Clonal turnover of enterohemorrhagic Escherichia coli O157:H7 in experimentally infected cattle

A total of 401 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates from two experimentally infected calves were analyzed using molecular biological methods. Genetic differences detected by pulsed-field gel electrophoresis were observed between the inoculated and recovered strains as early as 1 day post inoculation. The loss of the inoculated clone was observed in one calf. Replication and dissemination of the EHEC O157:H7 strains that mutated in cattle may result in the diversification of this organism among cattle populations.     

Validating the use of green fluorescent-marked Escherichia coli O157:H7 for assessing the organism behaviour in foods

AIMS: Monitoring bacterial kinetics in food is of great importance in food safety. The targeted micro-organism has to be identified accurately among competitive flora. Using green fluorescent protein (GFP)-transformed strains is a possible answer to such issues. However, quantitative studies require that this transformation does not alter the micro-organism behaviour: parent and transformed organisms were thus compared. METHODS AND RESULTS: Three Escherichia coli O157:H7 strains were transformed using a GFP-plasmid expressing. Parent and transformed strains were compared according to their genetic characteristics and serotypes. Growth ability was also assessed in constant and fluctuating temperature profiles. Cardinal values of pH, water activity and temperature were computed. No differences were observed between parent and transformed strains for all these experiments. The plasmid was satisfactorily maintained within transformed strains throughout the studies. Growth was eventually monitored in beef meat. CONCLUSIONS: Using the GFP marker is of great value, as it allows easier enumeration of E. coli O157:H7 in food in the presence of natural microflora. Using transformed strains is legitimate: their behaviour does not differ from that of their parent strains. SIGNIFICANCE AND IMPACT OF THE STUDY: GFP transformation appears to be a valuable and reliable tool for challenge testing studies and predictive microbiology.     

Sequence analysis of Escherichia coli O157:H7 bacteriophage ΦV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen

Bacteriophage ΦV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the ΦV10 genome is 39 104 bp long and contains 55 predicted genes. ΦV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage ɛ15 and a prophage from E. coli APEC O1. The attachment site of ΦV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. ΦV10 encodes an O -acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block ΦV10 superinfection, indicating that the O157 antigen is most likely the ΦV10 receptor.     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

Escherichia coli O157:H7 colonization in small domestic ruminants

Enterohaemorrhagic Escherichia coli O157:H7 was first implicated in human disease in the early 1980s, with ruminants cited as the primary reservoirs. Preliminary studies indicated cattle to be the sole source of E. coli O157:H7 outbreaks in humans; however, further epidemiological studies soon demonstrated that E. coli O157:H7 was widespread in other food sources and that a number of transmission routes existed. More recently, small domestic ruminants (sheep and goats) have emerged as important sources of E. coli O157:H7 human infection, particularly with the widespread popularity of petting farms and the increased use of sheep and goat food products, including unpasteurized cheeses. Although the colonization and persistence characteristics of E. coli O157:H7 in the bovine host have been studied intensively, this is not the case for small ruminants. Despite many similarities to the bovine host, the pathobiology of E. coli O157:H7 in small domestic ruminants does appear to differ significantly from that described in cattle. This review aims to critically review the current knowledge regarding colonization and persistence of E. coli O157:H7 in small domestic ruminants, including comparisons with the bovine host where appropriate.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

Earthworms as vectors of Escherichia coli O157:H7 in soil and vermicomposts

Survival and movement of Escherichia coli O157:H7 in both soil and vermicompost is of concern with regards to human health. Whilst it is accepted that E. coli O157:H7 can persist for considerable periods in soils, it is not expected to survive thermophilic composting processes. However, the natural behavior of earthworms is increasingly utilized for composting (vermicomposting), and the extent to which earthworms promote the survival and dispersal of the bacterium within such systems is unknown. The faecal material produced by earthworms provides a ready supply of labile organic substrates to surrounding microbes within soil and compost, thus promoting microbial activity. Earthworms can also cause significant movement of organisms through the channels they form. Survival and dispersal of E. coli O157:H7 were monitored in contaminated soil and farmyard manure subjected to earthworm digestion over 21 days. Our findings lead to the conclusion that anecic earthworms such as Lumbricus terrestris may significantly aid vertical movement of E. coli O157 in soil, whereas epigeic earthworms such as Dendrobaena veneta significantly aid lateral movement within compost. Although the presence of earthworms in soil and compost may aid proliferation of E. coli O157 in early stages of contamination, long-term persistence of the pathogen appears to be unaffected.     

