Department of Pharmacology, Microbiology and Food Hygiene, The Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo, Norway1
Author for correspondence: Sigrid Brynestad. Tel: +47 22597052. Fax: +47 22964850. e-mail: sigrid.brynestad{at}veths.no
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ABSTRACT |
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Keywords: Stx2-encoding phages, STEC, VTEC
Abbreviations: STEC, Shiga-toxin-producing Escherichia coli; Stx, Shiga toxin
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INTRODUCTION |
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The Shiga toxins (Stxs) are the main virulence factors in the disease progression of haemorrhagic colitis and haemolytic uraemic syndrome caused by STEC O157:H7 and other STEC. The toxin-encoding genes (stx) are located on lysogenic lambdoid double-stranded DNA phages, which form a heterogeneous family of stx converting phages (OBrien et al., 1984 ). Three of the stx2 phages (933W, VT2-Sa and VT2-Sakai) have so far been completely sequenced (Plunkett et al., 1999
; Makino et al., 1999
; Miyamoto et al., 1999
). VT2-Sa and VT2-Sakai are prophages from different E. coli O157:H7 isolates from the Sakai outbreak in Japan, and are very similar. The only major difference is an IS629 insertion in VT2-Sakai. The sequences of these two phages showed considerable variation in the regions encoding replication and early regulation functions as compared to 933W.
phages are typically mosaic in structure, and it is proposed that they share a common gene pool from which DNA can be frequently exchanged (Muniesa et al., 2000
; Hendrix et al., 2000
). From the complete genome sequence of the E. coli O157:H7 type strain EDL933, 18 multigenic regions related to known bacteriophages were identified (Perna et al., 2001
). Only the stx2 phage 933W appeared to be a fully functional phage, while the other
-like sequences showed a mosaic structure, indicating extensive exchange of genes. Another STEC O157:H7 isolate that is currently being sequenced also has many phage-like sequences in its genome (Yokoyama et al., 2000
), and a similar phenomenon has also been observed in Shigella dysenteriae (McDonough & Butterton, 1999
). The general position of the stx genes in Stx-encoding phages appears to be conserved, as they are located between the genes encoding the transcription antiterminator Q protein and the holin S gene of the lysis cassette (Plunkett et al., 1999
; Unkmeir & Schmidt, 2000
). Upstream from Q is the gene encoding the cI repressor, which determines immunity, and the genes for the replication proteins O and P. It is assumed that this organization is necessary for controlled expression and release of the toxins together with the infectious phage particles (Neely & Friedman, 1998
; Plunkett et al., 1999
; Muniesa et al., 2000
). The stx phages should therefore not only be viewed as passive vectors for the dissemination of stx, but as genetic entities where the characteristics of the phage itself influence toxin production and thus virulence of the host bacteria (Wagner et al., 1999
).
Pulsed field gels of the Norwegian STEC isolates used in this study have previously been blotted and hybridized with stx phage gene probes (Johansen et al., 2000 ). The results showed that the bacteria tested contained a single copy of stx2, that the P gene homologous to that from the phage 933W was present in one to three copies and was associated with stx1 but not with stx2, and that there were 12 or more copies of IS1203-like elements in the bacterial genomes. The findings indicated that the stx2 phages in these isolates were not identical to the published sequences of stx2 phages, and that the phages could play a role in the genome changes observed in the PFGE types. We were interested in examining the diversity and genetic composition of the stx phages in these bacteria, and in determining the possible association between the phage type and the toxicity of the host bacteria.
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METHODS |
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Southern blot hybridization of RFLP fragments.
DNA fragments from the RFLP agarose gels were transferred to nylon membranes (Hybond N+; Amersham Pharmacia Biotech) according to the manufacturers instructions. The localization to RFLP fragments of the genes for stx2, IS1203-like DNA and integrase (int) was determined by hybridization using the ECL direct nucleic acid labelling and detection system (Amersham Pharmacia Biotech) according to the manufacturers instructions with labelled PCR products. The following primers were used to generate the probes: stx2-1 and stx2-2 for the stx2 probe, IS1 and IS2 for the IS probe and UintF and UintR for the
-int probe (Table 2
).
