H7 Isolates Research Articles

Pheno-genotypic characterisation of Escherichia coli O157:H7 isolates from domestic and wild ruminants

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognised as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle and deer faeces and from unpasteurised goat milk in Spain over a period of 11 years. Characterisation was performed by polymerase chain reaction (PCR), phage typing and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and β-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx2 gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34 and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep and deer. Majority of the isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7, which may not be identified by routine culture methods or by biochemical tests used to characterise serotype O157:H7.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA)

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additonal research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.

Occurrence of Escherichia coli O157:H7 in faeces, skin and carcasses from sheep and goats in Ethiopia

To determine the occurrence and proportion of Escherichia coli O157:H7 in faeces, skin swabs and carcasses before and after washing, from sheep and goats in Ethiopia. Individual samples were enriched in modified tryptic soy broth with novobiocin, concentrated using immunomagnetic separation (IMS) and plated onto cefixime-tellurite containing sorbitol MacConkey agar. Presumptive colonies were confirmed by biochemical tests and subjected to latex agglutination tests. A PCR was performed on isolates for the detection of stx(1), stx(2) and eae genes. Escherichia coli O157:H7 was isolated from faeces (4.7%), skin swabs (8.7%) and carcasses before washing (8.1%) and after washing (8.7%) and on water samples (4.2%). The proportion of carcasses contaminated with E. coli O157:H7 was strongly associated with those recovered from faecal and skin samples. Both stx(1) and stx(2) genes were identified from one E. coli O157:H7 isolate from a goat carcass. Even though the numbers of samples examined in this study were limited to one abattoir, sheep and goats can be potential sources of E. coli O157:H7 for human infection in the country. Control measures to reduce the public health risks arising from E. coli O157:H7 in reservoir animals need to be addressed at abattoir levels by reducing skin and faecal sources and carcass contaminations at different stages of slaughter operations. Escherichia coli O157:H7 was detected from carcasses before and after washing during slaughtering operations, and one O157 isolate was positive for verotoxins.

Development of Real Time RT-PCR Assays for Detection of Type A Influenza Virus and for Subtyping of Avian H5 and H7 Hemagglutinin Subtypes

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Analysis of the Clonal Relationship of Serotype O26:H11 Enterohemorrhagic Escherichia coli Isolates from Cattle

Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.

Prevalence and antimicrobial resistance profiles of Escherichia coil O157:H7 and Salmonella isolated from feedlot lambs.

The present study examined the incidence of Escherichia coli O:H7 and Salmonella in feedlot lambs. Fifty-six feedlot lambs from eight sheep farming operations were grouped in a single drylot pen, fed, and managed as is typical in the southwestern United States. Fecal samples were collected on days 0, 46, 87, and 122 of the feeding period via rectal palpation. Wool samples (ventral midline) were collected one time only at the feedlot, immediately prior to shipping to the processing plant, and carcass swabs were collected following slaughter. All samples were cultured for E. coli O157:H7, Salmonella, and fecal coliforms, and select isolates were examined for antimicrobial susceptibility. Overall, the percentages of fecal and wool samples positive for E. coli O157:H7 averaged 9 and 18%, respectively. One carcass swab was E. coli O157:H7 positive. Of the 155 fecal samples collected, 11 (7%) were Salmonella positive. Salmonella was detected in nearly 50% of the wool samples collected prior to slaughter, while none of the carcasses were Salmonella positive 24 h postslaughter. All isolates (E. coli O157:H7, Salmonella, and fecal coliforms) were susceptible to ceftiofur, enrofloxacin, and trimethoprim-sulfamethoxazole. One E. coli O157:H7 isolate cultured from a carcass swab was resistant to seven antibiotics, and seven wool E. coli O157:H7 isolates were multidrug resistant. Results of this research demonstrate that feedlot sheep are naturally colonized with E. coli O157:H7 and Salmonella and wool can be a source of carcass contamination; however, in-plant processing procedures and intervention strategies were largely effective in preventing carcass contamination.

