Escherichia Coli O157 Research Articles

Increased adherence and expression of virulence genes in a lineage of Escherichia coli O157:H7 commonly associated with human infections.

BackgroundEnterohemorrhagic Escherichia coli (EHEC) O157:H7, a food and waterborne pathogen, can be classified into nine phylogenetically distinct lineages, as determined by single nucleotide polymorphism genotyping. One lineage (clade 8) was found to be associated with hemolytic uremic syndrome (HUS), which can lead to kidney failure and death in some cases, particularly young children. Another lineage (clade 2) differs considerably in gene content and is phylogenetically distinct from clade 8, but caused significantly fewer cases of HUS in a prior study. Little is known, however, about how these two lineages vary with regard to phenotypic traits important for disease pathogenesis and in the expression of shared virulence genes.Methodology/Principal FindingsHere, we quantified the level of adherence to and invasion of MAC-T bovine epithelial cells, and examined the transcriptomes of 24 EHEC O157:H7 strains with varying Shiga toxin profiles from two common lineages. Adherence to epithelial cells was >2-fold higher for EHEC O157:H7 strains belonging to clade 8 versus clade 2, while no difference in invasiveness was observed between the two lineages. Whole-genome 70-mer oligo microarrays, which probe for 6088 genes from O157:H7 Sakai, O157:H7 EDL 933, pO157, and K12 MG1655, detected significant differential expression between clades in 604 genes following co-incubation with epithelial cells for 30 min; 186 of the 604 genes had a >1.5 fold change difference. Relative to clade 2, clade 8 strains showed upregulation of major virulence genes, including 29 of the 41 locus of enterocyte effacement (LEE) pathogenicity island genes, which are critical for adherence, as well as Shiga toxin genes and pO157 plasmid-encoded virulence genes. Differences in expression of 16 genes that encode colonization factors, toxins, and regulators were confirmed by qRT-PCR, which revealed a greater magnitude of change than microarrays.Conclusions/SignificanceThese findings demonstrate that the EHEC O157:H7 lineage associated with HUS expresses higher levels of virulence genes and has an enhanced ability to attach to epithelial cells relative to another common lineage.

Causes, prevention and treatment ofEscherichia coliinfections

Escherichia coli is a normal inhabitant of the human gastrointestinal tract and can cause healthcare-associated infections. The organism is most frequently responsible for urinary tract infections and it is the bacterium most often implicated in the cause of diarrhoea in people travelling overseas. In recent years, a strain called Ecoli O157 has gained notoriety for causing foodborne infection, which can have severe health consequences, especially in young children. This article describes the range of different infections caused by Ecoli in healthcare settings and the community and discusses the characteristics of the different strains of the bacteria that explain variations in their pathogenicity.

Prevalence, Characterization, and Genotypic Analysis of Escherichia coli O157:H7/NM fromSelected Beef Exporting Abattoirs of Argentina

In Argentina, Escherichia coli O157:H7/NM (STEC O157) is the prevalent serotype associated with hemolytic uremic syndrome (HUS), which is endemic in the country with more than 400 cases per year. In order to estimate the prevalence and characteristics of STEC O157 in beef cattle at slaughter, a survey of 1,622 fecal and carcass samples was conducted in nine beef exporting abattoirs from November 2006 to April 2008. A total of 54 samples were found positive for STEC O157, with an average prevalence of 4.1% in fecal content and 2.6% in carcasses. Calves and heifers presented higher percentages of prevalence in feces, 10.5 and 8.5%, respectively. All STEC O157 isolates harbored stx(2) (Shiga toxin 2), eae (intimin), ehxA (enterohemolysin), and fliC(H7) (H7 flagellin) genes, while stx(1) (Shiga toxin 1) was present in 16.7% of the strains. The prevalent (56%) stx genotype identified was stx(2) combined with variant stx(2c (vh-a)), the combination of which is also prevalent (>90%) in STEC O157 post-enteric HUS cases in Argentina. The clonal relatedness of STEC O157 strains was established by phage typing and pulsed-field gel electrophoresis (PFGE). The 54 STEC isolates were categorized into 12 different phage types and in 29 XbaI-PFGE patterns distributed in 27 different lots. STEC O157 strains isolated from 5 of 21 carcasses were identical by PFGE (100% similarity) to strains of the fecal content of the same or a contiguous bovine in the lot. Five phage type-PFGE-stx profiles of 10 strains isolated in this study matched with the profiles of the strains recovered from 18 of 122 HUS cases that occurred in the same period.

