination of an enterotoxin-associated serotype O antigen and H antigen was more likely to be enterotoxigenic than strains possessing only the specific O antigen or H antigen or neither. Five E. coli strains belonging to undocumented enterotoxin-associated serotypes did contain a combination of previously reported enterotoxin-associated serotype O and H antigens and did produce enterotoxin.     

Localized adherence and attaching-effacing properties of nonenteropathogenic serotypes of Escherichia coli.

Traditional enteropathogenic Escherichia coli serotypes demonstrate a plasmid-mediated localized adherence in cultured HeLa or HEp-2 cells and induce an attaching-effacing intestinal lesion, both of which are considered pathognomonic and causes of diarrhea. This study describes three E. coli strains from infantile diarrhea which share these properties but belong to serotypes (O2:H2, O2:H25 and O15:H2) not considered enteropathogenic.     

Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children.

A clinicoepidemiological study was undertaken to determine if enterohemorrhagic Escherichia coli (EHEC) was associated with hemolytic-uremic syndrome (HUS) in children in Santiago, Valdivia, and Temuco, Chile. Prospective surveillance detected 20 hospitalized cases of HUS in children less than 4 years of age in these cities from March 1988 to March 1989. Each HUS patient was matched (by sex and age) with two control children (hospitalized elective-surgery patients). To detect EHEC, DNA from stool culture isolates of E. coli was detected by hybridization with biotin-labelled DNA probes specific for the EHEC virulence plasmid, Shiga-like toxin I (SLT-I) or SLT-II. Stool cultures from 6 of 20 cases (30%) and from 2 of 38 controls (5.3%) yielded EHEC (P = 0.0158). EHEC isolates from all HUS cases hybridized with the EHEC plasmid probe and with probes for SLT-I or -II (or both). The serogroups of the isolates included O157, O26, and O111. EHEC causes HUS in Chile, and the biotinylated gene probes are practical diagnostic tools for epidemiologic studies.     

Synergistic rotavirus and Escherichia coli diarrheal infection of mice.

Experimental or naturally acquired subclinical infections with rotavirus caused a significant increase in mortality of infant mice after challenge with enterotoxigenic Escherichia coli B44.     

Serum resistance in Escherichia coli strains causing acute pyelonephritis and bacteraemia.

The capacity of Escherichia coli to resist the bactericidal action of serum was examined in 367 clinical isolates obtained from children with acute pyelonephritis (n = 57), adults with acute pyelonephritis (n = 55), non-diabetic patients with bacteraemia (n = 101), diabetic patients with bacteraemia (n = 65) and from the faecal flora of healthy controls (n = 89). The incidence of serum-resistant E. coli strains was significantly higher in pyelonephritogenic strains from children and adults (93% and 82%) as compared to faecal control strains (57%, p less than 0.001 and p less than 0.005 respectively). Strains causing bacteraemia in non-diabetic and diabetic patients were more often serum resistant (72% and 80%) as compared to control strains (p less than 0.05 and p less than 0.001 respectively). The frequency of serum-sensitive strains was similar in diabetic patients with decreased renal function or proteinuria compared to those with normal renal function. There were no significant correlations between serum resistance of E. coli and expression of P fimbriae, type I fimbriae or mannose-resistant haemagglutination, cell surface hydrophobic properties, production of aerobactin, haemolysin or cytotoxic necrotizing factor in 53 pyelonephritogenic strains from adult patients.     

Interaction of drug resistance plasmids and bacteriophages with diarrheagenic strains of Escherichia coli.

Seven transfer-derepressed plasmids from different incompatibility groups in Escherichia coli K-12 were tested for their ability to enter 43 strains of diarrheagenic E. coli (mostly enteropathogenic E. coli clinical isolates) representing 12 serogroups and including rough and semirough mutants (characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Strains in some serogroups were more competent as recipients of plasmids than were those in others. Five test plasmids in an E. coli K-12 (rough) donor transferred significantly less efficiently to two smooth strains than to their rough or semirough isogenic derivatives. When the same smooth and rough strains were used as donors, the plasmids transferred to E. coli K-12 equally well. These results suggested that the O-antigenic lipopolysaccharide side chains of diarrheagenic E. coli isolates shielded the outer membrane receptors for conjugative pili, thus preventing plasmid entry. The different receptors for eight bacteriophages were also covered by O side chains. In addition, a limited survey of clinical isolates for drug resistance markers and resident plasmids was carried out.     

Actin accumulation associated with clustered and localized adherence in Escherichia coli isolated from patients with diarrhea.

Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence. Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation. After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence. When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation. Capsule-like, accumulated actin was also observed with enteropathogenic E. coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern. Four other strains of F1845 DNA probe-positive, diffusely adhering E. coli were negative for actin accumulation. Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes. In addition, clustered D2 cells were found inside tissue culture cells. The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E. coli. Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.