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Applied and Environmental Microbiology, September 1999, p. 3855-3861, Vol. 65, No. 9
Institut für Hygiene und Mikrobiologie
der Universität Würzburg, 97080 Würzburg,
Germany,1 and Institute of Medical
Microbiology, The 2nd Medical Faculty, Charles University, 15006 Prague, Czech Republic2
Received 19 May 1999/Accepted 7 July 1999
We investigated the ability of a detoxified derivative of a Shiga
toxin 2 (Stx2)-encoding bacteriophage to infect and lysogenize enteric
Escherichia coli strains and to develop infectious progeny from such lysogenized strains. The stx2 gene of
the patient E. coli O157:H7 isolate 3538/95 was replaced by
the chloramphenicol acetyltransferase (cat) gene from
plasmid pACYC184. Phage
Shiga toxin (Stx)-producing
Escherichia coli (STEC) strains have emerged as a serious
foodborne pathogen that has caused large-scale outbreaks of intestinal
diseases in the developed countries (10). Whereas E. coli O157:H7 is the predominant STEC serotype isolated from
patients in the United States, Canada, and Japan (3, 10), STEC strains belonging to other serotypes (non-O157 STEC) have been
responsible for outbreaks and sporadic cases of human disease in
continental Europe (6). Pathogenic STEC strains share
several determinants implicated in pathogenesis. These include Stx and a pathogenicity island termed LEE, the latter encoding proteins responsible for the intimate adherence of STEC to the intestinal mucosa
and for the subsequent characteristic destruction of the microvilli
termed "attaching and effacing" lesions (15, 18).
Stx are the major pathogenicity factors of these organisms, and there
is evidence that they are involved in the development of
extraintestinal complications, such as the hemolytic-uremic syndrome
(HUS) (25). Stx of E. coli are a family of
proteins which are cytotoxic to eucaryotic cells expressing the
glycolipid receptor globotriaosylceramide (Gb3) (17). They
function as rRNA-N-glycosidases and inhibit protein
biosynthesis in target cells (9). Moreover, it has been
demonstrated that Stx may induce apoptosis in renal tubular cells
(37). The members of the Stx family of E. coli
share genetic, structural, and functional features and consist of Stx1,
Stx2, and variants of Stx2 (18). Stx1 is nearly identical to
Stx of Shigella dysenteriae type 1, but it shares only 55%
amino acid sequence identity with Stx2 (18). In addition,
Stx1 and Stx2 are not neutralized by heterologous antisera (34,
36).
Stx1 and Stx2 are encoded in the genomes of several lambdoid prophages
(8, 13, 22, 36). It has been shown that such Stx-encoding
phages are heterogeneous in their genetic structure and size
(39). The Stx1-converting bacteriophages H19J
(24) and H19B (35), originally isolated from
Stx-producing O26:H11 strain H19, and the Stx2-converting bacteriophage
933W derived from E. coli O157:H7 EDL933 (24)
have been characterized intensively (12, 21, 35, 36). It has
been demonstrated that the stx1 and
stx2 genes in STEC O157:H7, O26, and O111
are closely associated with a p-like gene which is similar
to the p gene of bacteriophage Recently, Stx2-converting bacteriophages originating from E. coli O157:H7 outbreak strains isolated in Japan were
characterized. The authors described one phage, Stx2 Stx-encoding phages may be the major cause for the spread of
stx genes among wild-type E. coli and for the
emergence of new STEC types. Evidence for this has been provided by
Acheson et al. (1) who showed that a derivative of
bacteriophage H19B was able to transduce an E. coli
recipient laboratory strain in the murine gastrointestinal tract.
In this study, we constructed a detoxified derivative of a
Stx2-converting
phage Bacterial strains, plasmids, and bacteriophages.