Contents and antibacterial activity of flavonoids extracted from leaves of Psidium guajava

Four antibacterial flavonoids (morin-3-O-lyxoside, morin-3-O-arabinoside, quercetin, and quercetin-3-O-arabinoside) were isolated from fresh and driedPsidium guajava leaves, and their concentrations were determined. Among them, quercetin and morin-3-O-arabinoside were the most and the least abundant, respectively. Studies on inhibitory effects of the flavonoids on spoilage and foodborne pathogenic bacteria revealed that they had bacteriostatic mode of action against all tested spoilage and foodborne pathogenic bacteria includingBacillus stearothermophilus, Brochothrix thermosphacta, Escherichia coliO157:H7, Listeria monocytogenes, Pseudomonas fluorescens, Salmonella enterica, Staphylococcus aureus and Vibrio cholerae. Key words: Antibacterial activity, flavonoids, guava, Psidium guajava.

Modelling the interactions between Pseudomonas putida and Escherichia coli O157:H7 in fish‐burgers: use of the lag‐exponential model and of a combined interaction index

The objective of the current study was to examine the interactions between Pseudomonas putida and Escherichia coli O157:H7 in coculture studies on fish-burgers packed in air and under different modified atmospheres (30 : 40 : 30 O(2) : CO(2) : N(2), 5 : 95 O(2) : CO(2) and 50 : 50 O(2) : CO(2)), throughout the storage at 8 degrees C. The lag-exponential model was applied to describe the microbial growth. To give a quantitative measure of the occurring microbial interactions, two simple parameters were developed: the combined interaction index (CII) and the partial interaction index (PII). Under air, the interaction was significant (P < 0.05) only within the exponential growth phase (CII, 1.72), whereas under the modified atmospheres, the interactions were highly significant (P < 0.001) and occurred both in the exponential and in the stationary phase (CII ranged from 0.33 to 1.18). PII values for E. coli O157:H7 were lower than those calculated for Ps. putida. The interactions occurring into the system affected both E. coli O157:H7 and pseudomonads subpopulations. The packaging atmosphere resulted in a key element. The article provides some useful information on the interactions occurring between E. coli O157:H7 and Ps. putida on fish-burgers. The proposed index describes successfully the competitive growth of both micro-organisms, giving also a quantitative measure of a qualitative phenomenon.

Growth Inhibition of Escherichia coli 987P by Neutralizing IgY Antibodies

Egg yolk antibodies, called IgY, were isolated from the egg yolks of chickens immunized with enterotoxigenic Escherichia coli (ETEC) 987P. Specific binding activity of IgY against ETEC 987P was found. Whole cell titers remained relatively high during the immunization period (up to 19 weeks). The IgY antibodies cross-reacted with other members of Enterobacteriaceae, Escherichia coli O157:H7, Salmonella enteritidis, and S. typhimurium by 28.3%, 21.1% and 19.7%, respectively. Specific IgY with a concentration higher than 0.54 mg/ml was found to inhibit the growth of ETEC 987P in a liquid medium. The difference in bacterial growth between the specific and the non-specific IgY was 6.1 log CFU/ml after 8 hr incubation. The specific binding activity of IgY to bacteria was further evaluated by immunofluorescence and immunoelectron microscopy, showing the binding of specific IgY to the bacterial surface. The immunoelectron microscopic observation revealed structural alterations on the bacterial surface bound by specific IgY. This proves that specific binding activity of IgY against ETEC 987P whole cells resulted in growth inhibition of effect ETEC 987P in vitro.

Detection ofEscherichia coliO157 andEscherichia coliO157:H7 by the immunomagnetic separation technique andstx1andstx2genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.

Detection and identification of Escherichia coli O157:H7 by multiplex real-time PCR

