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.     

The LT1 and LT2 variants of the enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) are associated with major ETEC lineages

The heat-labile toxin (LT) is one of the major virulence factors of enterotoxigenic Escherichia coli (ETEC). We recently described that 20 polymorphic LT variants are present in ETEC strains isolated globally. Two of the variants, LT1 and LT2, are particularly common and we found that they were associated with clonal ETEC lineages that express the colonization factors (CFs), CFA/I, CS1+CS3, CS2+CS3, and CS5+CS6. ETEC expressing these CFs are frequently found among ETEC strains isolated from cases with diarrhea. ETEC expressing the colonization factors CS1+CS3, and CS2+CS3 are found in 2 discrete clonal lineages and express the LT1 variant and heat stable toxin (STh). Although they clearly are virulent they neither produce, nor secrete, high amounts of LT toxin. On the other hand ETEC strains expressing LT, STh, CFA/I and LT, STh, CS5+CS6, carry the LT2 variant and produce and secrete significantly more LT toxin. Despite differences in toxin production, LT1 and LT2 are found in ETEC lineages that have managed to spread globally confirming that these variants are important for ETEC virulence.     

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.     

Human antibody response to longus type IV pilus and study of its prevalence among enterotoxigenic Escherichia coli in Bangladesh by using monoclonal antibodies

Mouse monoclonal antibodies (MAbs) were derived against longus (CS20), a type IV pilus expressed by human enterotoxigenic Escherichia coli (ETEC). One MAb (ICA39) detected longus in 56 (8.5%) of 662 ETEC isolates obtained from a routine surveillance of diarrheal stools from children and adults. Five patients with diarrhea from whom longus-positive ETEC were isolated were also recruited. Of these 61 isolates, 50 were positive for other colonization factors (CFs; 61% for CFA/II and 21% for CFA/I), and 11 were negative for any of the other 8 CFs that were tested. They were either positive for the heat-stable enterotoxin (ST; n=29) or for the heat-labile enterotoxin (LT) and ST (n=32). All longus-positive ETEC were confirmed by polymerase chain reaction to harbor lngA, the longus structural pilin gene. Sera and/or fecal extracts from the patients reacted with the 22-kDa pilin polypeptide in immunoblots and ELISA. These studies show that longus is prevalent among ETEC in Bangladesh and that longus gives rise to IgA antibody responses in patients.     

Enterotoxins and adhesins of enterotoxigenic Escherichia coli: are they risk factors for acute diarrhea in the community?

A cohort of 228 Mexican children less than 5 years old was followed during the enterotoxigenic Escherichia coli(ETEC) season. The incidence of ETEC diarrhea-associated and asymptomatic infections was determined, and E. coli strains isolated from stool samples were tested for heat-labile and heat-stable toxins and for expression of colonization factor antigens (CFA). Of the children, 61% had at least one ETEC infection. Children with ETEC isolated from stools were more likely to have diarrhea than were ETEC-free age-matched control children (odds ratio [OR] = 4.5; 95% confidence interval [CI] = 2.9-7.0). Strains carrying CFA/IV, CFA/I, or CFA/II were found in 23%, 18%, and 5% of ETEC infections, respectively. The risk of having diarrhea associated with a CFA-expressing versus a CFA-negative ETEC strain was the same (age-adjusted OR = 0.8; 95% CI = 0.4-1.6). These data should be considered in the development of a diarrhea vaccine containing only CFAs.     

