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.     

Bacterial pathogens associated with diarrhoea on the island of Crete

Stool specimens from 3,600 diarrhoeal patients from the island of Crete, Greece, were examined for bacterial pathogens, during a three-year period (1992-1994). One or more pathogens were identified in 826 patients (22.9%), more often from children. Salmonella spp. were the most frequently isolated organisms in 13.6% of the patients, followed by Campylobacter in 4.7%, and enteropathogenic Escherichia coli (EPEC) in 3.9%. Yersinia enterocolitica was found in 0.7%, Shigella spp. in 0.7% and Aeromonas hydrophila in 0.05%. Vibrio spp. and enterohaemorragic E. coli were not identified in the stools tested. Resistance to ampicillin was observed in 36% of the Salmonella, 62% of the Shigella, and 27% of the EPEC isolates. Cotrimoxazole resistance was observed in 42% of the Shigella and 12% of the EPEC isolates, while tetracycline and the quinolones were inactive against almost half and erythromycin against 20% of the Campylobacter isolates. This is the first study investigating bacterial pathogens associated with diarrhoea on the island of Crete.     

A case-control study of diarrhoea in children caused by Escherichia coli producing heat-stable enterotoxin (EAST-1)

Escherichia coli strains associated with diarrhoeal disease have been classified into several types according to the pathogenic mechanism. Among these, enteroaggregative E. coli strains (EAggEC) have been associated with persistent childhood diarrhoea. Some strains of EAggEC produce a heat-stable toxin (EAST-1) that differs from others described previously. The main goal of this case-control study was to determine the prevalence of EAggEC and EAST-1-producing E. coli strains as a cause of diarrhoea in children in Spain and to study their in-vitro susceptibility to 21 antimicrobial agents. In the case group (115 children) 22 (19%) isolates and four (3.5%) isolates were EAST-1-producing E. coli and EAggEC, respectively, whereas in the control group (79 children) four (5%) isolates produced EAST-1 (p = 0.005) and three (3.8%) isolates were EAggEC. The present study suggests that EAST-1-producing E. coli strains are associated with diarrhoeal diseases in Spanish children, whereas EAggEC strains are not. Moreover, EAST-1-producing E. coli strains showed a high susceptibility to all the antimicrobial agents tested except for ampicillin.     

Acute and chronic diarrhoea and abdominal colic associated with enteroaggregative Escherichia coli in young children living in western Europe

BACKGROUND: Enteroaggregative Escherichia coli (EAggEC or EAEC) can spread and cause disease in developing countries, but it is not presently known whether it spreads disease in industrialised countries. Therefore, we did a prospective study to assess the incidence and the clinical manifestations of infections due to EAEC in children in Germany. METHODS: 798 children with diarrhoea, admitted to hospital within a defined geographical area during a 24-month period, were included in the trial. EAEC were cultured from stool specimens, screened by PCR, and identified by colony hybridisation from DNA sequences found on the virulence plasmid. The findings were confirmed by aggregative adherence to HEp-2 cells. Stool samples from 580 children admitted to hospital without diarrhoea were also studied as controls. FINDINGS: EAEC were found in the stools of 16 (2%) of 798 children with diarrhoea, but in none of 580 children without diarrhoea. Only four of the EAEC-infected children had travelled to developing countries. Most EAEC infections were acquired in the summer months. Infection with EAEC was associated with acute, watery diarrhoea in 12 children, and with chronic diarrhoea of up to 5 months' duration in four. Five children had abdominal colic that lasted for 2-4 weeks as their main symptom. The incidence of EAEC infection was 7.7 patients admitted to hospital per 100,000 children in the general population aged younger than 16 years. INTERPRETATION: EAEC infection is associated with acute, watery diarrhoea and may be acquired in industrialised countries. Chronic diarrhoea or abdominal colic of unknown aetiology in young children may also be caused by EAEC infection.     

Mechanisms of gut damage by Escherichia coli

This chapter primarily concerns three main categories of diarrhoeagenic Escherichia coli, enteropathogenic (EPEC), enterohaemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. They have distinctive virulence factors and vary in the enteropathies they produce. The molecular biological approach has opened up the complex way in which they interact with the intestine. EPEC and EHEC show a subversive approach to colonization in that they adapt the host cell to their requirements in the formation of the attaching effacing lesion. EAEC appear to co-opt the host defence system to produce a biofilm-like colony and currently go unrecognized in routine laboratories.     

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.     

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.     

Isolation and identification of Aeromonas species from human stools

One thousand and three diarrhoeal stool samples were processed in our laboratory during the period 1996/1997 for the presence of enteric pathogens especially Aeromonas spp., which has emerged as a new agent causing diarrhoea. Ampicillin sheep blood agar was found to be the best medium for the isolation of Aeromonas spp. from stool specimens. Enteric pathogens were found in 200 (20%) stools, of which Aeromonas spp. was the second commonest pathogen isolated amounting to 21% of isolates. This study clearly indicates that Aeromonas spp. must be looked for in every diarrhoeal stool samples, specially in children below 10 years of age. Isolation and identification is cost effective and easy, if the given protocol is observed.     