Objective To develop a rapid, sensitive and specific assay based on multiplex real-time PCR for detecting and identifying Escherichia coli O157: H7. Methods The lipopolysaccharide gene (rJbE) and H7 flagellar antigen gene(fliC) of Escherichia coli O157:H7 was chosen as targets, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and HEX fluo-resceins respectively; the 3'end of probes was labeled with MGB. The PCR reaction was optimized systemati-cally. Then the specificity, sensitivity and reproducibility of multiplex real-time PCR were estimated. Final-ly, multiplex real-time PCR was applied to detected clinical specimens. Results Escherichia coil O157:H7 were detected by multiplex real-time PCR accurately and quickly, which could distinguish Escherichia coli O157:H7 from O157: non-H7. Meanwhile, none of other bacteria could be identified. The sensitivity was 10 CFU/ml in pure culture. The coefficient of variation of intra-assay and inter-assay was less than 5%. When this assay was applied directly to identify 66 clinical specimens, the results showed that t5 were positive to Escherichia coil O157:H7 and 2 were positive to Escherichia coil O157: non-H7, in which 16 was the same to the results obtained from the conventional assays. The coincidence was 98.49%. Conclusion It is showed that multiplex real-time PCR is a reliable, accurate and feasible assay for detecting and identifying Escherich-ia coli Oi57: H7, The assay reported here provided a tool for analysis and diagnosis in the field of detecting clinical pathogens, epidemiologic survey and food safety monitoring. Key words: Multiplex real-time PCR; Escherichia coli O157: H7; Detection

Yeast cell-wall products containing β-glucan plus ascorbic acid affect neonatal Bos taurus calf leukocytes and growth after a transport stressor1

The objectives were to ascertain whether a yeast cell-wall derivative that was 1.8% beta-glucan in combination with ascorbyl-2-polyphosphate could improve innate immunity and mediate transportation stress in neonatal calves, and to compare the 1.8% beta-glucan yeast cell-wall derivative with a more purified yeast cell-wall derivative (70% beta-glucan). Treatments were 1) an unsupplemented control (CNT); 2) 113 g of a 1.8% (approximately 2%) beta-glucan derivative of yeast cell walls plus 250 mg of l-ascorbic acid phosphate (BG2); or 3) 150 mg of a purified beta-glucan fraction from yeast cell walls (approximately 70% beta-glucan) plus 250 mg/feeding of l-ascorbic acid phosphate (BG70). Calves (n = 39) were transported for 4 h, placed in outdoor hutches, and randomly assigned to treatments. Treatments (mixed with a milk replacer) were individually fed twice daily for 28 d. Calves were offered calf starter, free choice, throughout the study. Weekly starter intake and BW were measured, and fecal samples were collected for Salmonella Typhimurium and Escherichia coli O157:H7 PCR analysis. Blood was collected immediately before transport (d 0) and on d 3, 7, 10, 14, 21, and 28 after transport. Starter intake and DMI were less (P < 0.05) at d 28 for the BG2 and BG70 treatments compared with the CNT treatment. Hematocrit percentages increased (P = 0.002) throughout the experiment. White blood cell counts (treatment x time interaction, P = 0.066) were less for the calves supplemented with BG70 than for those supplemented with BG2 (P = 0.01) or for CNT calves (P = 0.04) on d 28. Granulocyte counts changed (P = 0.04) throughout the experiment. A trend (P = 0.077) for a treatment x time interaction was detected for peripheral blood mononuclear cell counts (PBMC). Counts of PBMC were greater (P = 0.006) for the BG2 treatment compared with the CNT treatment on d 3. Calves given the BG70 supplement had fewer PBMC than those given the BG2 supplement on d 21 (P = 0.03) and 28 (P = 0.05). Fibrinogen concentrations were affected only by time (P = 0.002). Time effects were detected for phagocytosis (P = 0.005), oxidative burst (P < 0.001), expression of cluster of differentiation 18 (P = 0.001), and increased cluster of differentiation 18 (P = 0.006). Phagocytosis was less (P = 0.05) for calves in the BG70 group than for those in the CNT group. Percentage of calves positive for E. coli O157:H7 was greatest (P <or= 0.05) for those in the BG2 group on d 7 compared with those in the other treatments. The BG2 and BG70 supplements both increased feed intake, but only the BG2 supplement increased E. coli shedding on d 7, and the BG2 and BG70 supplements varied in modulating immune functions, indicating differences in yeast extract function.

