Diarrheagenic enteroaggregative Escherichia coli causing urinary tract infection and bacteremia leading to sepsis.

We report a case of a 55-year-old immunocompromised female who presented to the emergency department with severe diarrhea and vomiting following travel to the Philippines. Stool bacteriology revealed a mixed infection involving an enteropathogenic Escherichia coli and two distinct strains of enteroaggregative Escherichia coli (EAEC). During hospitalization, urine and blood culture tested positive for one of the diarrheagenic EAEC strains, necessitating urinary catheterization, intensive care, and antimicrobial treatment with trimethoprim-sulfamethoxazole, followed by meropenem. Although known to occasionally cause urinary tract infections, EAEC have not been previously associated with sepsis. Our report highlights the potential of EAEC to cause severe extraintestinal infections.

Detection and characterization of Shiga toxin-producing Escherichia coli in faeces and lymphatic tissue of free-ranging deer.

Shiga toxin-producing Escherichia coli (STEC) have led to outbreaks worldwide and are considered emerging pathogens. Infections by STEC in humans have been reported after consumption of mainly beef, but also deer. This study investigated the occurrence of STEC in deer in Germany. The virulence genes eae, e-hlyA and saa, the stx subtypes, pulsed-field gel electrophoresis (PFGE) patterns and serovars were studied. In total, 120 samples of 60 animals were screened by real-time polymerase chain reaction (PCR). The PCR results showed a high detection rate of stx genes (83%). Mainly faecal samples, but also some lymphatic tissue samples, tested stx-positive. All isolates carried stx2, were eae-negative and carried e-hlyA in 38% and saa in 9% of samples. Serovars (O88:[H8], O174:[H8], O146:H28) associated with human diseases were also identified. In some animals, isolates from lymphatic tissue and faecal samples showed undistinguishable PFGE patterns. The examined deer were shown to be relevant reservoirs of STEC with subtype stx2b predominating.

Survival and spread of Shiga toxin-producing Escherichia coli in alpine pasture grasslands.

AIMS: To determine the fate of Shiga toxin-producing Escherichia coli (STEC) strains defecated onto alpine grassland soils. METHODS AND RESULTS: During the summers of 2005 and 2006, the field survival of STEC was monitored in cowpats and underlying soils in four different alpine pasture units. A most probable number (MPN)-PCR stx assay was used to enumerate STEC populations. STEC levels ranged between 3.9 and 5.4 log(10) CFU g(-1) in fresh cowpats and slowly decreased until their complete decay (inactivation rates k < 0.04 day(-1)). PFGE typing of STEC strains isolated from faecal and soil samples assessed the persistence of various clonal types for at least 2 months in cowpats and their vertical dispersal down through the soil at a depth up to at least 20 cm. STEC cells counts in soil were always below 2 log(10) CFU g(-1), regardless of the pasture unit investigated. The soil became rapidly free of detectable STEC once the cowpat had decomposed. The eight STEC strains isolated during this study belonged to six distinct serotypes and tested positive for the gene(s) stx2, including the stx2g and stx2 NV206 variants. CONCLUSIONS: STEC were able to persist in cowpats and disseminate down through the soil but were unable to establish. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides useful information concerning the ecology of STEC in alpine pasture grasslands and may have implications for land and cattle management.

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli.

AIMS: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. METHODS AND RESULTS: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. CONCLUSIONS: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. SIGNIFICANCE AND IMPACT OF THE STUDY: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.

Characteristics of shiga toxin-producing Escherichia coli isolated from Swiss raw milk cheese within a 3-year monitoring program.

Food is an important vehicle for transmission of Shiga toxin-producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cow's, goat's, and ewe's milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx(1) was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx(2) group, mainly stx(2) and stx(2vh-a/b). Production of Stx(2) and Stx(2vh-a/b) subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.

Evaluation of the 'GeneDisc' real-time PCR system for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains according to their virulence markers and their O- and H-antigen-associated genes.