Divergent signal transduction responses to infection with attaching and effacing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is an attaching and effacing pathogen that causes hemorrhagic colitis and the hemolytic-uremic syndrome. Although this organism causes adhesion pedestals, the cellular signals responsible for the formation of these lesions have not been clearly defined. We have shown previously that STEC O157:H7 does not induce detectable tyrosine phosphorylation of host cell proteins upon binding to eukaryotic cells and is not internalized into nonphagocytic epithelial cells. In the present study, tyrosine-phosphorylated proteins were detected under adherent STEC O157:H7 when coincubated with the non-intimately adhering, intimin-deficient, enteropathogenic E. coli (EPEC) strain CVD206. The ability to be internalized into epithelial cells was also conferred on STEC O157:H7 when coincubated with CVD206 ([158 +/- 21] % of control). Neither the ability to rearrange phosphotyrosine proteins nor that to be internalized into epithelial cells was evident following coincubation with another STEC O157:H7 strain or with the nonsignaling espB mutant of EPEC. E. coli JM101(pMH34/pSSS1C), which overproduces surface-localized O157 intimin, also rearranged tyrosine-phosphorylated and cytoskeletal proteins when coincubated with CVD206. In contrast, JM101 (pMH34/pSSS1C) demonstrated rearrangement of cytoskeletal proteins, but not tyrosine-phosphorylated proteins, when coincubated with intimin-deficient STEC (strains CL8KO1 and CL15). These findings indicate that STEC O157:H7 forms adhesion pedestals by mechanisms that are distinct from those in attaching and effacing EPEC. Taken together, these findings point to diverging signal transduction responses to infection with attaching and effacing bacterial enteropathogens.     

Aetiology of acute diarrhoea in hospitalized children in Hong Kong

To determine the role of enteric pathogens in acute childhood diarrhoea in Hong Kong, 388 children with diarrhoea and 306 children of similar age without diarrhoea were evaluated in a hospital-based study during a one-year period from August 1994 to July 1995. Of the diarrhoeal cases, 55% were under 1 year and 95% were below 5 years of age. On admission, 22% had some dehydration but none was severely dehydrated. All children were well nourished. Oyer 60% of children with diarrhoea had one or more pathogens in their stool. Rotavirus was the most commonly isolated pathogen (34.6%), followed by Salmonella (23.3%), Campylobacter (4.7%) and Shigella (2.1%). Rotavirus was not assessed in the controls and was detected mainly during the winter months December to February. Bacterial pathogens were identified more commonly in diarrhoea patients (30%) than in controls (5.6%) (P < 0.001). Despite rapid recent socioeconomic development in Hong Kong, non-typhoidal Salmonella diarrhoea remains a significant local problem in infants under 1 year. Further detailed assessment of the transmission and prevention of this infection is required.     

Frequency and etiology of calf losses and calf diseases before weaning in cow-calf farms. II. Microbiological and parasitological diagnoses in diarrheic calves]

Diarrhea is the most frequently diagnosed disease in Swiss cow-calf farms. During an longitudinal study conducted in 105 cow-calf farms in Switzerland in 1993-95, blood and fecal samples were collected from diarrheic calves and from calves that died because of diarrhea. Campylobacter spp. were detected in 42%, E. coli (VTEC) in 32%, rotavirus in 33%, coronavirus in 13%, coccidia in 43% and helminths in 8% of the cases. In some samples Yersinia pseudotuberculosis were isolated. The BVD-virus antigen was not detected in any of the calves examined. In most cases concurrent infection with several enteropathogens was diagnosed. Specific causes of diarrhea were therefore difficult to establish. The bacteriological findings of this study have implication for food safety: VTEC, Campylobacter spp. Yersinia spp. and cryptosporidia are considered as potential causes on zoonoses.     

Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.     

Production and role of interleukin-10 in concanavalin A-induced hepatitis in mice

Coliform colonies from children whose stools were submitted for microbiologic analysis were studied prospectively to determine the frequency of shedding of enteropathogenic Escherichia coli (EPEC). In total, 2225 isolates from 445 patients were probed with eaeA (encoding intimin) and the EAF (EPEC adherence factor) probe, and adherence and actin-aggregating phenotypes were determined. Twenty-five patients (5.6%) shed non-O157:H7 eaeA+ EAF- E. coli. Of these 25 patients, isolates from 5 produced Shiga toxins and from 3 possessed bfpA (encoding the bundle-forming pilus) sequences. Non-O157:H7 eaeA+ E. coli from 21 (84%) of 25 patients adhered locally to and aggregated actin in HeLa cells. Four patients shed nonadherent EAF+ eaeA- E. coli. Non-O157:H7 eaeA+ and EAF- isolates belonged to diverse electrophoretic types and classical and nonclassical enteropathogenic serotypes. EPEC are relatively common in stools submitted for analysis in this North American pediatric hospital. Their etiologic role in childhood diarrhea warrants elucidation.     