STABILITY OF ANTIBACTERIAL PROPERTY OF THAI GREEN CURRY DURING CHILLED STORAGE

Stability of the antibacterial activity of Thai green curry paste during chilled storage for 30 days after various heat treatment methods was investigated. The antibacterial activities of the curry aqueous extracts were tested against eight bacteria species that are foodborne pathogens and also seafood spoilage organisms including Salmonella typhi, Shewanella putrefaciens, Vibrio cholerae non 01, Vibrio parahaemolyticus, Pseudomonas fluorescens, Escherichia coli O157:H7, Clostridium perfringens and Staphylococcus aureus. It was found that the heat treatment methods were able to reduce the total viable count by at least 0.78 and 0.62 log cycle in the pasteurized and blanched samples, respectively. The curry aqueous extract inhibited the growth of S. aureus, S. putrefaciens and P. fluorescens with inhibition zones ranging from 22 ± 5 mm. The heat treatment methods with the chilled temperature were able to keep stable the antibacterial activity of the curry throughout the period of storage. PRACTICAL APPLICATIONS Thai green curry is a very popular curry in Thailand. This research has provided information on the effect of the various pretreatments that could be done to the curry before chilled storage. The antibacterial effect of this curry on pathogenic organisms suggested that the curry possesses medicinal value. The stability of the curry during storage is an indication that the curry could serve as a good marination agent for seafoods.

Phytochemical composition of the essential oils from three Apiaceae species and their antibacterial effects on food-borne pathogens

Abstract Essential oils (EOs) of three Apiaceae species, including Bunium persicum , Cuminum cyminum and Carum copticum , extracted by hydrodistillation, were analyzed by gas chromatography (GC) and GC/mass spectrometry. The main components of EOs of B. persicum were γ-terpinene (44.2%), cuminaldehyde (16.9%), γ-terpinen-7-al (10.5%), and ρ-cymene (8%). The major constituents of Cu. cyminum were cuminaldehyde (30.2%), ρ-cymene (14.1%), γ-terpinene (12.8%), and safranal (9.4%), while those of Ca. copticum were thymol (48.4%), ρ-cymene (21.8%) and γ-terpinene (21.3%). The antibacterial effects of the EOs were assessed on several food-borne pathogens, namely Staphylococcus aureus , Bacillus cereus , Escherichia coli O157:H7, Salmonella enteritidis , and Listeria monocytogenes . The ranges of minimum inhibitory concentration (MIC) of the oils were 0.03–0.5, 0.18–3.0, and 0.37–3.0 mg/ml, respectively, for Ca. copticum , B. persicum and Cu. cyminum . Moreover, the combination of B. persicum and Cu. cyminum EOs confirmed synergistic and additive activities against the pathogens.

INVESTIGATION ON THE ANTIMICROBIAL EFFECTS OF ESSENTIAL OILS FROM DILL AND CORIANDER SEEDS ON STAPHYLOCOCCUS AUREUS, ESCHERICHIA COLI O157:H7 AND SALMONELLA TYPHIMURUIM

In this study the antimicrobial effects of essential oils from dill and coriander seeds on staphylococcus aureus, Escherichia coli O157:H7 and salmonella typhimuruim were investigated and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each essential oil were determinate. For this purpose 5 concentration of each essential oils (125, 250, 500, 1000, 2000 and 4000 ppm) were chosen. For microbial count, Broth Dilution Test with Mueller Hinton Agar and Broth were used. Results showed that Staphylococcus aureus had more susceptibility and Salmonella typhimuruim was the resistant one. Our results also showed that essential oil from coriander seed had more antimicrobial effect on the gram-negative bacteria. The essential oil from coriander seed had MIC and MBC equal to 1000ppm and the essential oil from dill seed had MIC equal to 500ppm and MBC equal to 1000 ppm against Staphylococcus aureus. Key word: Broth Dilution Test, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella typhimuruim, essential oils, dill seed, coriander seed

Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff.

Although protected for nearly a century, California’s sea otters have been slow to recover, in part due to exposure to fecally-associated protozoal pathogens like Toxoplasma gondii and Sarcocystis neurona. However, potential impacts from exposure to fecal bacteria have not been systematically explored. Using selective media, we examined feces from live and dead sea otters from California for specific enteric bacterial pathogens (Campylobacter, Salmonella, Clostridium perfringens, C. difficile and Escherichia coli O157:H7), and pathogens endemic to the marine environment (Vibrio cholerae, V. parahaemolyticus and Plesiomonas shigelloides). We evaluated statistical associations between detection of these pathogens in otter feces and demographic or environmental risk factors for otter exposure, and found that dead otters were more likely to test positive for C. perfringens, Campylobacter and V. parahaemolyticus than were live otters. Otters from more urbanized coastlines and areas with high freshwater runoff (near outflows of rivers or streams) were more likely to test positive for one or more of these bacterial pathogens. Other risk factors for bacterial detection in otters included male gender and fecal samples collected during the rainy season when surface runoff is maximal. Similar risk factors were reported in prior studies of pathogen exposure for California otters and their invertebrate prey, suggesting that land-sea transfer and/or facilitation of pathogen survival in degraded coastal marine habitat may be impacting sea otter recovery. Because otters and humans share many of the same foods, our findings may also have implications for human health.