Serologic correlates of protection against enterotoxigenic Escherichia coli diarrhea

BACKGROUND: We conducted a nested case-control study in 397 rural Egyptian children <36 months of age to assess the correlation between serum levels of antibodies against toxin and colonization factors (CFs) and the risk of homologous enterotoxigenic Escherichia coli (ETEC) diarrhea. METHODS: Active case detection was performed via semiweekly home visits, and blood was obtained at 3-month intervals. After each serosurvey, case subjects were selected from children experiencing a CF antigen (CFA)/I-, CFA/II-, CFA/IV-, or heat-labile enterotoxin (LT)-ETEC diarrheal episode during the subsequent 3 months. Up to 5 control subjects per case subject were selected from children who did not experience an ETEC diarrheal episode during the corresponding interval. Serum titers of immunoglobulin G antibodies against CFA/I, coli surface antigen (CS) 3, CS6, and LT were measured by enzyme-linked immunosorbant assay. RESULTS: The distribution of serum titers of LT, CS3, and CS6 antibodies did not differ between the case and control subjects. For children <18 months of age, serum titers of CFA/I antibody were inversely related to the risk of CFA/I-ETEC diarrhea; reciprocal serum titers of CFA/I antibody > or =76 were associated with a 77% reduction in the odds of CFA/I-ETEC diarrhea. CONCLUSION: Induction of reciprocal serum titers of antibodies against CFA/I within or above the 76-186 range should be further evaluated as a predictor for assessment of the ability of candidate vaccines to protect against CFA/I-ETEC diarrhea.     

Characterization of enterotoxigenic Escherichia coli strains in patients with travelers' diarrhea acquired in Guadalajara, Mexico, 1992-1997

The relationship between enterotoxigenic Escherichia coli (ETEC) and travelers' diarrhea was examined in a high-risk area in 1992-1997. Toxin patterns, colonization-factor antigens (CFAs), and in vitro antimicrobial susceptibility were determined. In total, 928 US students with diarrhea acquired in Guadalajara, Mexico, were screened for enteric pathogens. Diagnosis of ETEC infection was done with oligonucleotide probes. ETEC was isolated in 19.9% of the travelers with diarrhea. CFAs were identified in 51% of the ETEC strains. The highest CFA frequency was observed among heat-stable isolates. Ampicillin, furazolidone, and sulfisoxazole resistance of ETEC increased during the study period. ETEC isolation rates and CFA patterns varied little during the 6 years of the study, which has implications for immunoprophylactic strategies. The finding that differences in the results of ribotyping and plasmid analysis change over time suggests that multiple strains of ETEC were responsible for the illness in the region studied.     

Genetically modified enterotoxigenic Escherichia coli vaccines induce mucosal immune responses without inflammation

Objective

Enterotoxigenic Escherichia coli (ETEC) is a major cause of acute diarrhoea in children in the developing world, in travellers and in the military. Safe, effective vaccines could reduce morbidity and mortality. As immunity to ETEC is strain specific, the ability to create vaccines in vitro which express multiple antigens would be desirable. It was hypothesised that three genetically attenuated ETEC strains, one with a genetic addition, would be immunogenic and safe, and they were evaluated in the first licensed UK release of genetically modified oral vaccines.

Methods

Phase 1 studies of safety and immunogenicity were carried out at a Teaching Hospital in London. Varying oral doses of any of three oral vaccines, or placebo, were administered to volunteers of both sexes (n = 98). Peripheral blood responses were measured as serum antibodies (IgG or IgA) by ELISA, antibody‐secreting cell (ASC) responses by enzyme‐linked immunospot (ELISPOT), and antibody in lymphocyte supernatant (ALS) by ELISA. Mucosal antibody secretion was measured by ELISA for specific IgG and IgA in whole gut lavage fluids (WGLFs).

Results

Significant mucosal IgA responses were obtained to colonisation factors CFA/I, CS1, CS2 and CS3, both when naturally expressed and when genetically inserted. Dose–response relationships were most clearly evident in the mucosal IgA in WGLF. Vaccines were well tolerated and did not elicit interleukin (IL) 8 or IL6 secretion in WGLF.