Most of the
E. coli wild-type strains used in this study were from our
strain collection and were isolated during routine diagnostic work in
recent years from stools of patients with diarrhea or HUS. Pure
cultures of wild-type isolates were stored in stab cultures. All in
all, from the initial isolation up to the phage experiments, isolates
were passaged four times at most. They included 25 STEC (17 serotype
O157:H7 or O157:H
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transduction of Enteric Escherichia coli Isolates with
a Derivative of Shiga Toxin 2-Encoding Bacteriophage
3538 Isolated
from Escherichia coli O157:H7
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
3538(
stx2::cat) was
isolated after induction of E. coli O157:H7 strain 3538/95 with mitomycin. A variety of strains of enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC),
Stx-producing E. coli (STEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and
E. coli from the physiological stool microflora were
infected with
3538(stx2::cat),
and plaque formation and lysogenic conversion of wild-type E. coli strains were investigated. With the exception of one EIEC
strain, none of the E. coli strains supported the formation
of plaques when used as indicators for
3538(stx2::cat). However, 2 of 11 EPEC, 11 of 25 STEC, 2 of 7 EAEC, 1 of 3 EIEC, and 1 of 6 E. coli isolates from the stool microflora of healthy individuals integrated the phage in their chromosomes and expressed resistance to chloramphenicol. Following induction with mitomycin, these lysogenic strains released infectious particles of
3538(stx2::cat) that formed plaques on a lawn of E. coli laboratory strain
C600. The results of our study demonstrate that
3538(stx2::cat) was able to infect and lysogenize particular enteric strains of pathogenic and nonpathogenic E. coli and that the lysogens produced
infectious phage progeny. Stx-encoding bacteriophages are able to
spread stx genes among enteric E. coli strains.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(8). This
implicated that the stx genes of different Stx-converting bacteriophages were located in the same site. This was confirmed in a
later study which also demonstrated the presence of an ileX tRNA gene upstream of the stx2 gene in STEC
O157:H7, O26:H11, O103:H2 and O111:H2 strains
(33). In addition, Neely and Friedman (21)
characterized a 17-kb DNA segment of the Stx1-encoding phage H19B that
covers an analogue of the Q transcription activator, the
stx1 gene, and an open reading frame homologous
to the holin lysis gene. They suggested a role for the -like Q
protein and the holin lysis genes in the regulation of stx
expression and release of the protein from the bacterial cell. They
also showed that the Stx2-encoding phage 933W had a similar structure
in this region (21). The complete sequence of phage 933W was
recently published and also provides evidence for the linkage of phage induction and stx expression (27).
-I, which
resembled phage 933W and another phage, Stx2-II, which had
properties distinct from those of Stx2-I (38). Stx2-I
was demonstrated to use the FadL protein as a receptor and the receptor
for Stx2-II was the LamB protein (38).
3538(stx2::cat)which
conferred chloramphenicol resistance to assess the range of infectivity
of this bacteriophage and to study its postuptake integration,
stability, and inducibility.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and 8 non-O157 serotypes), 11 enteropathogenic E. coli (EPEC), 6 enteroaggregative
E. coli (EAEC), 5 enterotoxigenic E. coli (ETEC),
and 3 enteroinvasive E. coli (EIEC) strains. Six
E. coli strains were isolated from stools of healthy
individuals and do not belong to any of the enteric E. coli
pathogroups. E. coli O157:H7 strain 3538/95 was isolated
from a patient with HUS in Würzburg, Germany, in 1995. This
strain carried stx2, eae, and E-hly and was cytotoxic to Vero cells. The EAEC O111 strain
DEF 53 was kindly provided by Anna Giammanco, University of Palermo, Italy.
(29) was used as a host strain for
plasmids and phages and as an indicator strain. E. coli
SM10pir (thi-1 thr leu tonA lacY supE
recA::RP4-2-Tc::Mu Kmr pir)
served as a donor for conjugation experiments with plasmid pGP704
(11). The E. coli K-12 derivative C600 strain was
used as a positive control in transduction experiments with phage
3538(stx2::cat) and as an
indicator strain. Plasmid pACYC184 (7) was used as a source
of the chloramphenicol acetyltransferase (cat) gene. Plasmids pUC18 (Apr) and pGP704 (Apr
ori R6K mob RP4 MCS of M13tg131) were used as
cloning vectors; the latter delivered suicide functions.
Stx2-converting phage 933W was prepared from E. coli
C600(933W) (24) and used as a control in the plaque assays
with wild-type E. coli strains.