Direct PCR detection of Escherichia coli O157:H7

AIMS: This paper reports a simple, rapid approach for the detection of Shiga toxin (Stx)-producing Escherichia coli (STEC). METHODS AND RESULTS: Direct PCR (DPCR) obviates the need for the recovery of cells from the sample or DNA extraction prior to PCR. Primers specific for Stx-encoding genes stx1 and stx2 were used in DPCR for the detection of E. coli O157:H7 added to environmental water samples and milk. CONCLUSIONS: PCR reactions containing one cell yielded a DPCR product. SIGNIFICANCE AND IMPACT OF THE STUDY: This should provide an improved method to assess contamination of environmental and other samples by STEC and other pathogens.     

Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces

AIM: To estimate the distribution and prevalence of both Escherichia coli O157 and O157:H7-infecting bacteriophages within a 50,000 head commercial beef feedlot. METHODS AND RESULTS: Escherichia coli O157 was detected in approximately 27% of the individual samples, distributed across seven of the 10 pens screened. In a simple initial screen to detect O157:H7-infecting phages, none were detected in any pen or individual sample. In contrast, after a series of enrichment procedures O157:H7-infecting phages were detected in every pen and in the majority of the samples from most pens; virulent bacteriophages active against E. coli O157:H7 were detected post-enrichment from 39/60 (65%) of the feedlot samples, and 58/60 (approximately 97%) contained phage that infected E. coli B or O157:H7. CONCLUSIONS: The data we present here indicates that we may be grossly underestimating the prevalence of O157:H7-infecting phages in livestock if we simply screen samples and that enrichment screening is required to truly determine the presence of phages in these ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that O157:H7-infecting phages may play a role in the ecology and transient colonization of cattle by E. coli O157:H7. Further, this and previous data suggest that before starting in vivo pathogen eradication studies using phage or any other regime, test animals should be enrichment screened for phage to avoid erroneous results.     

Construction of mini-Tn10luxABcam/Ptac-ATS and its use for developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7

Mini-Tn10luxABcam/Ptac-ATS was constructed in order to develop a luciferase-transducing bacteriophage for detecting Escherichia coli O157:H7. The transposon was designed to deliver a 3.6-kb insertion that confers n-decanal-dependent bioluminescence and resistance to chloramphenicol and was constructed using mini-Tn10cam/Ptac-ATS in the plasmid pNK2884 and luxAB from Vibrio harveyi. PhiV10, a temperate bacteriophage infecting common phage types of Escherichia coli O157:H7, was mutagenized as a prophage in E. coli O157:H7 strain R508. PhiV10::luxABcamA1-23 was rescued from the strain by propagating it on a strain lacking the bacteriophage and the vector containing the transposon. The bacteriophage transduced n-decanal-dependent bioluminescence to E. coli O157:H7 strain R508 that was measurable approximately 1 h post infection.     

Development of method to quantify extracellular carbohydrate complexes produced by Escherichia coli O157:H7

AIMS: The aim of this study was to optimize conditions to separate extracellular carbohydrate complexes (ECC) produced by Escherichia coli O157:H7 and to standardize the amount of ECC produced on a per cell basis. METHODS AND RESULTS: ECC fraction I was removed from E. coli O157:H7 cells produced on tryptic soya agar and lettuce juice agar by centrifugation. To remove ECC fraction II, cells were heated at 100 degrees C for 10 min, then centrifuged. The sum of ECC fractions I and II was considered as the total ECC produced by E. coli O157:H7. A correlation between cell mass and turbidity (O.D. 750 nm) of cell suspensions was determined. Cell mass has a linear relationship (R2 = 0.93) with turbidity of cell suspensions from which ECC is removed. The amount of ECC produced on a per cell basis was calculated by dividing total amount of ECC (microgram ml-1) produced by the turbidity (O.D. 750 nm) of heated cell suspension after removal ECC fractions I and II. CONCLUSIONS: A method for separating ECC from cells of E. coli O157:H7 has been developed and conditions have been optimized. A standard method to estimate the amount of ECC produced on a per cell basis was also developed. SIGNIFICANCE AND IMPACT OF THE STUDY: Using these procedures to prepare extract of ECC from E. coli O157:H7 and to standardize values, production of ECC on a per cell basis can be estimated and a comparison of the amount of ECC produced by the pathogen grown under different environmental conditions can be accurately measured.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium in cow manure and cow manure slurry