AIMS: To evaluate the GeneDisc multiplex real-time PCR assay for detection of enterohaemorrhagic Escherichia coli (EHEC) O26, O103, O111, O145 and O157 strains. METHODS AND RESULTS: GeneDiscs for detection of genes encoding Shiga toxins (stx), intimins (eae), E. coli O157 (rfbE(O157)) and H7 (fliC(H7)) antigens as well as genes specific for EHEC O26 (wzx(O26)), O103 (wzx(O103)), O111 (wbd1(O111)), O145 (ihp1(O145)) and O157 (ihp1(O157)) were evaluated. The assay was run with native bacteria in 1 h in a GeneDisc Cycler. All genotypes of stx and eae, except stx(2f) and eae-rho, were identified. Escherichia coli strains belonging to O-groups O26, O103, O111, O157 as well as EHEC O145:[H28] strains were specifically detected with this assay. The ihp1(O157) gene was not found specific for EHEC O157. O-rough mutants of EHEC and non-motile EHEC O157 strains were reliably identified with the GeneDisc assay. Two to three colonies of EHEC strains were still detectable in a lawn of 50 000 apathogenic E. coli from agar plates. CONCLUSIONS: The GeneDisc assay is a specific and reliable assay for detection of major EHEC strains. It is robust enough to detect few EHEC colonies in mixed cultures of bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is promising for its use in EHEC diagnostics and for EHEC monitoring with different kinds of samples.

[Tasks and duties of veterinary reference laboratories for food borne zoonoses]. / Aufgaben der veterinärmedizinischen Referenzlaboratorien im Bereich der lebensmittelbedingten Zoonosen.

Reference laboratories are of central importance for consumer protection. Field expertise and high scientific competence are basic requirements for the nomination of a national reference laboratory. To ensure a common approach in the analysis of zoonotic hazards, standards have been developed by the reference laboratories together with national official laboratories on the basis of Art. 33 of Directive (EG) No. 882/2004. Reference laboratories function as arbitrative boards in the case of ambivalent or debatable results. New methods for detection of zoonotic agents are developed and validated to provide tools for analysis, e. g., in legal cases, if results from different parties are disputed. Besides these tasks, national reference laboratories offer capacity building and advanced training courses and control the performance of ring trials to ensure consistency in the quality of analyses in official laboratories. All reference laboratories work according to the ISO standard 17025 which defines the grounds for strict laboratory quality rules and in cooperation with the respective Community Reference Laboratories (CRL). From the group of veterinary reference laboratories for food-borne zoonoses, the national reference laboratories are responsible for Listeria monocytogenes, for Campylobacter, for the surveillance and control of viral and bacterial contamination of bivalve molluscs, for E. coli, for the performance of analysis and tests on zoonoses (Salmonella), and from the group of parasitological zoonotic agents, the national reference laboratory for Trichinella.

Shedding of food-borne pathogens and microbiological carcass contamination in rabbits at slaughter.

To obtain microbiological data from rabbits at slaughter, 500 fecal samples and 500 carcasses samples were examined. All samples tested negative for Listeria and Salmonella. Campylobacter were detected in two fecal samples. Of the 500 fecal samples, 45.8% tested positive for eae (intimin), 1.2% for stx (Shiga toxin), and 1.8% for both eae and stx. By colony hybridization, 56 eae positive Escherichia coli strains were isolated. Among them, 27 strains (48.2%) were of the serotypes O178:H7 and O153:H7, whereas 15 strains (26.8%) belonged to a serogroup that has not yet been described (O(CB10681):H7). All strains possessed intimin beta1 and the translocated intimin receptor (tir) capable of being tyrosine phosphorylated. None of the strains harbored the genes for Shiga toxins, EAST1 (astA), bundlin (bfpA), or the EAF plasmid. Slaughter rabbits therefore constitute a reservoir for certain atypical enteropathogenic Escherichia coli. On rabbit carcasses, average total bacterial counts accounted for 3.3 log CFU cm(-2). Enterobacteriaceae and coagulase positive staphylococci (CPS) were detected on 118 (23.6%) and 153 (30.6%) carcasses, respectively. Enterobacteriaceae and CPS counts of positive samples were mainly <1.5 log CFU cm(-2). Among 153 selected CPS isolates, 98.7% were identified as Staphylococcus aureus. None of the 151 isolated strains harbored the gene for methicillin resistance (mecA). Genes for staphylococcal enterotoxins (SE) were detected in 102 strains. The combinations of seg and sei (53 strains) and sed, seg, sei, and sej (27 strains) dominated.