Culture techniques. Mutants of E. coli O157:H7 strain 3538/95 resistant to nalidixic acid were obtained by sequential incubation of this strain in Luria-Bertani (LB) broth containing increasing concentrations of nalidixic acid (Sigma-Aldrich, Deisenhofen, Germany) ranging from 0.5 to 40 µg/ml. A single colony resistant to 40 µg of nalidixic acid per ml was stored and designated 3538/95 (Nalr).
Filter mating of E. coli SM10pir/pHS3 and E. coli O157:H7 3538/95 (Nalr) was performed
according to the method of Miller (19) in a ratio of 1:4
with minor modifications. The LB agar plate containing the mating
mixture was incubated for 8 h at 37°C. Selection of transconjugants was performed on agar plates containing 30 µg of
chloramphenicol per ml (Sigma-Aldrich) and 50 µg of nalidixic acid
per ml that were incubated for 48 h at 37°C.
MICs of chloramphenicol were determined with E-test strips (AB Biodisk,
Solna, Sweden) according to the manufacturer's instructions.
Plaque assay and preparation of stock lysates of
3538(stx2::cat).
A
log-phase culture (optical density at 600 nm of 0.5) of strain
3538/95(stx2::cat) in tryptone
soya broth (Oxoid, Basingstoke, Hampshire, United Kingdom) containing 5 mM CaCl2 was adjusted to a final mitomycin (Sigma-Aldrich)
concentration of 0.5 µg/ml, and the cultivation was continued
overnight. Bacterial cells were sedimented at 5,000 × g for 10 min, and the supernatant was filtered through a
0.2-µm-pore-size membrane filter (Schleicher & Schuell GmbH, Dassel,
Germany). In the plaque assay, 100 µl of the filtrate, 100 µl of an
overnight culture of E. coli C600, and 125 µl of 0.1 M
CaCl2 solution were mixed with 3 ml of LB soft agar (0.7%) at 48°C and poured onto an LB agar plate containing 2% agar. Plaques were observed after overnight incubation at 37°C. A high-titer stock
lysate of 3538(stx2::cat)
was prepared from a single plaque according to the method described by
Sambrook et al. (29) for phage . This stock lysate
contained 108 PFU/ml.
Infection of enteric E. coli strains with
3538(stx2::cat).
One
hundred microliters of log-phase cultures of enteric E. coli
containing 107 CFU was mixed with 100 µl of a 1:1,000
dilution of 3538(stx2::cat) stock lysate (104 PFU), and the ability of wild-type
E. coli to form plaques and to develop lysogens was
investigated. The plaque assay was performed as described above, with
the exception that the LB basal layer contained 0.5 µg of
mitomycin per ml.
3538(stx2::cat) for 4 h at 37°C. Then they were transferred to 4 ml of LB broth containing
30 µg of chloramphenicol per ml, and selective enrichment of
lysogenic (chloramphenicol resistant [Camr])
bacteria was performed for 48 h at 37°C and 180 rpm. Bacteria were then harvested by centrifugation at 5,000 × g for
30 min and transferred to LB agar plates containing 30 µg of
chloramphenicol per ml (Cam-agar) by single-colony streaking. Colonies
grown on Cam-agar after overnight incubation were confirmed to be
lysogens by PCR with stx2-specific primers HSB1
and HSB3. The appearance of a 1.57-kb PCR product proved the presence
of the cat gene linked to the pieces of
stx2 (Fig. 1D) and
thus lysogenization of the strains with
3538(stx2::cat).
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General recombinant DNA techniques. Plasmids were purified with the Nucleobond AX100 Preparation kit (Macherey-Nagel, Düren, Germany). Restriction, ligation, and cloning experiments as well as the preparation of phage DNA were performed according to standard methods (29). Total DNA was prepared as described by Datz et al. (8).
