Cytolethal distending toxins in Shiga toxin-producing Escherichia coli: alleles, serotype distribution and biological effects.
ABSTRACT To assess the prevalence of cytolethal distending toxin (CDT) among Shiga toxin-producing Escherichia coli (STEC), 202 STEC strains were investigated using PCRs targeting various cdt alleles (cdt-I to cdt-V). Seven of the 202 strains contained cdt-III and an additional seven contained cdt-V. All 14 cdt-positive strains produced biologically active CDT, as demonstrated by a progressive distension of cultured Chinese hamster ovary cells. The CDT-positive STEC belonged to eight different serotypes, including sorbitol-fermenting O157 : NM (non-motile). The data demonstrate that CDT is present in some STEC serotypes only. However, more studies are required to evaluate whether CDT presence is associated with severe disease.
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ABSTRACT: Calf diarrhea is a major economic concern in bovine industry all around the world. This study was carried out in order to investigate distribution of virulence genes, pathotypes, serogroups and antibiotic resistance properties of Escherichia coli isolated from diarrheic calves.Biological research 01/2014; 47(1):28. · 1.13 Impact Factor
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ABSTRACT: A total of 73 Shiga toxin-producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (hemorrhagic coli pilus), elfA (E. coli laminin-binding fimbriae), sfpA (sorbitol fermenting EHEC O157 fimbriae plasmid-encoded) and of the toxigenic gene cdt-V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analyzed isolates possess besides stx genes, some adherence genes. Several of the isolates contained also multiple toxins genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt-V toxin in LEE-negative STEC strains that occur in foods, and this traits could increase their pathogenic potential. © 2013 The Authors Letters in Applied Microbiology © 2013 The Society for Applied Microbiology.Letters in Applied Microbiology 03/2013; · 1.63 Impact Factor
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ABSTRACT: Cytolethal distending toxins (CDT) are potent cytotoxins of several Gram-negative pathogenic bacteria, including Escherichia coli, in which five types (CDT-I to CDT-V) have been identified so far. CDT-V is frequently associated to shigatoxigenic E. coli (STEC), enterohaemorrhagic E. coli (EHEC) O157 strains and to strains not fitting any established pathotypes. In this study, we were the first to sequence and annotate a 31.2-kb long, non-inducible P2-like prophage carrying the cdt-V operon from an stx and eae negative E. coli O157:H43 strain of bovine origin. The cdt-V operon is integrated in the place of the tin and old phage immunity genes (termed the TO region) of the prophage, and the prophage itself is integrated into the bacterial chromosome between the housekeeping genes cpxP and fieF. The presence of P2-like genes (n=20) was investigated in a further 5 CDT-V positive bovine E. coli O157 strains of various serotypes, 3 EHEC O157:NM strains, 4 strains expressing other variants of CDT, and 8 CDT-negative strains. All but one CDT-V positive atypical O157 strains uniformly carried all the investigated genomic regions of P2-like phages, while the EHEC O157 strains missed three regions, and the CDT-V-negative strains carried only a few P2-like sequences. Our results suggest that P2-like phages play a role in the dissemination of cdt-V between E. coli O157 strains, and that after integration into the bacterial chromosome, they adapted to the respective hosts and became temperate.Applied and Environmental Microbiology 06/2013; · 3.95 Impact Factor
Cytolethal distending toxins in Shiga toxin-
producing Escherichia coli: alleles, serotype
distribution and biological effects
Dorothea Orth, Katharina Grif, Manfred P. Dierich and Reinhard Wu ¨rzner
Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University and
Austrian Reference Laboratory for Enterohaemorrhagic Escherichia coli, Scho ¨pfstr. 41, A-6020
Received 6 April 2006
Accepted 9 August 2006
To assess the prevalence of cytolethal distending toxin (CDT) among Shiga toxin-producing
Escherichia coli (STEC), 202 STEC strains were investigated using PCRs targeting various cdt
alleles (cdt-I to cdt-V). Seven of the 202strains contained cdt-III and an additional seven contained
cdt-V. All 14 cdt-positive strains produced biologically active CDT, as demonstrated by a
progressive distension of cultured Chinese hamster ovary cells. The CDT-positive STEC belonged
to eight different serotypes, including sorbitol-fermenting O157:NM (non-motile). The data
to evaluate whether CDT presence is associated with severe disease.