Serotypes and virutypes of enteropathogenic and enterohaemorrhagic Escherichia coli strains from stool samples of children with diarrhoea in Germany.

AIMS: To investigate the prevalence of traditional and emerging types of enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) strains in stool samples from children with diarrhoea and to characterize their virulence genes involved in the attaching and effacing (A/E) phenotype. METHODS AND RESULTS: Serological and PCR-based methods were used for detection and isolation of EPEC and EHEC strains from 861 stool samples from diarrhoeic children. Agglutination with traditional EPEC and EHEC O-group-specific antisera resulted in detection of 38 strains; 26 of these carried virulence factors of EPEC or EHEC. PCR screening for the eae gene resulted in isolation of 97 strains, five carried genes encoding Shiga toxins (stx), one carried the bfpA gene and 91 were atypical EPEC. The 97 EPEC and EHEC strains were divided into 36 O-serogroups and 21 H-types, only nine strains belonged to the traditional EPEC O-groups O26, O55, O86 and O128. In contrast, EPEC serotypes O28:H28, O51:H49, O115:H38 and O127:H40 were found in multiple cases. Subtyping the virulence factors intimin, Tir and Tir-cytoskeleton coupling effector protein (TccP)/TccP2 resulted in further classification of 93.8% of the 97 strains. CONCLUSIONS: Our findings show a clear advantage of the eae-PCR over the serological detection method for identification of EPEC and EHEC strains from human patients. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular detection by the eae-PCR followed by serotyping and virutyping is useful for monitoring trends in EPEC and EHEC infections and to discover their possible reservoirs.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level.

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzerland represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows', goats', and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx(1) and stx(2) variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx(2), 6 strains for stx(2d2), 2 strains for stx(2-O118), 1 strain for stx(2-06), 1 strain for stx(2g), 1 strain for stx(2) and stx(2d2), and 1 strain for stx(2) and stx(2g). Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.

Dissemination and persistence of Shiga toxin-producing Escherichia coli (STEC) strains on French dairy farms.

Some Shiga toxin-producing Escherichia coli strains (STEC), and in particular E. coli O157:H7, are known to cause severe illness in humans. STEC have been responsible for large foodborne outbreaks and some of these have been linked to dairy products. The aim of the present study was to determine the dissemination and persistence of STEC on 13 dairy farms in France, which were selected out of 151 randomized dairy farms. A total of 1309 samples were collected, including 415 faecal samples from cattle and 894 samples from the farm environment. Bacteria from samples were cultured and screened for Shiga toxin (stx) genes by polymerase chain reaction (PCR). STEC isolates were recovered from stx-positive samples after colony blotting, and characterized for their virulence genes, serotypes and XbaI digestion patterns of total DNA separated by pulsed-field gel electrophoresis (PFGE). Stx genes were detected in 145 faecal samples (35%) and 179 (20%) environmental samples, and a total of 118 STEC isolates were recovered. Forty-six percent of the STEC isolates were positive for stx1, 86% for stx2, 29% for intimin (eae-gene) and 92% for enterohemolysin (ehx), of which 16% of the STEC strains carried these four virulence factors in combination. Furthermore, we found that some faecal STEC strains belonged to serotypes involved in human disease (O26:H11 and O157:H7). PFGE profiles indicated genetic diversity of the STEC strains and some of these persisted in the farm environment for up to 12 months. A large range of contaminated samples were collected, in particular from udders and teats. These organs are potential sources for contamination and re-contamination of dairy cattle and constitute an important risk for milk contamination.

Virulence profiles of Shiga toxin 2e-producing Escherichia coli isolated from healthy pig at slaughter.