For Southern blot hybridization experiments, either plaques from LB plates or DNA fragments from agarose gels were transferred to nylon membranes (Zeta-probe GT; Bio-Rad, Munich, Germany) by standard procedures (29). DNA was fixed with a cross-linker (Stratalinker UV Crosslinker 1800; Stratagene, Heidelberg, Germany) by using the autocross-link mode (120,000 µJ/cm2). Stringent hybridization was achieved with the DIG (digoxigenin) DNA labelling and detection kit (Boehringer GmbH, Mannheim, Germany) according to the manufacturer's instructions. A 1,343-bp BsaAI-HincII fragment of pACYC184 containing the cat gene was randomly labeled with digoxigenin-11-dUTP and used as a probe. Nucleotide sequences were determined by Taq cycle sequencing with universal and reverse primers for pUC/M13 vectors and HSB1 and HSB3 complementary to the sequences of the stx2 gene as described previously (30). Nucleotide sequence analysis was performed with the Lasergene program package (Dnastar, Madison, Wis.).PCR. Primers HSB1 (5'-CCC GGT ACC ATG AAG TGT ATA TTA TTT AAA TGG-3') and HSB3 (5'-CCC GCA TGC TCA GTC ATT ATT AAA CTG CAC-3') were designed to amplify the entire stx2 gene, yielding a 1,259-bp PCR product containing KpnI and SphI restriction sites at the respective 5' ends. PCRs were performed with the GeneAmp PCR System 9600 (Perkin-Elmer-Applied Biosystems, Weiterstadt, Germany) in a volume of 50 µl containing each deoxynucleoside triphosphate at 200 µM, 30 pmol of each primer, 5 µl of 10-fold-concentrated polymerase synthesis buffer, 3 µl of 25 mM MgCl2 solution, and 2.0 U of AmpliTaq DNA polymerase (Perkin-Elmer, Applied Biosystems). The DNA was denatured at 94°C for 30 s, annealed at 53°C for 60 s, and then extended for 60 s at 72°C. After 30 cycles were completed, a final extension step of 5 min at 72°C was conducted.
Construction of an stx2-negative
derivative of 3538 which encodes chloramphenicol resistance.
From sequence studies, we did not expect alteration of essential phage
functions by mutagenesis in the stx region. Therefore, the
stx gene was chosen as a target for mutagenesis of the
phage. The entire stx2 gene of strain 3538/95
was amplified with primers HSB1 and HSB3. The resulting 1,259-bp PCR
product (Fig. 1A) was separated on a 0.6% agarose gel, excised from
the gel, and then purified with the Prep-a-Gene kit (Bio-Rad, Munich,
Germany). After restriction with KpnI and SphI,
the fragment was again purified and ligated in plasmid pUC18, which was
digested with the same enzymes (Fig. 1B). This plasmid was designated
pHS1. Plasmid pHS1 was then digested with EcoRV and
HincII, which cleaved at positions 146 and 1168 in the
stx2 gene (Fig. 1A), and electrophoresed on a
0.8% agarose gel. One of the resulting DNA fragments with a size of
2,875 bp was excised from the gel. It contained plasmid vector pUC18
with the remaining short fragments of 138 bp (A subunit gene) and 87 bp
(B subunit gene) of the stx2 gene (Fig. 1C). A 1,343-bp BsaAI-HincII fragment prepared from
pACYC184 was ligated with the 2,875-bp fragment resulting in plasmid
pHS2 (Fig. 1C). This plasmid now conferred resistance to
chloramphenicol and carried short stx2 sequences
(Fig. 1C) on both sides of the cat gene. The whole
stx2::cat construct was then
excised from pHS2 with KpnI and SphI and ligated
in suicide vector pGP704, digested with the same enzymes. This yielded
pHS3 (Fig. 1D). The ligation samples were transformed in E. coli SM10 pir. Transformants were checked to carry the
stx2::cat construct by PCR
with primers HSB1 and HSB3. A 1,567-bp product was detected, proving
the presence of the cat gene. Nucleotide sequence analysis
with primers HSB1 and HSB3 also confirmed that the
stx2::cat cassette was
inserted correctly.
protein, pHS3 got lost in subsequent culture
passages; only those recipients which had integrated the cat
gene in their chromosome by homologous recombination could grow. One
such isolate was characterized. PCR with primers HSB1 and HSB3 showed
that the stx2::cat fusion was
present in the E. coli O157:H7 recipient. In addition,
plasmids the size of pHS3 could not be detected by preparation methods. The lack of tetracycline resistance also gave evidence that the suicide
plasmid was absent. The transconjugant was then confirmed to be a
derivative of its parental strain by serotyping, PCR, and sequencing.