Detection of enterohemorrhagic Escherichia coli O157:H7 by loop-mediated isothermal amplification

To develop a loop-mediated isothermal amplification (LAMP) method for rapidly diagnosing of Escherichia coli (EHEC) O157:H7 in pathogen detection department or small-scale laboratories. Primers for LAMP test were designed by targeting the antigen coding rfbE of EHEC O157:H7, the Shiga-like toxin stx2 and the fliC encoding gene of H7 flagella antigen, respectively. The reaction condition and reaction system of LAMP were optimized. 2 EHEC O157:H7 type strains, 17 local strains and 33 other enterobacteria were analyzed to evaluate the LAMP's specificity and sensitivity. The results of the LAMP reaction were also compared with routine PCR method. The amplification products of O157 which had the corresponding target genes turned green by visual inspection and had ladder-like pattern on the gel, but products of other enterobacteria remained orange by visual examination and had no band on the gel. The detection results of LAMP were the same as of routine PCR method. The reaction time of the LAMP method was only 1.5 hours and the detection limit of LAMP assay was 26 CFU/reaction. In addition, the LAMP results could be determined only by visual inspection. LAMP assay is rapid, specific, and sensitive for the detection of EHEC O157:H7. This method might not only reduce the dependence of complicated equipments but also be a potential method for wider use in pathogen detection department, small-scale laboratory, emergency motor vehicle or field survey.

Gene and Antigen Markers of Shiga‐toxin Producing E. coli from Michigan and Indiana River Water: Occurrence and Relation to Recreational Water Quality Criteria

The relation of bacterial pathogen occurrence to fecal indicator bacteria (FIB) concentrations used for recreational water quality criteria (RWQC) is poorly understood. This study determined the occurrence of Shiga-toxin producing Escherichia coli (STEC) markers and their relation to FIB concentrations in Michigan and Indiana river water. Using 67 fecal coliform (FC) bacteria cultures from 41 river sites in multiple watersheds, we evaluated the occurrence of five STEC markers: the Escherichia coli (EC) O157 antigen and gene, and the STEC virulence genes eaeA, stx1, and stx2. Simple isolations from selected FC cultures yielded viable EC O157. By both antigen and gene assays, EC O157 was detected in a greater proportion of samples exceeding rather than meeting FC RWQC (P < 0.05), but was unrelated to EC and enterococci RWQC. The occurrence of all other STEC markers was unrelated to any FIB RWQC. The eaeA, stx2, and stx1 genes were found in 93.3, 13.3, and in 46.7% of samples meeting FC RWQC and in 91.7, 0.0, and 37.5% of samples meeting the EC RWQC. Although not statistically significant, the percentage of samples positive for each STEC marker except stx1 was lower in samples that met, as opposed to exceeded, FIB RWQC. Viable STEC were common members of the FC communities in river water throughout southern Michigan and northern Indiana, regardless of FIB RWQC. Our study indicates that further information on the occurrence of pathogens in recreational waters, and research on alternative indicators of their occurrence, may help inform water-resource management and public health decision-making.

Acute Bloody Diarrhea: A Medical Emergency for Patients of All Ages

Acute bloody diarrhea should be considered a medical emergency. Its causes are frequently serious or actionable or both and are usually identified. However, acute bloody diarrhea as a stand-alone clinical presentation has received little scholarly attention in the past several decades. Although the range of possible causes of acute bloody diarrhea is broad, infectious considerations are paramount and should always be prioritized in the evaluation of such patients. History, examination, and laboratory testing should be focused on minimizing time to diagnosis (and, by extension, to implementing appropriate therapy). Strategically chosen tests and imaging, avoidance of extraneous diagnostic pursuits, and provision of supportive care while awaiting diagnostic clarity are central to the adroit management of patients with acute bloody diarrhea. Diagnostic considerations differ somewhat between adults and children but have many elements in common, including the need for vigilance in detecting Escherichia coli O157:H7 infection. In this review, we discuss diagnostic approaches (emphasizing the importance of rapid, accurate, and thorough microbiologic investigation) and measures that can be taken to support patients while awaiting information that determines the cause of their disease. These topics are discussed in the context of the medical care that is available to children and adults with bloody diarrhea in most institutions in developed nations.

Inhibitory effect of commercial green tea and rosemary leaf powders on the growth of foodborne pathogens in laboratory media and oriental-style rice cakes.

