A possible murine model for investigation of pathogenesis of sudden infant death syndrome.

Several studies have indicated a possible causative role of toxigenic bacteria in sudden infant death syndrome (SIDS). This study examined the effect of toxigenic E. coli on pregnant and infant mice to determine if these animals could be used as a model for SIDS pathogenesis. Strains of E. coli from the intestinal contents of infants who have died of SIDS or other causes and from the faeces of healthy infants were collected over a broad time scale. The isolates were tested for their ability to produce then known toxins of E. coli and were serotyped (O and H antigens). Certain serotypes (e.g. O1:H- and O25:H1) emerged significantly more frequently from cases of SIDS than from healthy infants and isolates of these types were generally toxigenic in Vero-cell cultures but whose verotoxicity was not related to classical Shiga or other known toxins. This mouse model was developed to test the effects of these toxigenic and also non-toxigenic strains. Four apparently healthy pups aged between 17 and 21 days died unobserved overnight but no pups of the 54 control mice died suddenly (P = 0.0247, Fisher's exact test). These were considered to represent sudden unexpected deaths. Pathological effects compatible with those in SIDS were observed in mouse pups exposed to toxigenic strains indicating this model may be suitable for further study into the pathogenesis of unexpected deaths in infancy. Providing an animal model of SIDS would promote a much better avenue for studying the pathogenesis of this enigmatic condition.

Serotypes of Escherichia coli in sudden infant death syndrome.

AIM: To examine the diversity of Escherichia coli serotypes found in the intestinal contents of infants who died of Sudden Infant Death Syndrome (SIDS) compared with that in comparison infants. METHODS AND RESULTS: Over the 3-year period, 1989-1991, in South Australia and Victoria (Australia), a total of 687 E. coli isolates from 231 patients with SIDS (348 isolates), 98 infants who had died from other causes (144 isolates) and 160 healthy infants (195 isolates) were studied. The isolates from patients with SIDS were found to represent 119 different serotypes; the isolates from 'other cause' infants represent 97 different serotypes; and the isolates from healthy infants represent 117 different serotypes. The seven common serotypes isolated most frequently from infants with SIDS belonged to those associated with extra-intestinal infections in humans. Compared to healthy infants (6%), these were found in significantly higher proportions among infants who died of other causes (13%, P < 0.05) or infants with SIDS (18.7%, P = 0.0002). CONCLUSIONS: Despite these sources yielding a wide variety of serotypes of E. coli, a pattern of certain potential pathotypes of E. coli being associated with SIDS is apparent. SIGNIFICANCE AND IMPACT OF THE STUDY: While SIDS remains one of the most important diagnoses of postneonatal death, its causes are still unexplained. If E. coli has a role in the pathogenesis of SIDS (as suggested by the pathotypes identified on the basis of serotype), further studies may reveal novel virulence factors that may clarify the role of this bacterium in SIDS.

Detection of virulence genes in Escherichia coli of an existing metabolic fingerprint database to predict the sources of pathogenic E. coli in surface waters.

A collection of 366 Escherichia coli strains from 10 host groups and surface waters were tested for the presence of 15 virulence genes associated with strains causing intestinal and extra-intestinal infections. The virulence genes included eaeA, VT1, 2 and 2e, LT1, ST1 and 2, Einv gene, EAgg gene, CNF1 and 2, papC, O111 and O157 side chain LPS. Of the 262 strains obtained from nine different hosts, 39 (15%) carried one or more of these virulence genes. These included six strains from humans, two from horses, eight from dogs, two from ducks, five from cattle, seven from chickens, four from pigs, two from sheep and three from deer. Of the remaining 104 strains obtained from water samples, 10 (10%) also carried one or more of the tested virulence genes. Of these, six had identical biochemical phenotypes (BPTs) to strains isolated from humans (two strains), dogs (two strains), chickens (one strain) and sheep (one strain) with 4 BPTs also carrying same virulence genes. Our results indicate that the sources of clinically important E. coli strains found in surface waters due to faecal contamination can be predicted by using a combination of biochemical fingerprinting method and the detection of virulence genes. From the public health point of view this information will be of great importance for evaluating the risk associated with public use of the catchment.

Reliability of O157:H7 ID agar (O157 H7 ID-F) for the detection and isolation of verocytotoxigenic strains of Escherichia coli belonging to serogroup O157.