Recently, virulence patterns of Stx2e-producing Escherichia coli from pigs with edema disease and from humans were compared and strains from diseased pigs were reported to be unlikely human pathogens [Sonntag, A.K., Bielaszewska, M., Mellmann, A., Dierksen, N., Schierack, P., Wieler, L.H., Schmidt, M.A., Karch, H., 2005. Shiga toxin 2e-producing Escherichia coli isolates from humans and pigs differ in their virulence profiles and interactions with intestinal epithelial cells. Appl. Environ. Microbiol. 71, 8855-8863]. In the present study, 31 Shiga toxin-producing E. coli (STEC) strains harboring stx2e, which were previously isolated out of fecal samples from healthy pigs at slaughter [Kaufmann, M., Zweifel, C., Blanco, M., Blanco, J.E., Blanco, J., Beutin, L., Stephan, R., 2006. Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland. J. Food Prot. 69, 260-266], were characterized by phenotypic and genotypic traits. Nine of the thirty-one sorbitol-positive non-O157 STEC (stx2e) isolated from healthy pigs belonged to serotypes found in STEC isolated from humans, including two serotypes (O9:H-, O26:H-) reported in association with hemolytic-uremic syndrome. Otherwise, the serotypes were different from those isolated from cases of edema disease in pigs. The eae (intimin) gene, which is strongly correlated with severe human disease, was not detected. Moreover, all strains were lacking the genes for enterohemolysin (ehxA), porcine A/E associated protein (paa), STEC autoagglutinating adhesin (saa) and the serin protease EspI (espI). Nine strains tested positive for astA (EAST1), one O141:H17 strain for fedA (F18 fimbrial adhesin) and one O159:H- strain for terF (tellurite resistance). Similar to the Stx2e-producing E. coli isolated from humans, which are mainly lacking further virulence factors, genes of an iron uptake system on the high-pathogenicity island (irp2, fyuA) were detected in three ONT:H10 and ONT:H19 strains from healthy pigs. Consequently, although the isolated strains are unlikely to be associated with severe human diseases, healthy pigs cannot be excluded as a potential source of human infection with Stx2e-producing STEC.

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland.

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.

Heterogeneity of the amino-acid sequences of Escherichia coli Shiga-like toxin type-I operons.

PCR was used to amplify approx. 1470-bp segments of DNA containing complete Shiga-like toxin type I (sltI) operons from Escherichia coli strains belonging to serotypes O48:H21, O111:H- and OX3:H8. These fragments were cloned and DNA sequence analysis identified several variations, as compared with published sltI sequences. All three sltI genes analysed were more closely related to Shiga toxin-encoding genes (sht) of Shigella dysenteriae type 1, than to previously published E. coli phage-encoded sltI genes. The greatest deviation in deduced amino acid (aa) sequence was observed in the SltI protein from the OX3:H8 strain, which differed from the phage 933J-encoded SltI by 9 aa in the A subunit and 3 aa in the B subunit.

Isolation of enterohemolysin (Ehly2)-associated sequences encoded on temperate phages of Escherichia coli.

We have cloned and sequenced the enterohemolysin (ehl)-associated region from a temperate bacteriophage isolated from an Escherichia coli O26:H11 strain. Phage phi C3208 was isolated together with other temperate bacteriophages which transduce the enterohemolytic phenotype to non-hemolytic E. coli O26 strains. The nucleotide sequence of the 1245-bp phi C3208 DNA insert in plasmid pEO39, which mediates Ehly2 production in E. coli K-12, was determined and was found to be partially homologous to DNA of bacteriophage lambda but is completely unrelated to DNA sequences encoding the synthesis of Ehly1 [Stroeher et al. Gene 132 (1993), 89-94]. It was shown that part of this region can be used as an Hly2-associated specific DNA probe.

Characterization and sequence of a 33-kDa enterohemolysin (Ehly 1)-associated protein in Escherichia coli.

The nucleotide sequence of the 2.1-kb EcoRI-AccI fragment of the enterohemolysin (Ehly)-associated plasmid, pEO21, has been determined. A third of this sequence encodes a 29.6-kDa protein, and coincides with the location of Tn1725 insertions which inactivate the production of Ehly. A protein of similar size (33-35 kDa) was found to be produced in large amounts by JM83[pEO21] and found to be immunologically related to the approximately 65-kDa protein made by the parental strain, C3888. DNA sequences coding for the 29.6-kDa protein of phi C3888, now designated Ehly 1, were found only in some enterohemolytic Escherichia coli indicating the existence of multiple, genetically different Ehlys.