The antimicrobial effects of green tea and rosemary added to foods as antagonists to foodborne pathogens were determined in laboratory media and oriental-style rice cakes. The growth of each pathogen (Bacillus cereus, Salmonella Typhimurium, Enterobacter sakazakii, Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes) in tryptic soy broth or rice cake with or without addition of green tea or rosemary leaf powders before autoclaving or cooking, respectively, was investigated after inoculation. The addition of 1% green tea or rosemary produced similar results for inhibiting the growth of pathogens in tryptic soy broth. However, green tea was more effective than rosemary for inhibiting the growth of L. monocytogenes. Both botanicals had inhibitory effects against all pathogens tested in this study. Green tea was particularly effective against B. cereus, S. aureus, and L. monocytogenes, and rosemary was strongly inhibitory against B. cereus and S. aureus. The addition of 1 or 3% green tea or rosemary to rice cakes did not significantly reduce total aerobic counts; however, levels of B. cereus and S. aureus were significantly reduced in rice cakes stored for 3 days at room temperature (22 degrees C). The order of antimicrobial activities against B. cereus in rice cake was 1% rosemary < 1% green tea < 3% rosemary = 3% green tea. These results indicate that the use of natural plant materials such as green tea and rosemary could improve the microbial quality of foods in addition to their functional properties.

Detection of Escherichia coli O157:H7 by multiplex PCR-DHPLC

Objective To develop the PCR-denaturing high performance liquid chromatography (DHPLC) for detection of E. Coli O157: H7. Methods The virulence genes of Shiga-like toxin(SLT) and rfbE were specifically amplified by 2 sets of primers. The target gene fragments of the PCR assay were 224 bp and 499 bp, respectively. Results Analysis of 37 strains demonstrated that this PCR system was specific. The detection limit of the PCR was 4 CFU/ml. Conclusion These results indicated that the multiplex PCR-DHPLC assay can be used for specific and sensitive detection of E. Coli O157:H7. Key words: E.coli O157: H7; Multiplex PCR; Denaturing high performance liquid chromatogra-phy

Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

To develop a new, rapid and accurate reverse dot blot (RDB) method for the detection of intestinal pathogens in fecal samples. The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7, Clostridium botulinum, Bacillus cereus, Clostridium perfringens, Vibrio parahaemolyticus, Shigella spp., Yersinia enterocolitica, Vibrio cholerae, Listeria monocytogenes and Staphylococcus aureus. The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay. The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%. The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.

Risk Factors of Sporadic Human Infection of Shiga Toxin Producing Escherichia coli O157 and Non-O157 in Australia

Background: Shiga toxin producing Escherichia coli (STEC) causes acute gastroenteritis characterised by abdominal cramps and bloody diarrhoea. In Australia and elsewhere, serotype O157 is the predominant strain of STEC. To investigate risk factors for STEC O157 and non-O157 infection we conducted an Australia-wide case control study between 2003 and 2007. Methods: Patients infected with STEC that were notified to health departments were eligible for the study and three controls per case were selected from a bank of controls based on sex and five year age groups. Cases were recruited in South Australia from July 2003, with all other Australian states participating from 2005. A standardised questionnaire was used to collect information regarding demographic data, food, behavioural and environmental exposures. All factors associated with O157 and non-O157 infection at the univariate level (p < 0.1) were included in separate logistic regression models. Results: The O157 analysis included 43 cases and 117 controls; the non-O157 analysis included 71 cases and 187 controls. In multivariate analysis, STEC O157 infection was associated with eating hamburgers (OR = 6.6, 95% CI = 2.2–20.0), eating out of the home (OR = 4.8, 95% CI = 1.7–13.6) and living on or visiting a farm (OR = 4.6, 95% CI = 1.2–17.6). Non-O157 STEC infection was associated with consuming chicken deli meat (OR = 5.3, 95% CI = 1.5–18.5), working with animals (OR = 3.2, 95% CI = 1.2-8.3) and asthma (OR = 2.7, 95% CI = 1.1–6.3). Bush camping in Australia had an elevated odds ratio, although this was not significant (OR = 3.3, 95% CI = 0.9–11.9). Conclusions: This national case control study is the first to provide risk factors for both O157 and non-O157 STEC cases. That O157 was associated with farm exposure and eating hamburgers is consistent with that reported internationally. However, some of the risk factors for non-0157 cases have not been reported previously. It is important to consider these risk factors in developing prevention programmes for this serious illness.