Studies of Escherichia coli Cultured on RainbowTM Agar O157 with Particular Reference to Enterohaemorrhagic Escherichia coli (EHEC)

Rainbow^TM Agar O157 is designed for the rapid isolation and identification of enterohaemorrhagic Escherichia coli (EHEC), particularly O157, characterised by black colonies. Five-hundred-eighty-five E. coli strains, including O157, O111 and O113 serogroups from many sources were examined on Rainbow^TM Agar O157. EHEC O157 could readily be isolated and recognized uniquely by typical black colonies. Some other EHEC also stand out as blue-black, whereas O113 and some other EHEC strains were mauve, red or pink and indistinguishable from SLT-negative strains of E. coli.     

Heterogeneity in Expression of Lipopolysaccharide and Major Outer-Membrane Proteins by Strains of Escherichia coli O157 with Different H-Serotypyes

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long-chain lipopolysaccharide (LPS) and major outer-membrane proteins (OMPs) by means of SDS-PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga-like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze-substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H-serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H-serotype. These data support the existence of heterogeneous groups of O157 strains.     

Phenotypic and genotypic characterization of sorbitol-negative or slow-fermenting (suspected O157) Escherichia coli isolated from milk samples in Lombardy region

AIMS: To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS: Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS: The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY: The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.     

Growth of starved Escherichia coli O157 cells in selective and non-selective media.

Escherichia coli O157 strains starved in sterile deionized water (SDW) and filter-sterilized natural river water (SRW) were investigated with specific reference to their culturability in selective and non-selective media. Growth of the strains starved in both SDW and SRW were markedly suppressed with time in selective liquid media such as modified trypticase soy broth supplemented with novobiocin (mTSB+n) and modified E. coli broth supplemented with novobiocin (mEC+n). This suppression was more pronounced when incubated at 42 C than at 37 C, especially with mEC+n. By contrast, such growth suppression was seldom observed when cultured at 37 C in non-selective liquid media such as trypticase soy broth (TSB) and buffered peptone water. In mEC+n at 42 C, the non-starved cells from overnight cultures with an initial density of less than 10(3) colony-forming units (CFU)/ml grew to the density of over 10(7) CFU/ml after 24 hr incubation, whereas those starved for 6 weeks in SRW were only to maintain their initial density or died off after 24 hr incubation under the same culturing conditions. These results indicated that the isolation of starved cells of E. coli O157 from water samples would be most difficult with selective enrichment or direct plating on the selective plate media. It is thus highly recommended that a "resuscitation" of the cells with non-selective enrichment should be performed as a routine practice for maximum recovery of E. coli O157 from water systems.     

Isolation of Escherichia coli O157:H7 from dung beetles Catharsius molossus

In an epidemiological survey, Escherichia coli O157:H7 was isolated from the intestine 4 of 113 dung beetle Catharsius molossus captured below ground at Tongshan County, Jiangsu Province of China. In parallel, 10 strains of E. coli O157:H7 were isolated from fecal samples of 383 diarrhea patients from the same region. Most importantly, using pulsed field gel electrophoresis (PFGE) of chromosomal DNA restriction fragments and PCR method, we found that the PFGE pattern and virulence genes of beetle isolates were identical to those of the human isolates, such as Shiga-toxins (stx) and enterohemorrhagic Escherichia coli hemolysin A (EHEC-hlyA). Therefore, dung beetle might acquire pathogenic E. coli O157:H7 through contact with feces of domestic animals.     

Release of membrane vesicles containing endotoxic lipopolysaccharide in Escherichia coli O157:H7 clinical isolates

Membrane vesicles released by E. coli O157:H7 strains were investigated by immuno-electron microscopy using anti-O157 antibody. Anti-O157 antibody enhanced the negative-staining of vesicles and we found numerous small vesicles clearly formed around bacterial cells. An immunogold-electron microscopic examination confirmed that lipopolysaccharide (LPS) including the O-side chain is present on the surface of the vesicles. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the purified vesicles showed that the vesicles contained LPS consisting of a lipid-A and an O polysaccharide. In addition, the endotoxic activity of the vesicle was confirmed by a limulus test. These results suggest that the vesicles may play an important role in the pathogenesis of Escherichia coli O157:H7.     

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice

An immunoglobulin enriched bovine colostrum preparation, IMMULAC (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin-resistant EHEC O157:H7 strain (O157-SM(R)). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3-week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim-milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin-pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim-milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food-borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.     

Protective Effect of Japanese Green Tea Extract on Gnotobiotic Mice Infected with an Escherichia coli O157:H7 Strain

We examined the effect of Japanese green tea extract (JGTE) on enterohemorrhagic Escherichia coli (EHEC) O157:H7 infection in a gnotobiotic mouse model. Gnotobiotic mice inoculated with an EHEC strain developed neurologic and systemic symptoms, usually culminating in death. In contrast, none of mice receiving dietary JGTE showed clinical signs or death. This report describes the effect of JGTE, which includes the inhibition of bacterial growth in vivo. The Shiga-like toxin (SLT) level in the feces of the JGTE diet group was significantly lower than that of the control group.     