Escherichia coli O157:H7 Strains That Persist in Feedlot Cattle Are Genetically Related and Demonstrate an Enhanced Ability To Adhere to Intestinal Epithelial Cells

A longitudinal study was conducted to investigate the nature of Escherichia coli O157:H7 colonization of feedlot cattle over the final 100 to 110 days of finishing. Rectal fecal grab samples were collected from an initial sample population of 788 steers every 20 to 22 days and microbiologically analyzed to detect E. coli O157:H7. The identities of presumptive colonies were confirmed using a multiplex PCR assay that screened for gene fragments unique to E. coli O157:H7 (rfbE and fliC(h7)) and other key virulence genes (eae, stx(1), and stx(2)). Animals were classified as having persistent shedding (PS), transient shedding (TS), or nonshedding (NS) status if they consecutively shed the same E. coli O157:H7 genotype (based on the multiplex PCR profile), exhibited variable E. coli O157 shedding, or never shed morphologically typical E. coli O157, respectively. Overall, 1.0% and 1.4% of steers were classified as PS and NS animals, respectively. Characterization of 132 E. coli O157:H7 isolates from PS and TS animals by pulsed-field gel electrophoresis (PFGE) typing yielded 32 unique PFGE types. One predominant PFGE type accounted for 53% of all isolates characterized and persisted in cattle throughout the study. Isolates belonging to this predominant and persistent PFGE type demonstrated an enhanced (P < 0.0001) ability to adhere to Caco-2 human intestinal epithelial cells compared to isolates belonging to less common PFGE types but exhibited equal virulence expression. Interestingly, the attachment efficacy decreased as the genetic divergence from the predominant and persistent subtype increased. Our data support the hypothesis that certain E. coli O157:H7 strains persist in feedlot cattle, which may be partially explained by an enhanced ability to colonize the intestinal epithelium.

Pulsed-Field Gel Electrophoresis Profile Changes Resulting from Spontaneous Chromosomal Deletions in Enterohemorrhagic Escherichia coli O157:H7 during Passage in Cattle

A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.

Invasions by Eurasian Avian Influenza Virus H6 Genes and Replacement of Its North American Clade

The spread of highly pathogenic avian influenza virus (AIV) (H5N1) underlines the potential for global AIV movement through birds. The phylogenies of AIV genes from avian hosts usually separate into Eurasian and North American clades, reflecting limited bird migration between the hemispheres. However, mounting evidence that some H6 sequences from North America cluster with Eurasian subtype H6 sequences calls the strict hemispheric divide into question. We conducted a comprehensive phylogenetic analysis of the extent and timing of cross-hemisphere movements by the H6 gene. Results suggested that Eurasian H6 subtype has invaded North America several times, with the first invasions occurring 10 years before the first detection of invading isolates. The members of the North American clade decreased from 100% in the 1980s to 20% in the 2000s among H6 isolates from North America. Unraveling the reasons for this large-scale gene movement between hemispheres might identify drivers of global AIV circulation.

Analysis of the Genome of theEscherichia coliO157:H7 2006 Spinach-Associated Outbreak Isolate Indicates Candidate Genes That May Enhance Virulence

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.

O8 Lyme spirochete moving to the north: Borrelia burgdorferi found in Ixodes tick in Hazelton, BC (N 55 degree)

Objective: The gold standard for identification of the flagellar H7 antigen is by tube agglutination which is lengthy and labor intensive. Commercial serological slide agglutination assays for H7 perform comparably with tube agglutination, but availability can be limited. The objective of this study was to develop a real time PCR to identify H7 in E. coli O157. Methods: Characterized E. coli isolates (n = 134) including O157:NM, O157:H7, O157:non H7 and non O157 were included. All strains were grown overnight on blood agar and DNA was extracted using MagaZorb (Promega) and quantified. The primers targeted directly to the fliC gene was used for the H7 PCR real time SYBR green assay, to give an amplicon size of 247 bp. The PCR was performed using the LightCycler (Roche Diagnostics). The amount of magnesium chloride used for the assay ranged from 1mM, to 3mM. Ten fold dilutions of purified DNA from 100 ng to 0.01 ng along with different cycle number of amplification were used to determine the sensitivity of the assay. Sequencing of the whole fliC gene was also performed to confirm its presence in all E. coli O157:NM and selected O157:H7 and O157:non H7 isolates. Results: The amplification curves of all H7 positive and NM isolates using 2.5 ng of purified DNA as template clustered together with crossing point values less than 10 and the melting temperature was detected at 85oC. All the remaining isolates that were non O157, O157:non H7 also amplified but all their crossing points were greater than 25 with melting temperatures varying from 80oC to 90oC. No difference was observed with the different concentrations of magnesium chloride used for the amplification. Detection level of the real time assay was at 0.01 ng of purified DNA isolated from O157:NM and O157:H7 amplified for 28 cycles. Sequencing results of the fliC gene confirmed its presence in E. coli O157:NM isolates. Conclusions: A real time SYBR green assay was developed and the amplification was optimized at 3.0mM of magnesium with 1.0 ng of DNA for 28 cycles of amplification performed in the LightCycler. All E. coli O157:NM studied were positive for the fliC gene although the protein might not be expressed and therefore not detected in the laboratory.