AIMS: The reliability of the O157:H7 ID agar (O157 H7 ID-F) to detect verocytotoxigenic strains of Escherichia coli (VTEC) of serogroup O157 was investigated. METHODS AND RESULTS: This medium, designed to detect strains belonging to the clone of VTEC O157:H7/H-, contains carbohydrates and two chromogenic substrates to detect beta-d-galactosidase and beta-d-glucuronidase and sodium desoxycholate to increase selectivity for Gram-negative rods. A total of 347 strains of E. coli including a variety of serotypes, verocytotoxigenicity of human and animal sources were tested. The green VTEC O157 colonies were easy to detect among the other dark purple to black E. coli colonies. Of 63 O157:H7/H- strains, 59 (93.7%) gave the characteristic green colour. Three of the failed four strains of O157:H- were not verocytotoxigenic, missing only one VTEC O157. Three non-O157 strains gave the characteristic green colour on the medium and were VTEC OR:H- (2) and Ont:H- (1), possibly being degraded variants of the O157 enterohaemorrhagic E. coli clone. CONCLUSIONS: The O157:H7 ID agar (O157 H7 ID-F) was largely successful in isolating VTEC belonging to the O157:H7/H- clone. SIGNIFICANCE AND IMPACT OF THE STUDY: A medium, suitable for isolating strains of VTEC O157 was successfully evaluated and should be useful for the isolation of these pathogens.

The diversity of Escherichia coli serotypes and biotypes in cattle faeces.

AIM: To study the diversity of commensal Escherichia coli populations shed in faeces of cattle fed on different diets. METHODS AND RESULTS: Thirty Brahman-cross steers were initially fed a high grain (80%) diet and then randomly allocated into three dietary treatment groups, fed 80% grain, roughage, or roughage + 50% molasses. Up to eight different E. coli isolates were selected from primary isolation plates of faecal samples from each animal. Fifty-two distinct serotypes, including nine different VTEC strains, were identified from a total of 474 E. coli isolates. Cattle fed a roughage + molasses diet had greater serotype diversity (30 serotypes identified) than cattle fed roughage or grain (21 and 17 serotypes identified respectively). Cluster analysis showed that serotypes isolated from cattle fed roughage and roughage + molasses diets were more closely associated than serotypes isolated from cattle fed grain. Resistance to one or more of 11 antimicrobial agents was detected among isolates from 20 different serotypes. Whilst only 2.3% of E. coli isolates produced enterohaemolysin, 25% were found to produce alpha-haemolysin. CONCLUSIONS: Diverse non-VTEC populations of E. coli serotypes are shed in cattle faeces and diet may affect population diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the serotype diversity and phenotypic traits of predominant E. coli populations in cattle faeces, which could be sources of environmental contamination.

A newly discovered verotoxin variant, VT2g, produced by bovine verocytotoxigenic Escherichia coli.

A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H-.

Serotypes of Escherichia coli isolated from healthy infants in Berlin, Germany and Melbourne, Australia.

The characterization of Escherichia coli strains isolated from healthy infants under one year of age with respect to O:H serotype, K1 and K5 antigens in two disparate parts of the developed world was the purpose of this investigation. A total of 450 strains were examined, 264 from Berlin and 186 from Melbourne. Of all the 220 different O:H serotypes found, 179 were only isolated once, 90 in Berlin and 89 in Melbourne. However, 30 of the 41 O:H serotypes (73.2%) found more than once were isolated in both centers. The most commonly identified serotypes were found in both centers and included O1:H-; O1:H7; O2:H2; O2:H4; O2:H7; O4:H5; O6:H-; O6:H1; O15:H1; O18:H7; O25:H1; and 075:H-. Potentially pathogenic serotypes were found in both cities. Enteropathogenic E. coli (EPEC)-associated serotypes (O18:H7; O26:H-; O44:H34; O86:H-; O128:H2) were present in 11 cases and enterohaemorrhagic E. coli (EHEC)-associated types including O26:H11; O128:H2) were present in four cases. The distributions of serotypes found were similar in the two cities, strongly suggesting the wider applicability of these results.

Serotypes of non-O157 verocytotoxigenic Escherichia coli isolated from meat in New Zealand.

Verocytotoxigenic Escherichia coli (VTEC) were isolated from meat in New Zealand. They were tested for the presence of virulence factors associated with VTEC and serotyped. Some of the serotypes found were identical to ones reported from other parts of the world, but some serotypes were also found which had not been reported elsewhere. This study confirms the world-wide distribution of these emerging food-borne pathogens.

Development of a rapid method for the detection of verocytotoxin-producing Escherichia coli (VTEC).