However, it was not cytotoxic for Vero cells. The strain was designated
E. coli
3538/95(stx2::cat).
Induction of E. coli
3538/95(stx2::cat) with
mitomycin and subsequent plaque assay with DH5 as an indicator
confirmed the presence of infectious particles of
3538(stx2::cat). In
addition, 3538(stx2::cat)
converted its laboratory host strain DH5 to express resistance to chloramphenicol.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Transduction of various enteric E. coli strains with
3538(stx2::cat).
In order to investigate the ability of
3538(stx2::cat)
to spread among enteric E. coli strains, we performed
transduction experiments which exploited the fact that the phage
could convert its host to express chloramphenicol resistance. Before
starting these experiments, we determined the MIC of chloramphenicol
for all 57 wild-type strains. The MICs ranged from 0.5 to 4.0 µg/ml and were mostly 
3.0 µg/ml. The MICs for E. coli C600 and
DH5 were 1.5 and 4.0 µg/ml, respectively, and that for strain
3538/95(stx2::cat) was 128 µg/ml. Since the breakpoint for sensitivity of E. coli to
chloramphenicol is 8 µg/ml (20), all strains used in
this study except for
3538/95(stx2::cat) were
sensitive to chloramphenicol. The concentration of 30 µg/ml that
represented a 10-fold MIC for more than half of the wild-type strains
and inhibited growth of all of them was chosen for selection of
lysogens. The MICs for the relevant strains are shown in Table
1.
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3538(stx2::cat), and
development of plaques and production of lysogens were determined. Only
1 of 57 wild-type strains, EIEC 12860, formed plaques upon infection
with 3538(stx2::cat) (Table 1). None of the other wild-type strains used as indicator strains supported the formation of plaques. The same results were obtained when the Stx2-converting phage 933W and the original (unlabeled) phage 3538/95 were used as positive controls to infect these strains.
Since the phage-associated cat gene confers resistance to
chloramphenicol, the ability to grow on Cam-agar was used as a marker to screen for lysogens. The 57 wild-type strains were subjected to the
selective chloramphenicol enrichment procedure after having been
exposed to
3538(stx2::cat).
Camr colonies developed in 26 of 57 strains. PCR with
primers HSB1 and HSB3 performed with 15 pooled colonies of each of
these Camr strains confirmed that 17 of the 26 strains were lysogenized with
3538(stx2::cat). From
the remaining nine PCR-negative strains, up to 100 colonies were
subsequently investigated, but the presence of the phage could
not be demonstrated.
Control experiments were conducted under the same conditions with
wild-type strains that had not been exposed to
3538(stx2::cat). Only
two strains developed Camr colonies, with a frequency of
4 × 108 during the selective enrichment procedure.
In total, lysogenization with
3538(stx2::cat)
occurred in 2 of 11 EPEC strains, 2 of 7 EAEC strains, 1 of 3 EIEC
strains, and 1 of 6 physiological E. coli strains. None of
five ETEC strains was lysogenized. Among the STEC strains tested, 1 of
8 non-O157 strains and 10 of 17 O157 strains were lysogenized by
3538(stx2::cat). The 17 strains that produced lysogens are characterized in Table 1.
The laboratory strain E. coli C600 was used as a positive
control in
3538(stx2::cat)
transduction experiments. As demonstrated in Table 1, the strain formed
plaques upon infection with the phage and produced lysogens from which
infectious phage particles were induced by treatment with mitomycin.
Location of the cat gene in the lysogens.
In order
to determine whether the lysogens had inserted
3538(stx2::cat) into
the chromosome or into a plasmid, plasmid and total DNA were prepared
from five selected lysogens. Among these there were
stx-negative as well as stx-carrying strains. The
DNA preparations were restricted with EcoRV and
HincII and separated in parallel on agarose gels.