Evaluating the Effect of Environmental Factors on Pathogen Regrowth in Compost Extract

Pathogenic microorganisms may survive the composting process in low numbers and subsequently regrow to high levels under favorable conditions. The objective of this study was to investigate the regrowth potential of Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in dairy-based composts under different environmental conditions. Water extract of commercially available dairy compost was used as a model system. Cocktails of five rifampin-resistant strains of each pathogen previously grown in reduced nutrient media (1/2 or 1/10 strength of tryptic soy broth, TSB) were inoculated into water extract of compost of different ratios (1:2,1:5, and 1:10, w/v), and then stored at 35 degrees C or 22 degrees C for 7 days. The strains exhibiting greatest survival or regrowth were identified by pulsed-field gel electrophoresis (PFGE). At 22 degrees C, both E. coli O157:H7 and L. monocytogenes multiplied in all compost extracts, whereas Salmonella spp. regrew in both 1:2 and 1:5 compost extracts but not in 1:10. For all three pathogens, incubation at 22 degrees C provides better conditions for regrowth than at 35 degrees C. Both Salmonella and E. coli O157:H7 previously adapted to nutrient-limited broth (1/10 strength of TSB) regrew in compost extracts to higher populations than the control cultures grown previously in full strength of TSB. In the absence of indigenous microorganisms, all three pathogens regrew even in the most diluted sterile compost extract (1:10) with growth potentials ranging from 2.30 to 3.59 log CFU/ml. In nonsterile compost extract with ca. 5 log CFU/ml of background microorganisms, all three pathogens regrew only in the most concentrated compost extract (1:2) with much less population increases ranging from 0.70 to 1.43 log CFU/ml. Compost extract samples of all ages supported the regrowth of both Salmonella and E. coli O157:H7 with population increases ranging from 0.95 to 2.32 log CFU/ml. The PFGE patterns for E. coli O157:H7 isolates from sterile compost extracts matched with either the spinach outbreak strain or an avirulent B6914 strain. These results demonstrated that compost extract of dairy-based compost contained sufficient nutrients for pathogen regrowth. Cultures previously adapted to low nutrient media regrew to higher populations than control cultures; however, indigenous microflora suppressed the pathogen regrowth in compost extract, especially at 35 degrees C.

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin

To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin. Escherichia coli O157:H7 isolates were compared using (i) PFGE XbaI patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene. Six SNPs were detected in the vtx2A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid. Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.

Multistate Outbreak ofEscherichia coliO157:H7 Infection Associated with Consumption of Packaged Spinach, August–September 2006: The Wisconsin Investigation

Escherichia coli O157:H7 infection often causes hemorrhagic colitis and hemolytic uremic syndrome. In 2006, the Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene, in cooperation with other local, state, and federal partners, investigated an outbreak of E. coli O157:H7 infection. In September 2006, the Wisconsin Division of Public Health and the Wisconsin State Laboratory of Hygiene were able to link geographically dispersed E. coli O157:H7 isolates recovered from the stool samples of ill persons, all of which had the same pulsed-field gel electrophoresis pattern (i.e., outbreak pattern). Investigators conducted a case-control study with control subjects (n = 86) matched to case patients (n = 49) by age, sex, and residential location. All case patients' onsets of illness occurred during the period from 20 August through 14 September 2006. Illness was associated with spinach consumption (matched odds ratio, 82.1; 95% confidence interval, 14.7 to >1000). Of the 49 case patients, 26 (53%) recalled eating brand A spinach. On multibrand analysis, only brand A was associated with illness (undefined matched odds ratio; 95% confidence interval, 6.8-infinity). Wisconsin's agriculture laboratory isolated E. coli O157:H7 with the outbreak pattern from spinach in 2 brand A packages, both produced on 15 August 2006. The rapid multijurisdictional epidemiologic and laboratory response, including timely pulsed-field gel electrophoresis pattern analysis and PulseNet posting, facilitated prompt voluntary recall of brand A spinach.