Conclusions

Genetically modified ETEC vaccines are safe and induce significant mucosal IgA responses to important colonisation factors. Mucosal IgA responses were clearly seen in WGLF, which is useful for evaluating oral vaccines.Enterotoxigenic Escherichia coli (ETEC) infection is the single most frequent cause of bacterial diarrhoeal disease worldwide and is associated with two main clinical syndromes. In the developing world it is a major cause of weanling diarrhoea in children,^1,2 making a very large contribution to 1 800 000 deaths annually from diarrhoeal disease worldwide.³ In visitors to endemic areas, ETEC is the most common cause of traveller''s diarrhoea, with 20–60% of adults and children experiencing a diarrhoeal episode^4,5 and with ETEC implicated in up to 40% of cases.¹ Epidemics of diarrhoeal disease, again most commonly due to ETEC, also have a significant impact on the health and activity of military personnel on exercise or active duty in these regions.⁶In exposed individuals, mucosal immunity develops, but an immune subject can still shed virulent organisms in the stool. Therefore, in endemic areas, the environment becomes heavily contaminated with ETEC, with most infants encountering ETEC at weaning, but with older children and adults having low rates of clinical infection. Immunologically naïve adults, including travellers to the region, remain susceptible.ETEC causes diarrhoea principally via two enterotoxins, the heat‐labile (LT) and heat‐stable (ST) enterotoxins. Different strains can produce LT, ST, or both LT and ST. LT is similar to cholera toxin and is highly immunogenic, while ST is a small protein and does not appear to be immunogenic. ETEC also expresses a range of colonisation factor antigens (CFAs), which allow adherence to the mucosal surface and therefore colonisation of the intestine. Some CFAs are subdivided into coli surface (CS) antigens, giving a complex range of vaccination targets. CFA/I, CFA/II (comprising CS3 alone or with CS1 or CS2) and CFA/IV (CS6 alone or with CS4 or CS5) are the most common antigens encountered in natural ETEC infection.^7,8 An ideal vaccine against ETEC should colonise the intestinal mucosa without causing inflammation, and then stimulate a protective immune response. In order to cover the widest range of ETEC subtypes, any potential vaccine should therefore contain at least CFA/I, CFA/II and CFA/IV components.⁸ LT may also be required in a vaccine to achieve optimal immune protection.A spontaneous toxin deletion mutant of a CFA/II‐expressing (CS1/CS3) ETEC strain (E1392/75/2A) has been found to provide significant (75%) protection against subsequent ETEC challenge, but unfortunately caused mild diarrhoea in approximately 13% of recipients.⁹ Further attenuation by deleting the genes aroC, ompC and ompF reduced side effects without compromising immunogenicity.^10,11 In the studies reported here, three live genetically modified strains of ETEC have been tested in Phase 1 studies for potential inclusion in a polyvalent oral vaccine (ie, a vaccine containing multiple strains). This was the first environmental release of genetically modified oral vaccine strains in ambulant volunteers in the UK. As such, their release into the environment required approval from the Department of the Environment, Food and Rural Affairs (DEFRA). Approval was also obtained from the Medicines Control Agency (MCA) and the North East London Health Authority Research Ethics Committee. As these vaccines were administered orally, we compared responses in peripheral blood and in mucosal lavage fluid, and cytokine secretion into whole gut lavage fluid (WGLF) was measured to confirm that genetic modification did not induce inflammation.     

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.     

Prevalence of enteric pathogens among international travelers with diarrhea acquired in Kenya (Mombasa), India (Goa), or Jamaica (Montego Bay)

Stools from tourists from Europe and North America who acquired diarrhea in Mombasa (Kenya), Goa (India), or Montego Bay (Jamaica) were examined for enteric pathogens. Enterotoxigenic Escherichia coli (ETEC) was the most common pathogen (25%) identified in the 3 locations. Isolation of Shigella species was more frequent in Goa and Mombasa than in Montego Bay (10%, 9%, and 0.3%, respectively; P <.005). Viruses (rotaviruses and enteric adenoviruses) were found in 9% of travelers to the 3 areas. Of 275 ETEC isolates in this study, 158 (57%) produced a defined colonization factor antigen (CFA). Coli surface 6 (CS6) was the most frequent and was found in 41%-52% of CFA/CS-positive ETEC isolates. The frequency of resistance among bacterial enteropathogens to traditional antimicrobial agents was particularly high throughout the study period in all 3 regions. Quinolones were active against the bacterial enteropathogens in the 3 sites.     