AIMS: The object of this study was to adapt a new test kit to achieve the rapid identification of verocytotoxin-producing Escherichia coli (VTEC). METHODS AND RESULTS: The kit, which is based on reversed passive latex agglutination (RPLA), was combined with a method for identifying heat-labile enterotoxin-producing E. coli (LT-ETEC). An answer can thereby be obtained within a single working day. Over 200 strains, both VTEC and non-VTEC, of human and animal origin, belonging to a variety of serotypes were tested in this new rapid technique. Apart from three false-positive results, all other strains gave the correct answer by this rapid method. CONCLUSION: The method should enable the diagnosis of these important human pathogens to be made rapidly by most microbiological laboratories, and permit appropriate actions to be taken rapidly and therefore more effectively.

The common ovine Shiga toxin 2-containing Escherichia coli serotypes and human isolates of the same serotypes possess a Stx2d toxin type.

Shiga toxin 2 (Stx2) has been reported as the main Shiga toxin associated with human disease. In addition, the Stx2 toxin type can have a profound impact on the degree of tissue damage in animal models. We have characterized the stx(2) subtype of 168 Shiga toxin-producing Escherichia coli (STEC) isolates of which 146 were derived from ovine sources (principally feces and meat) and 22 were isolated from humans. The ovine STEC isolates were of serotypes that have been shown to occur commonly in the gastrointestinal tract of healthy sheep. The major stx(2) subtype in the ovine isolates was shown to be stx(2d-Ount) (119 of 146 [81.5%]) and was predominantly associated with serotypes O75:H(-)/H8/H40, O91:H(-), O123:H(-), O128:H2, and OR:H2. However, 17 of 18 (94.4%) ovine isolates of serotype O5:H(-) possessed a stx(2d-O111/OX3a) subtype. Furthermore, STEC isolates of serotypes commonly found in sheep and recovered from both clinical and nonclinical human infections also contained a stx(2d) (stx(2d-Ount/O111/OX3a)) subtype. These studies suggest that a specific stx(2) subtype(s) associates with serotype and may have important epidemiological implications for tracing sources of E. coli during outbreaks of STEC-associated diseases in humans.

Virulence properties and serotypes of Shiga toxin-producing Escherichia coli from healthy Australian slaughter-age sheep.

A group of 1,623 ovine fecal samples recovered from 65 geographically distinct mutton sheep and prime lamb properties across New South Wales, Australia, were screened for the presence of Shiga toxin-producing Escherichia coli (STEC) virulence factors (stx(1), stx(2), eaeA, and ehxA). A subset was cultured for STEC isolates containing associated virulence factors (eaeA and/or ehxA), which were isolated from 17 of 20 (85%) and 19 of 20 (95%) tested prime lamb and mutton sheep properties, respectively. STEC isolates containing stx(1), stx(2), and ehxA were most commonly isolated (19 of 40 flocks; 47.5%), and this profile was observed for 10 different serotypes. Among 90 STEC isolates studied, the most common serotypes were O91:H(-) (22 isolates [24.4%]), O5:H(-) (16 isolates [17.8%]), O128:H2 (11 isolates [12.2%]), O123:H(-) (8 isolates [8.9%]), and O85:H49 (5 isolates [5.6%]). Two isolates (2.2%) were typed as O157:H(-). A total of 78 of 90 STEC isolates (86.7%) expressed Shiga toxin in Vero cell culture and 75 of 84 ehxA-positive isolates (89.3%) expressed enterohemolysin on washed sheep blood agar. eaeA was observed in 11 of 90 (12.2%) ovine STEC isolates, including serotypes O5:H(-), O84:H(-), O85:H49, O123:H(-) O136:H40, and O157:H(-). Although only 2 of 90 isolates were typed as O157:H(-), the predominant serotypes recovered during this study have been recovered from human patients with clinical disease, albeit rarely.

The detection of Shiga toxin-producing Escherichia coli in diagnostic bovine faecal samples using vancomycin-cefixime-cefsulodin blood agar and PCR.

