Structure of CFA/I fimbriae from enterotoxigenic Escherichia coli

Adhesion pili (fimbriae) play a critical role in initiating the events that lead to intestinal colonization and diarrheal disease by enterotoxigenic Escherichia coli (ETEC), an E. coli pathotype that inflicts an enormous global disease burden. We elucidate atomic structures of an ETEC major pilin subunit, CfaB, from colonization factor antigen I (CFA/I) fimbriae. These data are used to construct models for 2 morphological forms of CFA/I fimbriae that are both observed in vivo: the helical filament into which it is typically assembled, and an extended, unwound conformation. Modeling and corroborative mutational data indicate that proline isomerization is involved in the conversion between these helical and extended forms. Our findings affirm the strong structural similarities seen between class 5 fimbriae (from bacteria primarily causing gastrointestinal disease) and class 1 pili (from bacteria that cause urinary, respiratory, and other infections) in the absence of significant primary sequence similarity. They also suggest that morphological and biochemical differences between fimbrial types, regardless of class, provide structural specialization that facilitates survival of each bacterial pathotype in its preferred host microenvironment. Last, we present structural evidence for bacterial use of antigenic variation to evade host immune responses, in that residues occupying the predicted surface-exposed face of CfaB and related class 5 pilins show much higher genetic sequence variability than the remainder of the pilin protein.     

Toxins and colonization factor antigens of enterotoxigenic Escherichia coli among residents of Jakarta, Indonesia

Infection caused by enterotoxigenic Escherichia coli (ETEC) poses a serious health problem among children and adults in developing countries. Colonization of the small intestinal mucosa by ETEC strains is mediated by antigenically specific fimbriae, also known as colonization factor antigens (CFA). The significance of this study arises from reports that active and passive immunization with ETEC strains harboring CFAs has previously been shown to induce protective immunity against diarrhea in animal models. The aim of this study was to determine toxin-associated CFAs of ETEC isolated from a diarrheal disease case-control study in Jakarta, Indonesia. Thirteen hundred and twenty-three diarrheic and control patients with lactose-fermenting colonies were screened by ganglioside GM1-enzyme-linked immunosorbent assay (GM1-ELISA) for heat-labile (LT) and heat-stable (ST) toxins. Two hundred and forty-six (19%) ETEC isolates identified by GM1-ELISA for the LT/ST toxins were screened for CFAs by Dot blot assay using monoclonal antibodies against CFA/I, II, and IV and against the putative colonization antigens (PCF) PCFO159, PCFO166, CS7, and CS17. Of the 246 ETEC isolates, 177 (72%) elaborated ST, 56 (23%) produced LT, while 13 (5%) elicited both the ST and LT toxins. CFA testing of the 246 ETEC isolates showed that 21 (8%) expressed CFA/I, 3 (1%) exhibited CFA/II, 14 (6%) elaborated CFA/IV, while 7 (3%) expressed PCFO159 and PCFO159 plus CS5. No CFAs or PCFs could be associated with 201 (82%) of the ETEC strains. This report documents the types of CFAs associated with ETEC strains in Jakarta, Indonesia. These data may help current research efforts on the development of CFA-based vaccines for humans against ETEC and provide additional information for future ETEC vaccine trials in Southeast Asia.     

Volunteer challenge with enterotoxigenic Escherichia coli that express intestinal colonization factor fimbriae CS17 and CS19

Human challenges with enterotoxigenic Escherichia coli (ETEC) have broadened our understanding of this important enteropathogen. We report findings from the first challenge studies using ETEC-expressing colonization factor fimbria CS17 and CS19. LSN03-016011/A (LT, CS17) elicited a dose-dependent effect, with the upper dose (6 × 10(9) organisms) causing diarrhea in 88% of recipients. WS0115A (LTSTp, CS19) also showed a dose response, with a 44% diarrhea rate at 9 × 10(9) organisms. Both strains elicited homologous antifimbrial and anti-LT antibody seroconversion. These studies establish the relative pathogenicity of ETEC expressing newer class 5 fimbriae and suggest suitability of the LT|CS17-ETEC challenge model for interventional trials.     