Lack of prophylactic efficacy of an enteric-coated bovine hyperimmune milk product against enterotoxigenic Escherichia coli challenge administered during a standard meal

Orally administered bovine immunoglobulins with specific activity against colonization factors of enterotoxigenic Escherichia coli (ETEC) could provide passive protection against ETEC challenge in volunteers. Twenty healthy adult volunteers ingested either a placebo or a partially enteric-coated preparation of bovine immunoglobulins with activity against the colonization factor antigens CFA/I, CS3, and CS6 and then were challenged with ETEC strain E24377A (CS1+, CS3+) administered with a standard meal. There was no difference in the incidence or severity of diarrhea among the 10 volunteers who received the bovine immunoglobulins and the 10 who received placebo. Either the specificity or titer of anti-colonization factor antibodies or the formulation of antibodies in this product was not adequate to provide passive protection against ETEC challenge.     

Administration of purified colonization factor antigens (CFA/I, CFA/II) of enterotoxigenic Escherichia coli to volunteers. Response to challenge with virulent enterotoxigenic Escherichia coli

Colonization factor antigens (CFAs) were administered orally to volunteers, and the mucosal immune response was assessed by measuring secretory immunoglobulin A (IgA) in saliva and intestinal secretions and by challenge with virulent enterotoxigenic Escherichia coli (ETEC). A combination of CFA/I and CFA/II (8 mg each) administered orally in four doses in milk failed to induce a mucosal IgA response and also failed to protect against challenge with CFA-positive ETEC. When CFA was administered orally (1.5 mg divided into three doses with bicarbonate) to volunteers without preexisting serum anti-CFA levels, it failed to elicit a serum IgG or intestinal secretory IgA response or to protect against challenge with virulent ETEC. The CFA is destroyed by acid gastric contents but it induced significant antibody titer rises in 8 of 11 (73%) volunteers with preexisting serum anti-CFA IgG levels after oral administration of 1 mg of CFA/I with sodium bicarbonate. Subcutaneous priming (50 micrograms CFA/I) did not induce a serum IgG response but, when followed by oral boosting (1 mg CFA/I in two divided doses with sodium bicarbonate), induced intestinal anti-CFA secretory IgA in 4 of 8 volunteers and protected against challenge with CFA/I-positive ETEC. These results, although preliminary, are encouraging and demonstrate that it may be possible to develop an effective oral vaccine based on soluble nonreplicating antigens such as purified CFAs.     

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.     

Seasonal variation of entertoxigenic Escherichia coli among children with diarrhoea in Bangkok

ETEC which produced both LT and ST or LT alone were isolated from 7.4 percent (18/244) of children with diarrhoea treated at two hospitals in Bangkok, Thailand between May 1, 1980 and April 30, 1981. These enteric pathogens were only isolated from children with diarrhoea during the dry and the beginning of the wet season. Eighty-three percent (15/18) children infected with ETEC were infected with antibiotic resistant isolates. One hundred percent of LT + ST + E. coli and 76 percent (50/66) of LT + ST - E. coli were resistant to two or more antibiotics. Fourteen of 15 (93%) ETEC transferred antibiotic resistance and nine of 14 (64%) isolates which transferred resistance in bacterial conjugation experiments also transferred toxigenicity. This experience suggests that the widespread use of antibiotics in Thailand could increase the prevalence of antibiotic resistant enterotoxigenic bacteria.     