Enteropathogenic, enterotoxigenic and enteroinvasive Escherichia coli isolated from acute gastroenteritis patients in Lagos, Nigeria

Enteropathogenic, enterotoxigenic and enteroinvasive Escherichia coli were isolated from 52 (4.8%) of 1,082 patients with acute gastroenteritis reporting at the Lagos University Teaching Hospital, Lagos, Nigeria between October 1979 and March, 1981. Of the 52 strains of E. coli isolated, 35 (67.3%) were enteropathogenic, 12 (23.1%) were enterotoxigenic and five (9.6%) were enteroinvasive. E. coli 0111 (25.7%) was the most predominant among the serotypes of the "classical" enteropathogenic strains found in this study. Diarrhoea associated with enteropathogenic E. coli occurred only in children aged less than five years, whereas enterotoxigenic and enteroinvasive E. coli were found primarily in adults. The study has highlighted for the first time the important role that enterotoxigenic and enteroinvasive E. coli strains could play in acute diarrhoeal diseases in Lagos, Nigeria.     

Giardiasis in children with chronic diarrhea. Incidence, growth, clinical symptoms and changes in the small intestine

During a six-year period, 29 children (aged 0.7-13.5 years, mean 3.3 years) suffering from chronic diarrhoea due to giardiasis were studied. The incidence of this illness was 81 per 1,000,000 per year among children aged 0- < 7 years. According to growth charts, relative height and weight of the patients decreased significantly (both approximately 0.5 SD) from before the onset of diarrhoea to the time of diagnosis and subsequently increased up to the end of catch-up growth. Small intestinal mucosal specimens were studied. Two patients had severe villous atrophy, 8 moderate abnormalities, 6 only slight changes and 13 biopsies were normal. D-xylose or lactose malabsorption was detected in 25% of the patients. The lactose malabsorption was due to hereditary low lactase levels. None of the patients with a Danish ethnic background showed lactose malabsorption. D-xylose absorption and the relative weight loss of the patients correlated with the degree of mucosal damage. Patients with persistent diarrhoea (n = 19) were younger and had a shorter duration of diarrhoeal illness and a more significant weight reduction than those with intermittent diarrhoea (n = 10). However, the age at onset of symptoms was similar in the two groups (medians 1.3 years). Seven patients contracted the disease abroad. They all developed persistent diarrhoea and had a more severe course of the illness than those who acquired the disease in Denmark.     

Cellular responses to enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC), first described in the 1940's and 1950's, remain an important cause of severe infantile diarrhoea in many parts of the developing world. EPEC do not produce enterotoxins and are not invasive; instead their virulence depends upon exploitation of host cell signalling pathways and the host cell cytoskeleton both as a means of colonizing mucosal surfaces of the small intestine and causing diarrhoea. Following initial mucosal attachment, EPEC secrete 'signalling' proteins and express a surface adhesin, intimin, to produce 'attaching & effacing' lesions in the enterocyte brush border membrane characterised by localised destruction of brush border microvilli, intimate bacterial adhesion and cytoskeletal reorganisation and accretion beneath attached bacteria. The pathophysiology of EPEC diarrhoea is also complex and probably results from a combination of epithelial cell responses including both electrolyte secretion and structural damage.     

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.     

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.     

Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain

Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5 Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1-7, 8-14, 15-21 and 22-30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22-30 days and all other age groups, and between the age group 1-7 days and the age groups 8-14 days and 15-21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1-7, 8-14, 15-21 and 22-30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p < 0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.     

A clinical and epidemiological study of gastro-enteritis

Over a 13-year period, 2,903 Asian, Coloured and White children with gastro-enteritis were admitted to the Wilkins Infectious Diseases Hospital, Salisbury. During a pilot study of 250 patients conducted over the first 2 years, a definite seasonal variation in the occurrence of gastro-enteritis, with a peak incidence in winter, was noticed, and this led to a more detailed study of 2,653 patients over the next 11 years. Bacterial pathogens were recovered from 27% of patients, strains of Shigella accounting for 12% Salmonella for 7% and Escherichia coli for 8%. A severe outbreak of E. coli infection occurred between October 1971 and December 1972, and 4 of a total of 9 deaths occurred during this period, all in children suffering from E. coli 0111/B4 infections. The bacterial recovery rate was highest in the summer (40%) and lowest in the winter (12%). Winter diarrhoea broke out over 3-month periods during each year and accounted for 37% of the cases, but none of the children who contracted it died. The clinical picture of winter diarrhoea differed from the others and the most serious illness was that caused by enteropathogenic strains of E. coli. The over-all mortality in the series was 0,27%.