An Engineered R-Type Pyocin Is a Highly Specific and Sensitive Bactericidal Agent for the Food-Borne Pathogen Escherichia coli O157:H7

Some strains of Pseudomonas aeruginosa produce R-type pyocins, which are high-molecular-weight phage tail-like protein complexes that have bactericidal activity against other Pseudomonas strains. These particles recognize and bind to bacterial surface structures via tail fibers, their primary spectrum determinant. R-type pyocins kill the cell by contracting a sheath-like structure and inserting their hollow core through the cell envelope, resulting in dissipation of the cellular membrane potential. We have retargeted an R-type pyocin to Escherichia coli O157:H7 by fusing a tail spike protein from an O157-specific phage, phiV10, to the pyocin tail fiber. The phiV10 tail spike protein recognizes and degrades the O157 lipopolysaccharide. This engineered pyocin, termed AVR2-V10, is sensitive and specific, killing 100% of diverse E. coli O157:H7 isolates but no other serotypes tested. AVR2-V10 can kill E. coli O157:H7 on beef surfaces, making it a candidate agent for the elimination of this pathogen from food products. All rare AVR2-V10-resistant mutants isolated and examined have lost the ability to produce the O157 antigen and are expected to have compromised virulence. In addition, E. coli O157:H7 exposed to and killed by AVR2-V10 do not release Shiga toxin, as is often the case with many antibiotics, suggesting potential therapeutic applications. The demonstration that a novel R-type pyocin can be created in the laboratory by fusing a catalytic tail spike from the family Podoviridae to a tail fiber of a member of the family Myoviridae is evidence that the plasticity observed among bacteriophage tail genes can, with modern molecular techniques, be exploited to produce nonnatural, targeted antimicrobial agents.

Detection and characterization of avian influenza and other avian paramyxoviruses from wild waterfowl in parts of the southeastern United States

Cloacal swabs were taken from migratory hunter-killed, nonmigratory, nesting waterfowl and migratory shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Samples were processed in embryonated eggs followed by hemagglutination (HA), Directigen, and real-time reverse transcription-PCR tests. Sequence analysis of the hemagglutinin (H) gene of the H10N7 Alabama isolate revealed that it was closely related (98%) to recent isolates from Delaware and Canada, but only 90% related to an H10N7 isolated 30 yr ago. Four isolates had 94 to 97% similarity to published H1N1 isolates including one from swine. No H5 or H7 isolates were found. One sample was highly pathogenic in embryos, produced a high HA titer, and was positive for both avian influenza (AIV) and Newcastle disease virus or avian paramyxovirus (APMV)-1. In recent (2008) sampling, more (14%) AIV, APMV, or both were isolated than in 2006 to 2007 (1% isolation rate). The higher isolation rate during 2008 may be attributed to optimized sample collection, storage in dry ice, new egg incubator, healthier eggs, time or habitat for isolation, species sampled, migratory status of birds, and more experience with detection procedures. An additional egg passage resulted in increased viral titer; however, no HA-negative samples became HA positive. The chance of transmission of APMV or low-nonpathogenic AIV from wild waterfowl to commercial poultry is possible. However, the chance of transmission of H5 or H7 AIV isolates from waterfowl to commercial farms in Alabama, Georgia, or Florida is unlikely. Therefore, continual testing of these birds is justified to ensure that H5 or H7 AIV are not transmitted to commercial poultry.