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.     

Enzyme-linked immunosorbent assays for the detection of adhesion factor antigens of enterotoxigenic Escherichia coli

Two hundred forty-four specimens of Escherichia coli isolated in Bangladesh and Thailand and identified as enterotoxin producers were tested for the presence of adhesion antigens by mannose-resistant hemagglutination, immunodiffusion, and enzyme-linked immunosorbent assays (ELISAs). Specific antisera to the antigens colonization factor antigen (CFA)/I, CFA/II (consisting of coli surface antigens [CS] 1, 2, and 3), and putative colonization factor antigen (PCF) 8775 (consisting of CS4, 5, and 6) were used in immunodiffusion tests and ELISAs. The results showed that the antigens could be detected in more strains by ELISA than by immunodiffusion. Twenty-nine percent of specimens of E. coli from Thailand and 47% from Bangladesh carried an adhesion antigen. Many of the strains had lost the ability to produce enterotoxins. Forty percent of strains from Thailand and 64% from Bangladesh that were still enterotoxigenic carried adhesion factors. These antigens were found on strains with heat-stable or heat-stable and heat-labile enterotoxin but not on strains producing only heat-labile enterotoxin. PCF8775 antigens were associated mainly with strains from Bangladesh, where 10 strains that produced only CS6 were detected.     

Serologic responses to somatic O and colonization-factor antigens of enterotoxigenic Escherichia coli in travelers.

To improve the retrospective diagnoses of enterotoxigenic Escherichia coli (ETEC) as a cause of travelers' diarrhea, as well as to determine the presence of colonization-factor antigens in these infections, a study of serologic responses to antigens of ETEC was done. Paired sera from 60 United States students in Cholula, Puebla, Mexico, were analyzed for rises in titer of antibody to heat-labile toxin, eight somatic antigen O serogroups associated with ETEC, and two colonization-factor antigens, CFA/I and CFA/II. Only 9% had a response to O antigens, while 20% had responses to the colonization-factor antigens. Response to the colonization-factor antigens correlated significantly with response to the heat-labile toxin and with culture evidence of ETEC infection. Serologic studies confirmed that colonization-factor antigen has a role in naturally acquired cases of travelers' diarrhea and that it can be used as an additional determinant of infection with ETEC.     

Enterotoxins,colonization factors,serotypes and antimicrobial resistance of enterotoxigenic Escherichia coli (ETEC) strains isolated from hospitalized children with diarrhea in Bolivia

Enterotoxigenic Escherichia coli (ETEC) is recognized as the main cause of bacterial diarrhoea among children in Asia, Africa and Latin America but less investigated in Bolivia. Objective: To determine the relation between enterotoxins, CFs and serotypes as well as the antimicrobial resistance patterns in a set of ETEC isolates collected from hospitalized children with acute diarrhea. In the present study we characterized 43 ETEC strains isolated from 2002 to 2006 from hospitalized children (0–5 years) with acute diarrhea in Bolivia. The strains were analyzed for heat-labile (LT) and heat-stable (ST) enterotoxins and colonization factor (CF) profiles, as well as for serogroups and antimicrobial resistance using phenotypic (ELISA, dot blot, slide agglutination and disc diffusion) and genotypic (Multiplex PCR) methods. Among the ETEC isolates tested, 30 were positive for LT, 3 for STh and 10 for LT/STh. Sixty-five percent (28/43) of the strains expressed one or more CF. The most common CFs were CS17 (n = 8) and CFA/I (n = 8). The phenotypical and genotypical results for toxins and CFs were congruent except for CS21 that was amplified in 10 of the strains by multiplex PCR, but CS21 pili was only detected phenotypically in four of these strains. The ETEC strains had diverse O and H antigens and the most common types were O8:H9 LT CS17 (n = 6; 14%) and O78:HNM LT-ST CFA/I (n = 4; 9%). The analysis of antibiotic resistance showed that 67% (n = 29/43) of the strains were resistant to one or several of the antimicrobial agents tested. Presence of CFs was associated with antibiotic resistance. Conclusion: The most common toxin profile was LT 70%, LT/STh 23% and STh 7%. High antimicrobial resistance to ampicillin among serogroups O6, O8 and O78 were the most common.     