Detection of Verotoxin-Producing Escherichia coli O157:H7 by Multiplex Polymerase Chain Reaction

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin, Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.     

Molecular subtyping for Escherichia coli O157: H7 isolated in Taiwan

Enterohaemorrhagic Escherichia coli O157: H7 is an important pathogen these days. Outbreaks of its infection have been reported all over the world, in Australia, Canada, Japan, the United States, south Africa, and various countries in Europe. In the summer of 2001, the first clinical infection by E. coli O157: H7 was identified in Taiwan. In this study, the standard procedures for molecular subtyping were applied to several strains collected in Taiwan as well as from elsewhere. The two molecular subtyping methods we used are pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The isolates from the U.S.A., Canada, Japan, and Taiwan each showed a unique molecular fingerprinting pattern. The environmental strains isolated in Taiwan showed closer relationships with each other, and their similarity was in the range of 75-85%. The first clinical strain isolated in Taiwan in 2001 was similar to the strains from North America but not closely related to the Taiwanese environmental strains. Our surveys showed that some local E. coli O157: H7 strains did exist in Taiwan, but there had been only one official case report of the infection by local E. coli O157: H7. The eating habits of the people and the geographic distribution of the pathogen are considered crucial risk factors in Taiwan. The establishment of a database of our own and joining the global network database are important tasks if we want to control such agricultural and food-borne pathogens, and reduce the number of victims and amount sufferings, as well as the economic losses due to the infection.     

玉溪市大肠埃希菌O157菌株的流行率、表型与毒力因子特征

目的:检测并分析大肠埃希菌O157(Escherichia coli O157)在玉溪市奶牛、猪、鸡和腹泻病人中的带菌率及其特征,为E.coli O157感染症的防治和该菌分离鉴定技术的建立提供科学依据.方法:将玉溪市最大的一个奶牛场、1个猪屠宰场、2个生禽市场和市医院检验科作为监测点,采集牛、猪、鸡、腹泻病人粪便标本数分别为70、250、350和400例,用免疫磁性分离法和免疫色层技术进行E.coli O157菌株的分离培养,纯化菌株经3种鉴别培养基培养以及法国生物梅里埃公司全自动微生物鉴定系统VITEK 32 GNI 或肠杆菌科鉴定系统API 20 E试条、血清学、噬菌体型、亚碲酸盐抗性、聚合酶链反应等检测与分析.结果:牛、猪、鸡、腹泻病人E.coli O157的带菌率分别为1.4%、2.0%、0和1.2%,分离到4株E.coli O157∶H7、6株E.coli O157∶Hund和1株E.coli O157∶HNM,认识了11株E.coli O157的形态学、生物化学、血清学、噬菌体型、亚碲酸盐抗性、毒力因子等特征.结论:单一动物群或人群可同时携带多株E.coli O157,甚至同一个体也会携带2株E.coli O157;E.coli O157菌株间的表型与毒力因子特征存在一定差异,基因指纹技术可确认并证实菌株间的差异性.     

Production of shiga toxin by a luxS mutant of Escherichia coli O157:H7 in vivo and in vitro

Quorum sensing is a type of bacterial communication mediated by chemical signaling molecules called autoinducers (AIs). The production of AI-2 and AI-3 is dependent on the luxS gene in Escherichia coli O157:H7. A luxS mutation caused a minimal decrease (about 2-fold) in Shiga toxin (Stx) production in in vitro cultures using Luria-Bertani broth. The effect of a luxS mutation on the virulence of E. coli O157:H7 was examined by using germfree mice. There were no differences between the luxS mutant and the wild-type in the bacterial counts in feces shedding, Stx production, or the survival of the mice. The treatment of ciprofloxacin decreased the bacteria in feces but increased the Stx production. However, even treatment with ciprofloxacin did not make any difference between the luxS mutant and the wild-type in animal experiments.     