The prevalence of complex Shiga toxin-producing Escherichia coli (STEC), i.e. STEC containing accessory virulence factors intimin (eaeA) and/or enterohaemorrhagic E. coli haemolysin (ehxA) and their serotypes were determined in diagnostic bovine faecal samples processed during a 3 months period. The presence of complex STEC was determined using PCR and vancomycin-cefixime-cefsulodin blood agar (BVCCA) using a dual approach which involved (i) direct culture of faecal samples on BVCCA followed by mutiplex PCR of BVCCA positive colonies and (ii) culture of faecal samples enriched in modified EC (mEC) broth (with a complex STEC profile determined by PCR) on BVCCA followed by multiplex PCR of BVCCA positive colonies. Using both techniques complex STEC were isolated from 23 (18.7%) of the 123 faecal samples. Complex STEC were isolated from 14 faecal samples by direct culture on BVCCA and 13 faecal samples yielded complex STEC by culture of mEC broths with a complex STEC profile on BVCCA. Only four samples were positive using both techniques. The serotypes isolated included O5:H-, O26:H-, O26:H11, O91:H21, O111:H-, O111:H8, O104:H11, O113:H21 and O157:H8. This study confirms that non-O157 STEC can be isolated from bovine faeces and that they carry types associated with human disease. This work also demonstrates that the use of a dual approach is advisable to increase the likelihood of isolating complex STEC.

Simultaneous isolation of verotoxin-producing strains of Escherichia coli O128:H2 and viruses in gastroenteritis outbreaks.

Three outbreaks of gastroenteritis from which the Verotoxin producing Escherichia coli serotype O128:H2 was isolated are reported. In addition Norwalk-like viruses were detected in patients from two of the outbreaks and astrovirus in the third outbreak. While it cannot be specifically determined which of these agents played the major role in these outbreaks, the findings suggest that the viral agents need to be considered in investigations of gastroenteritis outbreaks, regardless of whether bacterial enteropathogens have also been isolated. This study points to a strong need to investigate gastroenteritis outbreaks for both bacterial and viral agents and to review in detail the asymptomatic carriage rate of Verotoxin-producing bacteria and gastroenteritis-associated viral agents; these areas of public health significance have been largely neglected.

Multiple outbreaks of Norwalk-like virus gastro-enteritis associated with a Mediterranean-style restaurant.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

Occurrence and virulence factors of non-O157 Shiga toxin-producing Escherichia coli in retail meat in Dunedin, New Zealand.

Retail raw meat was sampled for the presence of Shiga toxin-producing Escherichia coli (STEC) using enrichment culture and Vero cell assay. The STEC obtained were serotyped and tested for enterohaemolysin (Ehly) production and the eae gene. The presence of Shiga toxin genes (stx) was confirmed by polymerase chain reaction. A total of 18 STEC were isolated accounting for 12% of beef, 17% of lamb and 4% of pork samples. Five isolates produced Ehly but none possessed the eae gene. Five isolates were identified which possessed the stx2 gene and belonged to serotypes associated with severe infection.

Escherichia coli 'O' group serology of a haemolytic uraemic syndrome (HUS) epidemic.

This is the first comprehensive serological analysis of a haemolytic uraemic syndrome (HUS) outbreak. A wide range of 'O' group Escherichia coli antibody responses in patients and controls was examined. The study provides a unique insight into the epidemiology of such epidemics, points a way to the most appropriate investigation of these and indicates possible answers to a number of issues related to severity of disease. In order to be able to test for a wide variety of E. coli 'O' antigens, a microagglutination assay was used to examine E. coli 'O' group serological responses of 22 children admitted to hospital with HUS and 14 contemporaneous age-matched controls. A total of 51 'O' serogroup strains were used. These included 'O' groups reported to be associated with cases of HUS, with 6 isolates from patients associated with the Adelaide outbreak (O26, O111, O123 and O157), environmental Verocytotoxigenic/Shiga-toxin producing Escherichia coli (VTEC/STEC) strains and common human commensal strains. Sixteen clinically confirmed HUS cases (72.7%) of 22 seroconverted to 1 or more serogroups of which 11 (50%) seroconverted to O111 (the serogroup isolated from 16 patients). In addition, 11 (50%) and 10 (45.5%) developed antibody to O137 and O145, respectively, although no stool isolates of these serogroups were made. Seventeen (77.3%) of 22 HUS patients had antibody to serogroup O157, with 11 (50%) seroconversions, however, O157:H- was isolated from only 2 of these. Overall, titres ranged from 100 to 6400, some of the highest in 3 patients were against O157, whose faeces yielded only Enterohaemorrhagic E. coli (EHEC) O111, and only 1 developed O111 antibody. Mixed infection was demonstrated serologically by microagglutination (confirmed by Western blot) and was consistent with the findings of multiple serogroups of VTEC found in the mettwurst incriminated as the source, and suggests further strains (not found in the source or in patients' faeces) were probably also involved. In HUS associated with EHEC infection, multiple strain infection may be the rule rather than the exception. A relationship with clinical severity deserves further investigation. Non-O157 EHEC (in addition to O157) should be sought in all future outbreaks of EHEC disease.