Hybridization with the cat probe demonstrated a signal with
a size of approximately 2,800 bp in the total DNA preparations of all
strains tested. However, no hybridization signals were present in the
respective plasmid preparations (data not shown). These experiments
clearly proved the association of the cat gene with the
bacterial chromosome.
Induction of
3538(stx2::cat) from
the lysogens.
Sixteen of 17 lysogens were further investigated for
their ability to produce infectious particles of phage
3538(stx2::cat) after
induction with mitomycin. The EAEC strain 4140-86 was excluded from
this study because we were not able to isolate lysogens from the
PCR-positive colony pool (Table 1). As demonstrated in Table 1, 15 of
16 lysogens could be induced with mitomycin and produced phage progeny
that formed plaques on a lawn of the indicator strain E. coli C600.
3538(stx2::cat)-associated
plaques ranged from 0.1% to 82% (Table 1). To ensure that the
hybridization signals were specific, control hybridization experiments
were performed. All 15 parental E. coli strains (not
transduced with
3538(stx2::cat) were
also induced, and the supernatants were investigated for plaque
formation. Plaques were observed with supernatants of 12 of the
15 strains. Plaque hybridization with the cat probe
was performed and was negative in all cases, demonstrating that no cat-like sequences were present in the parental E. coli strains. Moreover, hybridization of chromosomal DNA of all 57 wild-type strains with the same probe was also negative.
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. After mitomycin induction, we were able to
isolate phages containing the cat gene from all lysogens. Laboratory strain DH5 was transduced with these phages, and the cat-harboring phages could be reisolated from such
transductants. Control experiments were performed with
mitomycin-induced supernatants of the parental strains not harboring
3538(stx2::cat).
However, DH5 could not be converted to chloramphenicol resistance by
any of the supernatants.
In order to study putative recombination events that could have
occurred in the phage during the alternating lysogenic and lytic
cycles, we compared restriction fragment profiles of the original phage
3538(stx2::cat) and the
phages that we have isolated from the DH5 transductants as described
above. It could be demonstrated (Fig. 3)
that the phages had identical restriction patterns. Hybridization experiments demonstrated that the same restriction fragment of the
phage DNAs hybridized with the cat probe.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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STEC strains demonstrate a broad range of genotypic and phenotypic heterogeneity. Until now, the mechanisms by which this heterogeneity has evolved have been poorly understood. Besides recombination events and spontaneous mutations, mobile genetic elements could play a role. Although Stx are known to be encoded by temperate, lambdoid prophages, the role of these phages in the spread of stx genes has not been well elucidated.
STEC strains belong to more than 100 serotypes (14), and transduction of stx genes by phages to other enteric E. coli strains may contribute to the observed heterogeneity in STEC serotypes. Acheson et al. (1) demonstrated that it was possible to infect and lysogenize an E. coli laboratory strain with an Stx1-converting phage in the murine gastrointestinal tract. In addition, transductants which produced infectious phage particles were isolated. Although these experiments were the first evidence for in vivo transduction activity of Stx-encoding phages, wild-type isolates were not used as recipients.
Similar experiments were performed with CTX, a filamentous
Vibrio cholerae bacteriophage which encodes cholera toxin
(16). Using a model with suckling CD1 mice, Lazar and Waldor
showed transduction of CTX from a lysogen to a recipient strain in
the small intestine (16). It was also reported that
lysogenic conversion from a nonpathogenic Corynebacterium
diphtheriae strain to a toxinogenic strain could occur in the
upper respiratory tract (26).
Recently, Beutin et al. (4) demonstrated that Stx produced by a Shigella sonnei isolate was encoded by a bacteriophage which could be transduced to a nontoxinogenic S. sonnei reference strain and E. coli laboratory strains.
In the present study, we demonstrated that the detoxified phage
3538(stx2::cat), which
was isolated from a pathogenic STEC O157:H7 strain, was able to
infect and lysogenize certain wild-type strains of E. coli.
Moreover, lysogenic transductants produced infectious virus particles
upon induction with mitomycin.
In total, we were able to detect 17 of 57 wild-type E. coli strains transduced with the phage, and these belonged to a broad spectrum of various enteric pathogroups as well as to the physiological intestinal microflora.