Relative Cytotoxicity ofEscherichia coliO157:H7 Isolates from Beef Cattle and Humans

Differences and similarities between Escherichia coli O157:H7 isolates from beef cattle and those from sporadic human outbreaks are not fully elucidated. Here, we compared 44 O157:H7 isolates of bovine and human origins (22 isolates of each) to better understand their cytotoxic potential. The Shiga toxin genes stx1, stx2, or both were detected in the 44 isolates, and all elicited Vero cell cytotoxicity. The greatest cytotoxicity was caused by bovine isolates having only stx2 and which represented the majority of such isolates (81.8%). However, no correlation was found between the level of stx gene transcription and cytotoxicity. All human and bovine isolates possessed variant type stx2 and stx2c, respectively, as determined by PCR-restriction fragment length polymorphism. Isolates harboring both stx1 and stx2 genes were much more frequent in human isolates (86.4%). The combination stx1-stx2c found in only four bovine isolates was less cytotoxic. It is clear that cytotoxicity alone cannot account for the apparent inability of O157:H7 bovine isolates to cause diseases in humans. We have found that stx1-stx2-containing or stx1-stx2c-containing isolates were less cytotoxic than several bovine isolates having only stx2c, suggesting that the stx gene combination or other virulence genes in specific genetic lineages may affect the disease outcome.

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producingEscherichia coliO157:H7

To evaluate host range and lytic capability of four bacteriophages (rV5, wV7, wV8 and wV11) against Escherichia coli O157:H7 (STEC O157:H7) from cattle and humans. Four hundred and twenty-two STEC O157:H7 isolates (297 bovine; 125 human) were obtained in Alberta, Canada. The four phages were serially diluted and incubated for 5 h with overnight cultures of STEC O157:H7 to estimate their multiplicity of infection (MOI). All bovine STEC O157:H7 were subjected to pulsed-field gel electrophoresis (PFGE) and phage typing (PT). Phage wV7 lysed all human and bovine isolates irrespective of PFGE genotype or PT phenotype and exhibited the lowest MOI (0.004-0.006, P < 0.0001) of all phages. Phages rV5 and wV11 exhibited a lower MOI (0.002-0.04, P < 0.0001) than did phage wV8 (25-29) and they had a narrower host range than wV7 or wV8. Phages rV5, wV11 and wV8 lysed 342 (81.0%), 321 (76.1%) and 407 (96.4%), respectively, of the 422 isolates. Susceptibility of bovine STEC O157:H7 to rV5, w11 and wV8 was influenced by PFGE genotype and/or PT phenotype. Phages exhibited activity against the majority of bovine and human STEC O157:H7 isolates. PFGE genotype and/or PT phenotype of the host-target influenced their vulnerability to phage attack. Susceptibility of bovine STEC O157:H7 to phage may also differ among farms. Both lytic capability and host range should be considered in the selection of therapeutic phage for on-farm control of STEC O157:H7. The present work indicates that a four-phage cocktail should be equally effective at mitigating STEC O157:H7 isolates both of bovine and of human origin. Given that some STEC O157:H7 exhibited resistance to some but not all phages, a phage cocktail is the logical approach to efficacious on-farm therapy.

Escherichia coli O157:H7 strains isolated from environmental sources differ significantly in acetic acid resistance compared with human outbreak strains.

A number of studies on the influence of acid on Escherichia coli O157:H7 have shown considerable strain differences, but limited information has been reported to compare the acid resistance based on the different sources of E. coli O157:H7 isolates. The purpose of this study was to determine the survival of E. coli O157:H7 strains isolated from five sources (foods, bovine carcasses, bovine feces, water, and human) in 400 mM acetic acid solutions under conditions that are typical of acidified foods. The isolates from bovine carcasses, feces, and water survived acetic acid treatment at pH 3.3 and 30 degrees C significantly (P < or = 0.05) better than did any food or human isolates. However, resistance to acetic acid significantly increased as temperature decreased to 15 degrees C for a given pH, with little (P > or = 0.05) difference among the different isolation sources. All groups of E. coli O157:H7 strains showed more than 1.8- to 4.5-log reduction at pH 3.3 and 30 degrees C after 25 min. Significantly reduced (less than 1-log reduction) lethality for all E. coli O157:H7 strain mixtures was observed when pH increased to 3.7 or 4.3, with little difference in acetic acid resistance among the groups. The addition of glutamate to the acetic acid solution or anaerobic incubation provided the best protection compared with the above conditions for all groups of isolates. These results suggest that temperature, pH, and atmospheric conditions are key factors in establishing strategies for improving the safety of acidified foods.