Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios > or = 10(2) terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10(10) PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be > or = 10(2). In addition, phages were maintained at 10(6) PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

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.     

Application of Bacteriophages To Control Intestinal Escherichia coli O157:H7 Levels in Ruminants

A previously characterized O157-specific lytic bacteriophage KH1 and a newly isolated phage designated SH1 were tested, alone or in combination, for reducing intestinal Escherichia coli O157:H7 in animals. Oral treatment with phage KH1 did not reduce the intestinal E. coli O157:H7 in sheep. Phage SH1 formed clear and relatively larger plaques on lawns of all 12 E. coli O157:H7 isolates tested and had a broader host range than phage KH1, lysing O55:H6 and 18 of 120 non-O157 E. coli isolates tested. In vitro, mucin or bovine mucus did not inhibit bacterial lysis by phage SH1 or KH1. A phage treatment protocol was optimized using a mouse model of E. coli O157:H7 intestinal carriage. Oral treatment with SH1 or a mixture of SH1 and KH1 at phage/bacterium ratios ≥10² terminated the presence of fecal E. coli O157:H7 within 2 to 6 days after phage treatment. Untreated control mice remained culture positive for >10 days. To optimize bacterial carriage and phage delivery in cattle, E. coli O157:H7 was applied rectally to Holstein steers 7 days before the administration of 10¹⁰ PFU SH1 and KH1. Phages were applied directly to the rectoanal junction mucosa at phage/bacterium ratios calculated to be ≥10². In addition, phages were maintained at 10⁶ PFU/ml in the drinking water of the phage treatment group. This phage therapy reduced the average number of E. coli O157:H7 CFU among phage-treated steers compared to control steers (P < 0.05); however, it did not eliminate the bacteria from the majority of steers.     

Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37 degrees C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10(-6) CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10(-4) CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10(-6) CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.     

Characterization of bacteriophages with a lytic effect on various Salmonella serotypes and Escherichia coli O157:H7

Four phages isolated from cattle and poultry feces were analyzed for their ability to lyse Salmonella serotypes and Escherichia coli O157:H7. The phage one-step growth curves, morphology, and genetic characteristics were determined. All phages showed a lytic effect on various Salmonella serotypes and E. coli O157:H7, which lysed at least 70% of the 234 strains tested. The phages had latent periods ranging from 10 to 15 min and generation times of 30 to 45 min, while burst size fluctuated between 154 and 426 PFU/cell. Phages morphology showed isometric and elongated heads and rigid contractile tails, consistent with morphology of the Myoviridae family. Phages' DNA dendrograms showed a distinctive RFLP when digested by HindIII and EcoRV, and SDS-PAGE profile showed distinctive proteins expression as well. In vitro phage challenge showed a total reduction of E. coli O157:H7, Salmonella Typhimurium and Saintpaul counts at 2 h, whereas for Salmonella Montevideo a reduction and retardation growth, at a multiplicity of infection (MOI) of 100, was observed; however, under a MOI of 10 000, no viable cells were detected after 4 h. The wide host ranges of these phages suggested they could be used for simultaneous biocontrol of some Salmonella serotypes and E. coli O157:H7.     

Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7

Aims:  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.
Methods and Results:  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.
Conclusions:  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.
Significance and Impact of the Study:  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.     

Characterization of a virulent bacteriophage specific for Escherichia coli O157:H7 and analysis of its cellular receptor and two tail fiber genes

A virulent phage, named PP01, specific for Escherichia coli O157:H7 was isolated from swine stool sample. The phage concentration in a swine stool, estimated by plaque assay on E. coli O157:H7 EDL933, was 4.2x10(7) plaque-forming units per g sample. PP01 infects strains of E. coli O157:H7 but does not infect E. coli strains of other O-serogroups and K-12 strains. Infection of an E. coli O157:H7 culture with PP01 at a multiplicity of infection of two produced a drastic decrease of the optical density at 600 nm due to cell lysis. The further incubation of the culture for 7 h produced phage-resistant E. coli O157:H7 mutant. One PP01-resistant E. coli O157:H7 mutant had lost the major outer membrane protein OmpC. Complementation by ompC from a O157:H7 strain but not from a K-12 strain resulted in the restoration of PP01 susceptibility suggesting that the OmpC protein serves as the PP01 receptor. DNA sequences and homology analysis of two tail fiber genes, 37 and 38, responsible for the host cell recognition revealed that PP01 is a member of the T-even bacteriophages, especially the T2 family.     

Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P?< 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven?log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E.?coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.     

Occurrence of phages infecting Escherichia coli O157:H7 carrying the Stx 2 gene in sewage from different countries

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7. Recent studies have demonstrated a relatively high presence of Shiga toxin 2 phages in sewage from Spain, but no data on sewage from other areas were available. In order to evaluate the presence of such phages in sewage from diverse geographical origins, 33 sewage samples, including samples from eight different European countries as well as from New Zealand and South Africa were analysed. Using an experimental approach based on the detection of Stx 2 gene by a phage enrichment culture followed by PCR, bacteriophages infecting E. coli O157:H7 carrying the Shiga toxin 2 gene were detected in 15 of the samples studied. Results presented here show that the presence of phages carrying the Stx 2 gene is common in sewage from developed countries.     

Toward rational control of<Emphasis Type="Italic"> Escherichia coli</Emphasis> O157:H7 by a phage cocktail

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.     

Rapid detection of Escherichia coli O157:H7 by using green fluorescent protein-labeled PP01 bacteriophage

A previously isolated T-even-type PP01 bacteriophage was used to detect its host cell, Escherichia coli O157:H7. The phage small outer capsid (SOC) protein was used as a platform to present a marker protein, green fluorescent protein (GFP), on the phage capsid. The DNA fragment around soc was amplified by PCR and sequenced. The gene alignment of soc and its upstream region was g56-soc.2-soc.1-soc, which is the same as that for T2 phage. GFP was introduced into the C- and N-terminal regions of SOC to produce recombinant phages PP01-GFP/SOC and PP01-SOC/GFP, respectively. Fusion of GFP to SOC did not change the host range of PP01. On the contrary, the binding affinity of the recombinant phages to the host cell increased. However, the stability of the recombinant phages in alkaline solution decreased. Adsorption of the GFP-labeled PP01 phages to the E. coli cell surface enabled visualization of cells under a fluorescence microscope. GFP-labeled PP01 phage was not only adsorbed on culturable E. coli cells but also on viable but nonculturable or pasteurized cells. The coexistence of insensitive E. coli K-12 (W3110) cells did not influence the specificity and affinity of GFP-labeled PP01 adsorption on E. coli O157:H7. After a 10-min incubation with GFP-labeled PP01 phage at a multiplicity of infection of 1,000 at 4 degrees C, E. coli O157:H7 cells could be visualized by fluorescence microscopy. The GFP-labeled PP01 phage could be a rapid and sensitive tool for E. coli O157:H7 detection.     

Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10⁻⁶ CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10⁻⁴ CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10⁻⁶ CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.     

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.     

Genomic, proteomic and physiological characterization of a T5-like bacteriophage for control of Shiga toxin-producing Escherichia coli O157:H7