Presence of colonization factor antigens on fresh isolates of fecal Escherichia coli: a prospective study

In Dhaka, Bangladesh, fresh isolates of Escherichia coli from 197 patients with diarrhea were investigated for production of enterotoxin and possession of colonization factor antigen (CFA) I or II. Enterotoxigenic E. coli (ETEC) was isolated from 34% of the patients, and of the 67 enterotoxin-positive strains, 75% carried CFAs. Among 68 healthy control persons no strains positive for both enterotoxin and CFA were found. The CFAs in general were restricted to certain serotypes of E. coli. In a subgroup of patients, part of an ongoing surveillance study, mixed infection was seen in 23% of those from whom recognized pathogens were identified. There was a tendency to more severe dehydration when the two virulence factors, enterotoxin and CFA, were simultaneously present.     

Competitive inhibition of adherence of enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli and Clostridium difficile to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027

AIM: To observe competitive inhibition of adherence of enterotoxigenic Escherichia coli (ETEC), enteropathogenic Escherichia coli (EPEC) and Clostridium difficile (C. difficile) to intestinal epithelial cell line Lovo by purified adhesin of Bifidobacterium adolescentis 1027 (B. ado 1027). METHODS: The binding of bacteria to intestinal epithelial cell line Lovo was counted by adhesion assay. The inhibition of adherence of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo by purified adhesin of B. ado 1027 was evaluated quantitatively by flow cytometry. RESULTS: The purified adhesin at the concentration of 10 microg/mL, 20 microg/mL and 30 microg/mL except at 1 microg/mL and 5 microg/mL could inhibit significantly the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo. Moreover, we observed that a reduction in bacterial adhesion was occurred with increase in the concentration of adhesin, and MFI (Mean fluorescent intensity) was decreased with increase in the concentration of adhesin. CONCLUSION: The purified adhesin of B. ado 1027 can inhibit the adhesion of ETEC, EPEC and C. difficile to intestinal epithelial cell line Lovo in a dose-dependent manner.     

Characterization of a new putative colonization factor (CS17) from a human enterotoxigenic Escherichia coli of serotype O114:H21 which produces only heat-labile enterotoxin

Enterotoxigenic Escherichia coli (ETEC) of serotype O114:H21, which produced only heat-labile enterotoxin (LT), gave mannose-resistant hemagglutination (MRHA) with bovine erythrocytes. One strain, E20738A, was shown to possess fimbriae of approximately 7.5 nm diameter. On SDS-PAGE two possible fimbrial polypeptides of molecular masses 17.5 and 15.5 kDa were seen; the 17.5-kDa band was the most prominent. Loss of LT and MRHA together from strain E20738A was associated with loss of a 100-MDa plasmid. An absorbed anti-strain E20738A serum reacted specifically with the 17.5- and 15.5-kDa polypeptides and bound to the intact fimbriae. This antiserum reacted positively in an ELISA with LT-positive E. coli strains of serogroups O8, O15, O48, O114, and O146. The antiserum did not react with ETEC carrying known colonization factors. The term coli-surface-associated antigen (CS) 17 has been used to describe the fimbriae.     