The cytolethal distending toxin (CDT) was first described in
1987 as a novel toxin occurring in diarrhoeagenic
Escherichia coli (Johnson & Lior, 1987). During the years
following, closely related toxins were also detected in
other intestinal and extraintestinal pathogens, such as
Campylobacter spp. (Johnson & Lior, 1988a; Pickett et al.,
1996), Shigella spp. (Okuda et al., 1995), Salmonella typhi
(Haghjoo & Galan, 2004), Haemophilus ducreyi (Cope et al.,
1997), Actinobacillus actinomycetemcomitans (Mayer et al.,
1999) and Helicobacter spp. (Chien et al., 2000; Young et al.,
2000). The members of the CDT family are usually encoded
by a cluster of three genes (cdtA, cdtB, cdtC) which encode
2001a; Lara-Tejero & Galan, 2001). CDTs cause G1 or G2
cell cycle arrest in mammalian cells (Cortes-Bratti et al.,
2001b), leading to the inhibition of proliferation, the
characteristic distendedcellmorphology, andultimately cell
death (Bielaszewska et al., 2005a; Lara-Tejero & Galan,
2001). Because they interfere with the cell cycle, CDTs are
classified as cyclomodulins (Nougayrede et al., 2005).
Atotal offive differentcdtalleles(cdt-I,cdt-II,cdt-III,cdt-IV
and cdt-V) have been reported in E. coli (Janka et al., 2003;
Peres et al., 1997; Pickett et al., 1994; Scott & Kaper, 1994;
Toth et al., 2003), but there is only limited knowledge
available on the epidemiology of the strains harbouring
these genes and/or producing CDT. The strains have been
isolated from patients with diarrhoea (Albert et al., 1996;
Ansaruzzaman et al., 2000; Johnson & Lior 1988b; Okeke
et al., 2000) and non-intestinal diseases (Dobrindt et al.,
2003; Johnson & Stell, 2000; Johnson et al., 2002). In several
studies, the prevalence of cdt among E. coli isolated from
(Johnson & Stell, 2000) and meningitis (46%) (Johnson
et al., 2002), was higher than that in isolates from patients
with diarrhoea (1?6–6?4%) (Albert et al., 1996; Okeke et al.,
2000). In addition to human E. coli isolates, CDT
production and/or cdt genes have also been identified,
with varying frequencies, in E. coli isolated from both
diseased and healthy animals, including cattle (Clark et al.,
Mainil et al., 2003; Toth et al., 2003) and dogs (Mainil et al.,
2003; Starcic et al., 2002), as well as various species of wild
Sonntag et al., 2005a). The fact that different cdt alleles were
targeted in the various human and animal isolates makes it
difficult to draw any conclusions on the epidemiological
relationships among CDT+E. coli strains from the various
Although some E. coli isolates identified as cdt+/CDT+also
possess stx genes (Clark et al., 2002; Johnson & Lior 1988b;
Morabito et al., 2001; Sonntag et al., 2005a), such strains
originate mostly from animals, i.e. cattle (Clark et al., 2002)
and pigeons (Morabito et al., 2001; Sonntag et al., 2005a).
The presence of cdt genes in Shiga toxin (Stx)-producing E.
coli (STEC) from humans has rarely been observed
(Bielaszewska et al., 2004; Clark et al., 2002; Johnson &
Lior, 1988b). However, cdt-V has been detected in the
majority (87%)of sorbitol-fermenting (SF) STEC
Abbreviations: CDT, cytolethal distending toxin; CHO, Chinese hamster
ovary; HUS, haemolytic uraemic syndrome; NM, non-motile; NSF, non-
sorbitol fermenting; SF, sorbitol fermenting; STEC, Shiga toxin-
producing Escherichia coli.