There are some limitations to the techniques used for the detection of lysogens described in this paper. First, the selection of lysogens depended on both the initial MIC of chloramphenicol for the host strains and the ability to develop spontaneous chloramphenicol resistance during the selective enrichment procedure.
To address this, we determined the MICs for all strains used in this
study and found them to be low. All wild-type strains were sensitive to
chloramphenicol. In control experiments, two of the wild-type strains
which were not exposed to the phage
3538(stx2::cat) developed chloramphenicol resistance during the selective enrichment procedure. In these strains, the frequency of the appearance of Camr colonies was 4 × 108. This means
that spontaneous mutation to Camr is a relatively rare process.
In contrast, after exposure to the phage, we found 26 Camr
strains. From these, 17 strains contained phage
3538(stx2::cat) and 9 did not. The nature of the Camr in the latter strains is
not known.
Second, we were able to isolate lysogens from strains which did not support the formation of plaques. This may reflect the methodology used, lysogen isolation being more sensitive because of the selective chloramphenicol enrichment step. Without this step, we would not have been able to isolate any lysogen from wild-type isolates. In contrast, selective enrichment was not necessary for the isolation of lysogens from laboratory E. coli strains (31).
The lysogenized E. coli O157:H7/H strains
contained stx1,
stx2, and stx2c
genes alone or in combination. Since it was shown in earlier
studies that Stx1 and Stx2 are generally phage encoded (8, 21,
36), this was assumed for the E. coli O157 strains tested here. The fact that it was possible to lysogenize such strains
with 3538(stx2::cat)
indicates that the Stx-encoding prophages which were likely to be
present in these strains were different from
3538(stx2::cat).
Otherwise superinfection immunity would have prevented infection.
The presence of temperate Stx-converting phages that were not related
to 3538(stx2::cat)
could explain why in most of the lysogens, only a part of plaques
hybridized with the cat probe (Table 1). Natural E. coli strains frequently contain sequences which are related to
phage and presumed to be cryptic prophages (2). The most
intensively studied were those present in E. coli K-12,
whose chromosome contains, besides phage itself, four phage-related
elements, including DLP12, Rac, Qin, and e14 (5).
Cryptic prophages can alter the host phenotype and directly or
indirectly interact with active phages. They can mediate
superinfection immunity, recombine with active phage DNA, or modify the
host surface properties (5). Moreover, natural E. coli strains can harbor active lambdoid prophages, examples of
which are HK022, P2, and 434 (5). In order to successfully
infect a host cell, Stx-converting phages probably have to compete with
naturally occurring phages or phage-related elements. This could lead
to the failure of establishment of the superinfecting phage.
The obvious presence of several prophages in E. coli isolates could also contribute to the failure of the labeled phage to establish in the chromosome in a way that the insertion sites could be occupied, and therefore the phage could not replicate.
Another interesting aspect of our study concerns the genetic stability
of 3538(stx2::cat). We
could not detect any recombination of this phage with extant
phages of E. coli O157 strains, even under
chloramphenicol selection.
In conclusion, using a model of a detoxified derivative of an Stx2-converting bacteriophage, we have demonstrated that Stx2-converting bacteriophages are able to spread among enteric E. coli strains and may thus contribute to the genotypic and phenotypic heterogeneity observed in the STEC family. The findings described here suggest a role for Stx-encoding bacteriophages in the spread of stx genes among enteric E. coli strains.
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ACKNOWLEDGMENTS |
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We thank Beatrix Henkel for excellent technical assistance and Phil I. Tarr for helpful discussions.
This work was supported by grant Ka 717/3-1 from the Deutsche Forschungsgemeinschaft.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institut für Hygiene und Mikrobiologie der Universität Würzburg, Josef-Schneider-Str. 2, D-97080 Würzburg, Germany. Phone: 49/931/201-5160. Fax: 49/931/201-3445. E-mail: hschmidt{at}hygiene.uni-wuerzburg.de.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Applied and Environmental Microbiology, September 1999, p. 3855-3861, Vol. 65, No. 9
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