Comparison of rheumatological and gastrointestinal symptoms after infection with Campylobacter jejuni/coli and enterotoxigenic Escherichia coli

OBJECTIVES: To estimate the incidence of postinfectious joint complaints after Campylobacter jejuni/coli enteritis compared with enteritis caused by enterotoxigenic E coli (ETEC). To compare gastrointestinal symptoms, antibiotic treatment, and antibody levels among patients with and without joint symptoms. METHOD: Questionnaires were sent to 210 consecutive patients with Campylobacter infection and an equal number of patients with E coli (ETEC). Blood samples for anti-Campylobacter antibodies were collected after two weeks, three months, six months, and two years. RESULTS: Twenty seven of 173 (16%) patients with Campylobacter and 10/177 (6%) with E coli (ETEC) reported joint symptoms (p=0.004). In the Campylobacter group duration of diarrhoea was a median of 13 days for patients with arthralgia and seven days for those without joint pain (p=0.0058). Patients with E coli had diarrhoea of longer duration than patients infected with Campylobacter (14 days v seven days; p=0.0005). E coli patients had fewer gastrointestinal symptoms than Campylobacter patients (p=0.0001). Fifty nine per cent of Campylobacter patients with joint pain had received antibiotic treatment because of enteritis compared with 26% with enteritis only (p=0.03). Campylobacter species and serotypes were equally distributed in both groups and there was no difference in anti-Campylobacter antibody levels between the groups. CONCLUSION: There was a significantly increased risk of developing joint symptoms after contracting Campylobacter infection compared with E coli. Campylobacter patients with joint pain had more severe gastrointestinal symptoms and longer duration of diarrhoea. Antibiotic treatment does not seem to prevent reactive joint symptoms. Levels of anti-Campylobacter antibodies were the same in both groups.     

Trimethoprim/sulfamethoxazole remains active against enterotoxigenic Escherichia coli and Shigella species in Guadalajara, Mexico.

Bacterial isolates obtained from fecal cultures of U.S. adults in Guadalajara, Mexico, with diarrhea developing during the summers of 1987, 1988, and 1989 were tested for in vitro antimicrobial susceptibility. No resistance was seen among 220 enterotoxigenic Escherichia coli isolates nor among 89 Shigella strains to aztreonam, norfloxacin, ciprofloxacin, gentamicin, or furazolidone. High level (greater than 1000 micrograms/ml) resistance to trimethoprim/sulfamethoxazole (TMP/SMX) was found in 7% of E. coli and 3% of Shigella strains. Susceptibility patterns of 27 E. coli strains derived from fecal cultures of Mexican infants in Guadalajara with diarrhea during the same time period showed similar results, possibly reflecting the presence of a common microbial reservoir. The study serves as a baseline for newer antimicrobials (fluoroquinolones and aztreonam) where no resistance is currently seen and provides evidence of the continuing value of TMP/SMX for therapy of diarrhea among travelers to Gudalajara and perhaps other areas of Mexico.     

Zn(II)-induced cooperativity of Escherichia coli ornithine transcarbamoylase.

The steady-state reaction of ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) purified from the argI gene product of Escherichia coli strain K-12 exhibits Michaelis-Menten kinetics over an extended range of concentration for both L-ornithine and carbamoyl phosphate. In the presence of Zn2+, however, the saturation curve of L-ornithine becomes sigmoidal, revealing positive cooperativity for this anabolic enzyme. The kinetic data give a limiting Hill coefficient of 2.7 for this substrate at 0.3 mM Zn2+. The allosteric effect of Zn2+ on the enzyme is not altered by the concentration of carbamoyl phosphate, and the saturation curve of carbamoyl phosphate remains hyperbolic in the presence of the metal ion. At fixed substrate concentrations, initial velocity data obtained at 0.-0.3 mM Zn2+ indicate cooperative binding of the metal ion to ornithine transcarbamoylase; a Hill coefficient of 1.7 +/- 0.1 is found that is independent of the level of L-ornithine. These results suggest competitive and exclusive binding to the enzyme between L-ornithine and Zn2+ with conformational changes induced in the subunits of the enzyme only by the metal ligand. Neither Co2+ nor Cu2+ exerts an effect on the kinetic behavior of the enzyme. This finding reveals not only specific allosteric control of ornithine transcarbamoylase by Zn2+ but also the possibility of an interlocking metabolic regulation between the urea cycle and the pathway for pyrimidine biosynthesis.     

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.