Virulence traits associated with verocytotoxigenic Escherichia coli O157 recovered from freshwater biofilms

AIM: To investigate whether epilithic biofilms in freshwater streams in a mixed UK agricultural river catchment harbour Escherichia coli O157, and if so, whether they demonstrate an association with those excreted by grazing farm animals. METHODS AND RESULTS: Flint shingle, native to the study site, was used as a surface for biofilm development within cages of metal lath set into a stream bed at four locations on a chalkland farm. Shingle was collected from all sites once a month, as were pooled faecal samples from five farm animal populations. Subpopulations of E. coli, including E. coli O157 that demonstrated significant phenotypic and genotypic similarity with animal faecal isolates (t-test, P = 0.05) were isolated. Of 1002 E. coli isolates from biofilms and animal faeces, 48 were confirmed as the O157 strain by latex agglutination. The presence of five virulence traits associated with incidence of human disease was tested using PCR. Stx(2) was the most frequently isolated single gene (30 isolates), while stx(1) was the least frequently recovered (four isolates). CONCLUSION: Escherichia coli O157, expressing up to four virulence factors associated with human disease, reside within freshwater biofilms in this agricultural environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Aquatic biofilms may potentially act as a reservoir for these pathogens, and the implications of the findings for the protection of drinking water resources should be further investigated.     

Effect of antibiotics, levofloxacin and fosfomycin, on a mouse model with Escherichia coli O157 infection

There have been some reservations about the treatment of enterohemorrhagic Escherichia coli (EHEC) infection with antibiotics to prevent the occurrence of hemolytic uremic syndrome (HUS). However, the administration of antimicrobial agents for EHEC infection is under discussion. Therefore, we used an experimental mouse model to assess the advantage/disadvantage of two major antibiotics, levofloxacin (LVFX) and fosfomycin (FOM). Germ-free IQI mice were inoculated with EHEC O157 strain EDL931 or #7. Bacteria colonized feces at 10(9)-10(10) CFU/g, and Shiga toxins (STXs) were detected in the feces. From 1 day after infection, mice were assigned to LVFX (20 mg/kg) once daily or FOM (400 mg/kg) once daily. A significant decrease in overall mortality was observed after treatment of LVFX, with EHEC disappearing immediately from the feces of mice. FOM also reduced mortality for one strain, the STX level decreased gradually. LVFX exhibited higher therapeutic efficacy than FOM. Strain differences were observed in the model during the treatment.     

Characteristics of sheep-rumen isolates of Pseudomonas aeruginosa inhibitory to the growth of Escherichia coli O157

Screening facultative sheep-rumen bacteria which inhibit growth of Escherichia coli produced 11 strains of Pseudomonas aeruginosa. The isolates showed three different pulsed-field gel electrophoresis patterns and strains from different sheep produced pyocins that varied in strain specificity. Representative strains were resistant to ampicillin, methicillin, erythromycin, fusidic acid and augmentin, but not to tetracycline or nalidixic acid. Tested strains attached in large numbers to cultured rumen epithelial cells, potentially providing a means of survival in this ecosystem.     

Occurrence, virulence characteristics and antimicrobial resistance of Escherichia coli O157 in slaughtered cattle and diarrhoeic calves in West Bengal, India

AIMS: (i) To study the occurrence of Escherichia coli serotype O157 in cattle stool in West Bengal, India, and (ii) the virulence properties and antimicrobial resistance of the E. coli isolates. METHODS AND RESULTS: Following enrichment in modified EC broth and plating onto HiCrome MS.O157 agar, a total of 14 strains of E. coli serotype O157 was isolated from faecal samples from two (2.04%) slaughtered cattle and six (7.59%) diarrhoeic calves. By multiplex PCR, Shiga toxin genes were detected in all the isolates. The enterohaemolysin phenotype was found in all, but one strain. Among 14 strains, ten were resistant to at least one of the antimicrobial agents tested. Multiple antibiotic resistance was frequent. CONCLUSIONS: The study showed that occurrence of Shiga toxin-producing and multiple antibiotic-resistant E. coli O157 among cattle population in this region of India is significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering routine human contacts with cattle, a large human population in this region may be at risk for exposure to Shiga toxin-producing E. coli O157.     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

Occurrence of Escherichia coli O157 on hides of slaughtered cattle

AIMS: To obtain the first information on the occurrence of Escherichia coli O157 on hides of slaughtered cattle in Serbia. METHODS AND RESULTS: A total of 355 swabs were taken on the slaughterline from five areas of hide of each of the 71 cattle in a single commercial abattoir in Serbia. Using an ISO method incorporating enrichment and immunomagnetic separation steps, E. coli O157 was isolated from the hides of 20 animals (28 x 2%). With respect to different areas of the hides, the occurrence of the pathogen was, in decreasing order: hooves (11 x 3%), brisket (8 x 4%), rump (7 x 0%), neck (4 x 2) and flank (2 x 8%). In addition, factors that had more or less effects on the occurrence included visible dirtiness of the hide, cattle's age category, geographical origin of the animals and season. CONCLUSIONS: This study revealed the presence of E. coli O157 in the beef chain in Serbia and confirmed hide as an important potential source of related contamination of beef carcasses. Therefore, incorporation of preskinning hide decontamination treatments into HACCP-based slaughterline hygiene control measures could be very useful. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will enable further optimization of necessary measures along the beef chain to reduce the E. coli O157 risks in Serbia.     

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.