Local and systemic antibody responses to naturally acquired enterotoxigenic Escherichia coli diarrhea in an endemic area

Fifteen patients hospitalized with acute, watery diarrhea and with enterotoxigenic Escherichia coli (ETEC) detected from stool samples were studied to evaluate the extent to which natural ETEC diarrhea induces local and systemic antibody responses to E. coli heat-labile toxin (LT), homologous lipopolysaccharide (LPS), and colonization factors (CFA/I and CFA/II). Specific IgA and IgG antibodies to LT, CFA I and II, and each patient's homologous LPS were determined by ELISA in serum, saliva, breastmilk, and intestinal lavage fluid. The majority of patients had greater than a twofold rise in local levels of IgA antibodies in the intestine: 80% of LT+ patients responded to LT, 63% of CFA+ patients responded to CFA, and 78% of all toxin-positive patients responded to the LPS of their infecting strain. Local antibody responses in the intestine were associated with responses in breastmilk and saliva, but relationships were not clear-cut, and the usefulness of these secretions as proxy measures of local intestinal antibody production remains unclear. Antibody responses in serum also occurred in most patients and were significantly more frequent in cases than in controls. This study demonstrates that natural ETEC disease results in local IgA responses to LT, CFA, and LPS in the gut and also in immune responses in breastmilk, saliva, and serum.     

直接从粪便中检测致犊牛腹泻产肠毒素大肠杆菌的双重PCR方法的建立与应用

目的产肠毒素大肠杆菌(Enterototxigenic,Escherichia Coli,ETEC)是造成人和动物腹泻的主要病原之一,也是造成新生乳用犊牛腹泻的主要原因,一株ETEC可能产生一种或者多种肠毒素。根据产肠毒素大肠杆菌产生的两种主要毒素的基因序列,设计了两对引物,建立多重PCR方法。该方法仅用4.5小时即可检测出导致犊牛腹泻的产肠毒素大肠杆菌菌株,具有敏感度高、特异性强和检测速度快等特点。本试验的目的是用所建立的多重PCR方法来确定ETEC在导致犊牛腹泻中的作用。采用该方法对乳用犊牛的203个腹泻样品进行了检测,结果,用传统的分离培养方法得到的203株典型大肠杆菌中有135株为ETEC;其中LT阳性为105株,ST阳性为126株,LT+ST阳性的为96株,阳性率为66.5%。而采用直接从粪样中提取PCR模板的方法进行PCR,检测到146个犊牛粪样为ETEC阳性,其中LT阳性为112株,ST阳性为137株,LT+ST阳性为103株,阳性率为71.9%,明显高于传统的检测方法;并且所检测到的146个ETEC阳性的犊牛粪样完全包含用传统的分离培养方法得到的135个阳性粪样,且基因分型结果相同。     

Shifting prevalence of major diarrheal pathogens in patients seeking hospital care during floods in 1998, 2004, and 2007 in Dhaka, Bangladesh

Bangladesh experienced severe flooding and diarrheal epidemics in 2007. We compared flood data from 2007 with 2004 and 1998 for diarrheal patients attending the ICDDR,B hospital in Dhaka. In 2007, Vibrio cholerae O1 (33%), rotavirus (12%), and enterotoxigenic Escherichia coli (ETEC) (12%) were most prevalent. More severe dehydration was seen in 2007 compared with 2004 and 1998 (P < 0.001). In 2007, V. cholerae O1 Inaba (52%) and Ogawa (48%) were seen, whereas in 2004 and 1998 it was primarily Inaba and the Ogawa types, respectively (P < 0.001). In 2007, 51% of ETEC produced the heat labile toxin (LT) (P < 0.001 compared with 2004), 22% expressed the heat stable (ST) (P < 0.001), and 27% were ST/LT positive (P = 0.231). The CS7 colonization factor (CF) was the most prevalent in 2007 (20% compared with 6% in 2004; P = 0.05). Our findings demonstrate alterations in clinical features and phenotypic changes of major bacterial pathogens in the recent Bangladesh flood.     