PCR.
Using a BLAST search (Altschul et al., 1990 ; http://www.ncbi.nlm.nih.gov/blast/), regions that are conserved in the genome of as many as possible of the
-group of phages were determined. Primers were designed so that these conserved regions could be utilized to amplify the regions specific to stx1, stx2, IS1203-like DNA, different int, and genome sections of replicative and regulatory genes (O/P, cI and Q) of the individual bacteriophages (Table 2
). The presence of these genes in the host strains and in the isolated phages was determined by PCR carried out in a Minicycler (MJ Reseach) or Mastercycler gradient (Eppendorf). Bacterial DNA template prepared by suspending two bacterial colonies in 100 µl sterile water or isolated phage DNA were used as templates. Amplification was carried out in a total volume of 50 µl containing 10 pmol of each primer, 200 µM of each dNTP, 2 U DyNAzyme II DNA Polymerase (Finnzymes), 5 µl 10x buffer (Finnzymes) and 5 µl bacterial or 15 ng phage DNA template. After a denaturation step of 3 min at 94 °C, the template DNA was denatured at 94 °C for 1 min, annealed at the appropriate temperature for 1 min and extended for 1 min at 72 °C. This amplification was carried out for 30 cycles. The final step was an extension of 7 min. The amplified products were run on a 1% agarose gel (SeaKem GTG) and visualized by ethidium bromide staining.
Sequencing of phage fragments.
Partial sequencing of the PCR products obtained from the isolated phages was used to determine if the sequences showed homology to any known bacteriophage sequences. PCR products were ligated into pMOSBlue vector using a pMOSBlue blunt-ended cloning kit (Amersham Pharmacia Biotech) according to the manufacturers instructions. Plasmids were purified using a QIAprep Spin Miniprep Kit (Qiagen). Nucleotide sequencing was carried out with specific oligonucleotide primers on PCR products, or with universal primers on the vectors using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) according to the standard methods described by the manufacturer. An ABI PRISM 377 automatic sequencer (Perkin Elmer) was used.
Toxin production as evaluated by Vero cell assay.
Toxicity was determined using a Vero cell assay (Sandvig & Olsnes, 1982 ). E. coli strains were grown overnight in TSB at 37 °C with moderate agitation, and 50 µl culture supernatant was tested in each Vero cell well. Antibodies (Toxin Technology) against Stx1B and Stx2B were added individually and together to the samples (approx. 0·2 µg) to assess whether the cytotoxicity observed was caused by Stx. All experiments were performed in duplicate and repeated twice.
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RESULTS |
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RFLP of phage DNA
From each of the five isolates which produced stx2 phages, phages from three plaques were purified and designated by in front of, and A, B and C following, the bacterial strain number. The RFLP patterns with EcoRI, AvaI and PstI gave equivalent information, except that PstI did not digest the stx2 phages from isolates 1480 and 1607. Here, the EcoRI RFLP pattern is used to illustrate the results, and only one phage isolate is included in the figure and was used in further studies when all three phages from the bacteria resulted in identical patterns (Fig. 1
). Isolates 497 and 498 were from cattle from the same herd, and the phage RFLP patterns were indistinguishable from each other (
479-A and
498-A), but different from the other phage RFLPs. From isolates 1607, C21b and 1480, three, two and one phage types were detected, respectively (
1607-A/B/C;
C21b-A/C;
1480-A). None of the RFLP patterns from phages isolated from the mother isolate (1607) were identical to the RFLP pattern of the phage from the child isolate (1480) (Table 1
). Two phages were isolated from the EDL933 reference strain from CCUG and these were different from the pattern of
933HS-A phage. The results of blot hybridization of the RFLP gels are shown in Fig. 1
.
497-A and
498-A had an IS1203-like element located on a band of similar size, while
1607-A/B/C had the IS1203-like element on different bands and also on different bands to
1480-A. The stx2 probe hybridized to equivalent bands in the same RFLP pattern groups.