An exponential linear destruction was observed for Escherichia coli O157:H7 and Salmonella typhimurium in cattle manure and manure slurry stored at 4, 20 or 37 degrees C. The resulting decimal reduction times ranged from 6 days to 3 weeks in manure and from 2 days to 5 weeks in manure slurry. The main effects of time as well as temperature were pronounced with the most rapid destruction at 37 degrees C. The ammonia concentration in manure increased slightly during storage but did not exceed 0.1%. pH values in the deeper layers of manure remained constant except at 37 degrees C when the pH increased by 1 unit in 60 days. In the surface layers of manure, pH increased by 1.5-2 units, the oxidation-reduction potential of the manure declined rapidly to values below -200 mV. These changes do not seem to be reflected in changing rates of bacterial destruction. The observed order of destruction makes it possible to predict storage conditions (temperature and time) that will lead to a predetermined level of reduction of the two pathogens.     

Use of bioluminescent Escherichia coli O157:H7 to study intra-protozoan survival of bacteria within Tetrahymena pyriformis

A method was developed that enabled real-time monitoring of the uptake and survival of bioluminescent Escherichia coli O157 within the freshwater ciliate Tetrahymena pyriformis. Constitutively bioluminescent E. coli O157 pLITE27 was cocultured with T. pyriformis in nutrient-deficient (Chalkley's) and in nutrient-rich (proteose peptone, yeast extract) media. Non-internalised bacteria were inactivated by addition of colistin, indicated by a decline in bioluminescence. Protozoa were subsequently lysed with Triton X-100 which lead to a further drop in bioluminescence, consistent with release of live internal bacteria from T. pyriformis into the colistin-containing environment. Bioluminescence measurements for non-lysed cultures indicated that internalised E. coli O157 pLITE27 cells were only slowly digested by T. pyriformis, in both media, over the time period studied. The results suggest that bioluminescent bacteria are useful tools in the study of bacterial intra-protozoan survival.     

Evaluation of a PCR detection method for Escherichia coli O157:H7/H- bovine faecal samples

AIMS: Combinations of PCR primer sets were evaluated to establish a multiplex PCR method to specifically detect Escherichia coli O157:H7 genes in bovine faecal samples. METHODS AND RESULTS: A multiplex PCR method combining three primer sets for the E. coli O157:H7 genes rfbE, uidA and E. coli H7 fliC was developed and tested for sensitivity and specificity with pure cultures of 27 E. coli serotype O157 strains, 88 non-O157 E. coli strains, predominantly bovine in origin and five bacterial strains other than E. coli. The PCR method was very specific in the detection of E. coli O157:H7 and O157:H- strains, and the detection limit in seeded bovine faecal samples was <10 CFU g(-1) faeces, following an 18-h enrichment at 37 degrees C, and could be performed using crude DNA extracts as template. CONCLUSIONS: A new multiplex PCR method was developed to detect E. coli O157:H7 and O157:H-, and was shown to be highly specific and sensitive for these strains both in pure culture and in crude DNA extracts prepared from inoculated bovine faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex PCR method is suitable for the rapid detection of E. coli O157:H7 and O157:H- genes in ruminant faecal samples.     