Aetiological studies of diarrhoeal diseases in infants and young children in Iran

The aetiology of diarrhoeal diseases was investigated among 715 patients admitted to four Children's Hospitals in Tehran, during February 1986 to March 1987, and also among 443 patients attending the central Out-Patient Clinic in Sanandaj, State of Kordestan, during October 1986. Enteropathogenic Escherichia coli (EPEC) were the most common pathogens found in both areas. Almost 26.7% of the patients in Tehran and 20.1% of the patients in Sanandaj were infected with EPEC. Enterotoxigenic E. coli (ETEC) were the next most frequent groups found (17.1% both in Tehran and Sanandaj), with heat-stable enterotoxin (ST)-producing strains being dominant. Of 122 ETEC strains isolated in Tehran, 94 (77%) strains produced ST, 15 (12.3%) strains produced heat-labile enterotoxin (LT) and 13 (10.7%) strains produced both LT and ST. Almost the same pattern of toxigenicity was observed among ETEC strains isolated in Sanadaj. Of the 76 ETEC strains isolated in this area, 70 (92.1%) strains were ST producers, followed by those producing both LT and ST (five strains) and LT only (one strain). One strain of enteroinvasive E. coli (EIEC) was also isolated from a patient in Tehran. The rates of salmonella and shigella isolation were 8.8 and 5.7% in Tehran and 3.8 and 4% in Sanandaj respectively. Enterohaemorrhagic E. coli, Vibrio cholerae and V. parahemolyticus were not isolated but a mixture of two or more pathogens was found in 59 patients (8.2%) in Tehran and in 20 patients (4.5%) in Sanandaj. These findings suggest that diarrhoegenic E. coli are the most important cause of diarrhoeal diseases in infants and young children in these areas in Iran.     

Enterotoxin-producing bacteria isolated from Swedish travellers with diarrhoea

The isolation rate of bacterial enteropathogens of different species, particularly enterotoxin-producing Gram-negative bacteria, was determined in stool specimens from Swedish travellers with diarrhoea. Overall, bacterial enteropathogens were identified in 101 (47%) of the 217 travellers on their return home. The most common isolates were enterotoxin-producing bacteria (20%), Salmonellae (18%) and Campylobacter (8%), whereas Shigellae (3%) and Yersinia (0.5%) were rarely identified. Mixed infections were only found in 8 (4%) of the stool specimens. Enterotoxigenic bacteria of Escherichia coli (ETEC), Klebsiella, Morganella, Citrobacter, Pseudomonas and EF-group 10 species were identified. ETEC accounted for 37/43 (86%) enterotoxin-producing strains, and among them 54% produced heat-stable enterotoxin (ST) alone, 16% heat-labile enterotoxin (LT) alone and 30% both LT and ST. Four of the enterotoxin-producing non-E. coli strains produced ST and 2 produced LT alone. The isolation rate of enterotoxin-producing bacteria was somewhat higher in travellers visiting Africa, Asia and Latin America (24%) than in those travelling to Southern Europe (14%). Salmonellae, on the other hand, were identified in stools significantly more often after travel to Southern Europe (26%) than to various subtropical and tropical areas (12.5%).     

Vaccine for enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-linked to the B subunit of heat-labile toxin

Synthetically produced Escherichia coli heat-stable toxin (ST) was conjugated to the nontoxic B subunit of the heat-labile toxin (LT) by the carbodiimide reaction. Modifying the molar ratio of toxins mixed and the ratio of carbodiimide added to the toxins permitted synthesis of conjugates with any desired degree of proportional antigenicity for each toxin component. Immunization of rats by the parenteral/peroral routes with cross-linked vaccine containing 39% ST and 61% B subunit antigenicity, with 0.06% residual ST toxicity, evoked fourfold to sevenfold increases over control values of serum IgG and mucosal secretory IgA antitoxin titers to each of the component toxins, thus providing significant (P less than 0.001) protection against challenge with either LT or ST or with viable heterologous strains which produce these toxins. These observations show that cross-linking synthetic ST to the B subunit results in a nontoxic vaccine that provides protection against all types of enterotoxigenic E. coli.