H82-A and
H89-A were negative with both probes.
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ssbN region
The primers were selected from regions conserved in 933W, VT2-Sa, H-19B, HK97, HK022 and phi 21 phages in the -phage group (Franklin, 1985
). The homologies in the region between ssb and N correlated with the results for the cI gene, except for
1480-A and
1607-A, where an IS1203-like element was inserted in an otherwise 933W homologous DNA region (Fig. 3
).
Between P and Q
Primers based on conserved DNA sequences between P and Q (MPQR) and directly after the P gene (APF) (Table 2) were utilized for PCR.
C21b-A and
933HS-A gave bands of approx. 1700 bp, while
497-A,
498-A and 933W resulted in bands of approximately 2000 bp. No bands were obtained by PCR on
1480-A and
1607-A DNA.
Q genes
Primers based on Q (QR and QF) (Table 2) from the phages 933W, VT2-Sa and H-19B were used to amplify a 400 bp product inside the Q gene. PCR products of the expected size were found in all the phages except
497-A and
498-A, which were negative.
ileZstx2
PCR using an ileZ primer (ileZ) and internal stx2 primer (stx2-2) were positive in all the stx2 phages tested (Table 2).
Integrase genes
The phages 933W and VT2-Sa have nearly identical integrase genes, and isolate C21b and the reference strain bacterial cells tested positive in PCR for this integrase. The phages isolated from isolate C21b and the other phages isolated did not have a homologous integrase. Universal primers (UintF + UintR) (Table 2
) based on conserved integrase DNA sequences from
-group phages HK97, HK022, H19J,
and 434 (determined using BLAST) were tried in PCR without success on all the isolated phage DNA. These universal integrase primers were used to amplify integrase from
and this PCR product was used as a probe on RFLP blots, which also gave negative results. These results indicate that the phages studied here show no significant homology with integrases found in the
-group phages.
Presence of various O/P and cI in the host bacteria
To determine if there were other -group phages present in the host bacteria, which would provide a readily available gene pool for exchange and probable superimmunity of other phages, PCR on host bacterial DNA was performed for the O/P and cI genes found in this study, which were different to the isolated phage. As mentioned earlier, all bacterial isolates were positive for 933W (
) O/P, while isolates H82 and H89 were also positive for HK97 O/P. All isolates except 497 and 498 were positive for cI from HK97. Isolates H82 and H89 were positive for 933W cI, as were all bacteria from which the stx2 phage was isolated.
Vero cell toxicity assay
The reference strains and isolate C21b gave the highest level of activity in the Vero cell assay, while isolates 497, 498, 1480 and 1607 gave consistently lower values. The Stx1B antibodies reduced the toxicity levels in the stx1-positive strains, which had the highest activity, to the same levels as the stx1-negative strains. Neutralization with Stx2B antibodies reduced the effect of the supernatant on the Vero cells to very low levels (Table 3). From these studies it would appear that the levels of Stx2 production are in the same range for all the bacterial isolates where there is stx2 phage induction, and that Stx1 contributes to the total effect seen on Vero cells. Isolates H82 and H89, from which there was no stx2 phage induction detected, had values that were comparable to the Stx1 contribution in the other strains. This indicates that toxin production in bacteria that carry integrated, but apparently defective, phages is at a lower level than in bacteria from which phages can be induced.
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DISCUSSION |
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There have been very few clinical STEC cases in Norway, and the child-clinical case (1480) and mother-carrier (1607) isolates were assumed to come from the same, unidentified, source (Heir et al., 2000 ). While there was at least one band difference in the pattern from
1607-A/B/C RFLP as compared to
1480-A, hybridization indicated that there was one copy of stx2 in the same position in the stx2-positive phages. That the RFLP patterns for stx2 phages from the same bacterial isolate, and from the mother and child isolates, were different could explain the differences observed in the PFGE types of the bacterial isolates, and indicate that the gene exchange observed occurs relatively frequently.