Despite multiple control measures, Escherichia coli O157:H7 (STEC O157:H7) continues to be responsible for many food borne outbreaks in North America and elsewhere. Bacteriophage therapy may prove useful for controlling this pathogen in the host, their environment and food. Bacteriophage vB_EcoS_AKFV33 (AKFV33), a T5-like phage of Siphoviridae lysed common phage types of STEC O157:H7 and not non-O157 E. coli. Moreover, STEC O157:H7 isolated from the same feedlot pen from which the phage was obtained, were highly susceptible to AKFV33. Adsorption rate constant and burst size were estimated to be 9.31 × 10(-9) ml/min and 350 PFU/infected cell, respectively. The genome of AKVF33 was 108,853 bp (38.95% G+C), containing 160 open reading frames (ORFs), 22 tRNA genes and 32 strong promoters recognized by host RNA polymerase. Of 12 ORFs without homologues to T5-like phages, 7 predicted novel proteins while others exhibited low identity (<60%) to proteins in the National Centre for Biotechnology Information database. AKVF33 also lacked the L-shaped tail fiber protein typical of T5, but was predicted to have tail fibers comprised of 2 novel proteins with low identity (37-41%) to tail fibers of E. coli phage phiEco32 of Podoviridae, a putative side tail fiber protein of a prophage from E. coli IAI39 and a conserved domain protein of E. coli MS196-1. The receptor-binding tail protein (pb5) shared an overall identify of 29-72% to that of other T5-like phages, with no region coding for more than 6 amino acids in common. Proteomic analysis identified 4 structural proteins corresponding to the capsid, major tail, tail fiber and pore-forming tail tip (pb2). The genome of AKFV33 lacked regions coding for known virulence factors, integration-related proteins or antibiotic resistance determinants. Phage AKFV33 is a unique, highly lytic STEC O157:H7-specific T5-like phage that may have considerable potential as a pre- and post-harvest biocontrol agent.     

Abundance in Sewage of Bacteriophages That Infect Escherichia coli O157:H7 and That Carry the Shiga Toxin 2 Gene

Shiga toxin-converting bacteriophages are involved in the pathogenicity of some enteric bacteria, such as Escherichia coli O157:H7, but data on the occurrence and distribution of such phages as free particles in nature were not available. An experimental approach has been developed to detect the presence of the Shiga toxin 2 (Stx 2)-encoding bacteriophages in sewage. The Stx 2 gene was amplified by PCR from phages concentrated from 10-ml samples of sewage. Moreover, the phages carrying the Stx 2 gene were detected in supernatants from bacteriophage enrichment cultures by using an Stx 2-negative E. coli O157:H7 strain infected with phages purified from volumes of sewage as small as 0.02 ml. Additionally, the A subunit of Stx 2 was detected in the supernatants of the bacteriophage enrichment cultures, which also showed cytotoxic activity for Vero cells. By enrichment of phages concentrated from different volumes of sewage and applying the most-probable-number technique, it was estimated that the number of phages infectious for E. coli O157:H7 and carrying the Stx 2 gene was in the range of 1 to 10 per ml of sewage from two different origins. These values were approximately 1% of all phages infecting E. coli O157:H7.     

Escherichia coli serotype O55:H7 diversity supports parallel acquisition of bacteriophage at Shiga toxin phage insertion sites during evolution of the O157:H7 lineage

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene (stx)-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx-containing bacteriophages into current models.     

Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7

Artificial control of phage specificity may contribute to practical applications, such as the therapeutic use of phages and the detection of bacteria by their specific phages. To change the specificity of phage infection, gene products (gp) 37 and 38, expressed at the tip of the long tail fiber of T2 phage, were exchanged with those of PP01 phage, an Escherichia coli O157:H7 specific phage. Homologous recombination between the T2 phage genome and a plasmid encoding the region around genes 37-38 of PP01 occurred in transformant E. coli K12 cells. The recombinant T2 phage, named T2ppD1, carried PP01 gp37 and 38 and infected the heterogeneous host cell E. coli O157:H7 and related species. On the other hand, T2ppD1 could not infect E. coli K12, the original host of T2, or its derivatives. The host range of T2ppD1 was the same as that of PP01. Infection of T2ppD1 produced turbid plaques on a lawn of E. coli O157:H7 cells. The binding affinity of T2ppD1 to E. coli O157:H7 was weaker than that of PP01. The adsorption rate constant (ka) of T2ppD1 (0.17 x 10(-9)(ml CFU(-1) min(-1)) was almost 1/6 that of PP01 (1.10 x 10(-9)(ml CFU(-1) min(-1))). In addition to the tip of the long tail fiber, exchange of gene products expressed in the short tail fiber may be necessary for tight binding of recombinant phage.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Escherichia coli O157:H7 Shiga toxin-encoding bacteriophages: integrations,excisions, truncations,and evolutionary implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.