The coexistence of Escherichia coli serotype O157:H7 and its specific bacteriophage in continuous culture

For the development of phage therapy, systematic understanding mechanisms of bacteriophage resistance will be required. We describe a new strain of Escherichia coli O157:H7, named Mu(L), which stably co-exists with the O157:H7-specific lytic bacteriophage PP01. Chemostat cultures of E. coli O157:H7 infected with PP01 showed unchanging cell concentration, but phage concentrations which increased by approximately 10(8) PFU mL(-1). However, the latent period, burst size, and growth rate of Mu(L) were the same as in a PP01-susceptible strain. The binding rate of PP01 to the cell surface was diminished 8.5-fold in Mu(L). By observation of the binding of fluorescently labeled O157:H7-specific phage to individual Mu(L) cells, we found that clonal Mu(L) cultures were heterogeneous in their ability to bind bacteriophage. 15% of the Mu(L) population was completely resistant to PP01 infection. Mu(L) also co-existed with bacteriophages unrelated to PP01. Broad-range phage resistance by clonal heterogeneity represents a new class of bacteria-phage interactions.     

Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

Aims: The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real‐time PCR for the detection of viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells and bind to intracellular DNA, preventing its amplification via PCR. Methods and Results: Samples were stained with EMA for 5 min, iced for 1 min and exposed to bright visible light for 10 min prior to DNA extraction, to allow EMA binding of the DNA from dead cells. DNA was then extracted and amplified by TaqMan^® real‐time PCR to detect only viable E. coli O157:H7 cells. The primers and TaqMan^® probe used in this study target the uidA gene in E. coli O157:H7. An internal amplification control (IAC), consisting of 0·25 pg of plasmid pUC19, was added in each reaction to prevent the occurrence of false‐negative results. Results showed a reproducible application of this technique to detect viable cells in both broth culture and ground beef. EMA, at a final concentration of 10 μg ml^?1, was demonstrated to effectively bind DNA from 10⁸ CFU ml^?1 dead cells, and the optimized method could detect as low as 10⁴ CFU g^?1 of viable E. coli O157:H7 cells in ground beef without interference from 10⁸ CFU g^?1 of dead cells. Conclusions: EMA real‐time PCR with IAC can effectively separate dead cells from viable E. coli O157:H7 and prevent amplification of DNA in the dead cells. Significance and Impact of the Study: The EMA real‐time PCR has the potential to be a highly sensitive quantitative detection technique to assess the contamination of viable E. coli O157:H7 in ground beef and other meat or food products.     

B-cell epitope KT-12 of enterohemorrhagic Escherichia coli O157:H7: a novel peptide vaccine candidate

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is associated with hemorrhagic colitis, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome in humans. B-cell epitopes of intimin γ from EHEC O157:H7 were predicted and synthesized for evaluating their immunogenicity and protective effect and for screening a novel synthetic peptide vaccine. In the present study, five B-cell epitopes of IntC300 were predicted by Hopp-Woods, Chou-Fasman, Karplus-Schulz, Emini, Jameson-Wolf and Kolaskar-Tongaonakar analysis. One of them, KT-12 (KASITEIKADKT) was coupled with keyhole limpet hemocyanin, and used to immunize BALB/c mice three times by subcutaneous and intranasal injection. Mouse serum titers of IgG and IgA were assessed by indirect ELISA. Oral inoculation of EHEC O157:H7 resulted in infection and death of the mice. It was found that B-cell epitopes are located within or near the peptide segments 658-669, 711-723, 824-833, 897-914, 919-931. Both subcutaneous and intranasal immunization induced higher concentrations of IgG antibodies, as detected by indirect ELISA, and nasal-mucosal immunization induced the production of high concentrations of IgA antibodies. After infection with a lethal dose of EHEC O157:H7, the survival rate of mice that had received subcutaneous immunization was not significantly different from that of the control group (P > 0.05). On the other hand, mice that received intranasal immunization showed a better survival rate than the group that received subcutaneous immunization (P < 0.05). The synthesized antigenic peptide KT-12 induced mice to produce higher concentrations of IgG and IgA after immunization, but only intranasal immunization of KT-12 succeeded in protecting most mice from infection with EHEC O157:H7. This study suggests that the synthesized antigenic peptide KT-12 is be a potential vaccine candidate against EHEC O157:H7.