Induction of the isolates C21b, 1607 and EDL933 all resulted in more than one phage with different RFLP patterns. The major differences in the RFLP patterns from 1480 and
1607 were associated with IS1203-like elements, which could point to the IS element as a source of variation in this case. The IS elements found in phages
1480,
1607,
497 and
498 were located in non-protein coding DNA and did not seem to have a major effect on the phage functionality. Similar IS elements have been found in stx phages which have affected the function of the phage and inactivated the Shiga toxin (Yokoyama et al., 2000
; Kusumoto et al., 2000
). As many copies of IS1203-like elements have been found in the sequence of E. coli O157:H7, these must also be considered as part of the gene pool that are part of the gene exchange of phages (Perna et al., 2001
).
As we only examined phage isolated from plaques, only those gene exchanges that did not affect plaque formation were observed. Observations of defective prophages and many copies of viral genes in E. coli O157:H7 genomes indicates that less successful gene exchange can also influence the bacterial genome (Perna et al., 2001 ; Yokoyama et al., 2000
; Campbell, 1994
). The presence of different immunity genes in the bacteria could influence the immunity of the bacteria to new invasive phages. The availability of numerous phage genes in the bacteria could play an important part in the evolution of
-group phages and the development of stx phages, enabling the ability to produce toxin to spread into new bacteria (Hendrix et al., 2000
; Baker et al., 1991
).
We had hoped to be able to determine the chromosomal integration site of the various phages, but were hindered by the lack of homology of the integrase in our isolates to the -group of integrases. PCR using the WbrF and WbrR primers, spanning the site of 933W integration in the wrb gene, indicated that these phages integrate at sites other than those for 933W and VT2-Sa.
PFGE analysis has been established as the gold standard for typing of STEC (E. coli O157:H7 in particular) (Barrett et al., 1994 ). When analysing the PFGE patterns the possibility of rapid recombination among stx2 phage genes, which can possibly affect the clonal turnover of E. coli O157:H7 isolates and their genotypic patterns, should be kept in mind. Shifts in PFGE patterns of E. coli O157:H7 isolates during shedding from both humans and experimentally infected cattle have been been observed (Karch et al., 1995
; Akiba et al., 2000
).
A mosaic structure, typical for lambdoid double-stranded DNA phages (Campbell, 1994 ), has been found in the region between stx and the ssb genes in the phages we isolated. This supports the hypothesis that such phages share a common genetic pool with frequent exchange of genetic elements (Hendrix et al., 2000
; Baker et al., 1991
). This type of gene exchange may promote the evolution of mobile stx phages which, provided they show a gain in fitness, could contribute to an increased pathogenicity of their host bacterium. Consequently, the phage genotype may influence the transmission of stx between bacteria, and thus, the spread and emergence of STEC. Further characterization of the stx phage pool is required to determine the role played by these phages in STEC epidemiology and virulence.
Effects of phages on toxicity
The existence of two populations of E. coli O157:H7 in cattle, one, a large non-pathogenic population and the other, a smaller population pathogenic to man, has been suggested (Kim et al., 1999 ). This difference in pathogenicity between populations may, in part, rely upon differences in toxicity of strains. In our study, Vero cell testing showed that the stx-positive isolates produced Shiga toxins in appreciable amounts in isolates from which we found induced stx phage. The stx1 phages, although not induced with mitomycin C, contributed to total toxin production, as shown by the neutralization experiments. We suggest that the differences shown in pathogenicity in otherwise similar stx-positive strains (as discussed above), are associated with the burst size of the stx phages. Isolates in which the stx2 phages were inactivated produced less toxin. These results agree with a previous study (Wagner et al., 1999
) which showed that in a homogeneous bacterial background the amount of toxin produced was directly correlated with the number of phage induced. The stx phages themselves play a major part in toxin production, but the characteristics of the host cell, with its range of other phages and genomic factors, determine if the bacteria themselves survive in environments where they are able to act as pathogens. The pathogenic potential of an STEC strain is therefore partly a result of a complex interplay between the stx phages and the host cells, yet to be fully revealed.
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ACKNOWLEDGEMENTS |
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Received 20 February 2001;
accepted 16 March 2001.