Identification of Escherichia coli O-serogroups by restriction of the amplified O-antigen gene cluster (rfb-RFLP).

The precise serotyping of clinical Escherichia coli isolates is a crucial step for diagnostic and epidemiological purposes. Epidemiological knowledge associated with serotyping is so important that no alternative method may be considered if it does not correlate with serotyping. Unfortunately, E. coli are difficult to serotype. Genes specifically involved in O-antigen synthesis are clustered in E. coli, Shigella and Salmonella. Published oligonucleotide sequences complementary to JUMPstart and the gnd gene (the conserved flanking sequences upstream and downstream of O-antigen gene clusters, respectively) were used to amplify the O-antigen gene cluster of representative strains of 148 E. coli O-serogroups. A unique amplified fragment was observed for each serogroup (size ranging from 1.7 to 20 kbp). Clearly identifiable and reproducible O-patterns were obtained for the great majority of O-serogroups after MboII digestion of amplified products. The number of bands composing each pattern varied from five to 25. A database was built with the patterns obtained. A total of 147 O-patterns were obtained. Thirteen O-serogroups were subdivided into different O-patterns. However, each of 13 other O-patterns was shared by two or more O-serogroups. 0-serogroups of clinical isolates were deduced accurately from O-patterns in all cases, even for some rough or nonagglutinating isolates. The restriction method (rfb-RFLP) may prove to be better than serotyping since 100% of strains are typable, which is not the case with serotyping.

Comparative study of five different techniques for epidemiological typing of Escherichia coli O157.

A set of 47 Austrian human, food, and veterinary Escherichia coli O157:H7 isolates was used to evaluate five different epidemiological typing methods. Ribotyping using an automated microbial characterization system (RiboPrinter) was not suitable for detection of epidemiological relatedness. All but one E. Coli strain were typeable by phage typing. Random amplified polymorphic DNA-PCR fingerprinting was performed using primer M13 containing the sequence 5'-GAG GGT GGC GGT TCT-3' and primer 1247 (5'-AAGAGCCCGT-3'). Although both methods recognized only two clusters, both dendrograms grouped most of the EHEC O157 isolates into epidemiologically related subgroups. Pulsed-field gel electrophoresis of XbaI digested total DNA was a valuable subtyping system. We found that major differences can exist between results of multiple subtyping methods. E. coli O157 isolates should not be classified as epidemiologically related or nonrelated on the basis of a single typing method alone.

Detection of Escherichia coli alpha-haemolysin genes and their expression in a human faecal strain of Enterobacter cloacae.

Faecal samples of 200 infants were investigated for haemolytic Enterobacteriaceae. Forty infants were carrying alpha-haemolysin producing Escherichia coli, two carried haemolytic strains of Morganella morganii and one infant carried a haemolytic strain of Enterobacter cloacae. The M. morganii and E. cloacae strains were found to produce alpha-haemolysin which was tested with a specific monoclonal antibody and by DNA-hybridization with an alpha-haemolysin specific gene probe. To our knowledge this is the first report of alpha-haemolysin production found in a strain of E. cloacae.

Absence of DNA sequence homology with genes of the Escherichia coli hemB locus in Shiga-toxin producing E. coli (STEC) O157 strains.

By molecular cloning of chromosomal DNA of a human faecal Escherichia coli O6:non-motile strain, we identified a 1350-bp DNA segment which is commonly present in laboratory and wild-type E. coli strains but had no homology to DNA of Shiga-toxin producing E. coli O157, O145 and enteropathogens E. coli O55 strains. The nucleotide sequence of the 1350-bp segment cloned on plasmid pEO67 was determined (GenBank accession number AF087670) and a 97.2% sequence homology was found to a region of the E. coli hemB locus with an unknown gene function. The introduction of pEO67 into an STEC O157:H- strain had a stimulating effect on the growth of the recipient strain which was most expressed when bacteria were grown in iron depleted M9 medium with hemin added as the exogenous iron source. This growth effect was not observed with E. coli K-12 carrying pEO67. We suggest that the cloned gene is involved in iron uptake of E. coli and that the alteration in this part of the hemB locus is clonally inherited in genetically closely related STEC O157 and O55 strains.