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Journal of Medical Microbiology (2006), 55, 1487–1492
O157:NM (non-motile) strains in Germany (Janka et al.,
2003) and in four similar isolates in Austria (Orth et al.,
2006). In the present study, we assessed the prevalence of cdt
in STEC isolates belonging to a broad spectrum of serotypes
and originating from various sources.
Bacterial strains. Two hundred and two STEC strains from
humans (n=150), animals (n=38) and food (n=14) were investi-
gated in this study. One hundred and thirty-two strains were posi-
tive for the intimin-encoding eae gene, which is located at the locus
of enterocyte effacement (LEE); 70 strains were eae negative. The
isolates were recovered at the Austrian Reference Laboratory for
Enterohaemorrhagic Escherichia coli during routine diagnostic and
epidemiological investigations between 2003 and 2005. The isolation
of STEC strains from stools was performed as described previously
(Friedrich et al., 2002). The 150 STEC strains originating from
human stools were recovered from 12 patients with haemolytic urae-
mic syndrome (HUS), 101 patients with diarrhoea, 22 asymptomatic
individuals and 15 individuals with unknown clinical diagnosis. The
animal isolates all originated from cattle, except for one isolate that
was found in a goat. The food isolates were recovered from raw
meat (n=13) and raw milk (n=1). The 202 isolates belonged to 61
different serotypes (Table 1). Sixty strains contained stx1only, 67
strains stx1and stx2, and 75 strains stx2only, as detected by PCR
using primers KS7 and KS8, and LP43 and LP44 (Table 2).
PCRs. The PCR primers, target sequences and PCR conditions are
listed in Table 2. E. coli strains 6468/62 (O86:H34; cdt-I+) (Scott &
Kaper, 1994), 9142/88 (O128:H2; cdt-II+) (Pickett et al., 1994),
1404 (O78; cdt-III+) (Peres et al., 1997), 28c (O78; cdt-IV+) (Toth
et al., 2003), and 493/89 (O157:NM; cdt-V+) (Janka et al., 2003)
were used as positive controls. They were kindly provided by H.
Karch (University of Mu ¨nster), who had received the strains from
D. A. Scott (University of Maryland School of Medicine), C. L.
Pickett (University of Kentucky Medical Center) and E. Oswald
(E´cole nationale ve ´te ´rinaire de Toulouse), or were isolated in his
laboratory (strain 493/89).
CDT bioassay. CDT was assayed using Chinese hamster ovary
(CHO) cells and a modification of the procedure described by Scott
& Kaper (1994). Briefly, supernatants of bacterial cultures grown
overnight (37uC, 180 r.p.m.) in cell culture medium (Ham’s F12
with 10% fetal calf serum) were filter-sterilized (0?22 mm pore-size
filters; Corning), and 1 ml portions of twofold dilutions of the fil-
trates were added in duplicate to 16103CHO cells freshly seeded in
1?5 ml Ham’s F12 medium in six-well tissue culture plates (Falcon
3502; Becton Dickinson). The assay mixes were incubated for 4 days
at 37uC in 5% CO2and examined daily for typical cell distension
(Scott & Kaper, 1994). The CDT titre was defined as the highest fil-
trate dilution that caused distension, evaluated as described else-
where, in 50% of CHO cells (Bielaszewska et al., 2004, 2005a). SF
STEC O157:NM strain 493/89 (CDT-V) served as a positive con-
trol, and CHO cells not exposed to culture filtrates as a negative
RESULTS AND DISCUSSION
Frequency of cdt genes among STEC O157
We used a spectrum of PCR procedures to target various cdt
alleles, previously identified in E. coli strains (cdt-I, cdt-II,
cdt-III, cdt-IV and cdt-V), in STEC belonging to 61 different
serotypes. In the E. coli O157 group, none of the 72 non-
sorbitol-fermenting (NSF) E. coli O157:H7/NM strains
possessed cdt. Friedrich and colleagues have recently shown
that cdt-V-positive O157:H7 strains belong to particular
phage types (PTs), although the presence of cdt within these
PTs is not obligatory (Friedrich et al., 2006). Thus, they
conclude that the proportion of cdt-V-positive STEC
O157:H7 may depend on the PTs tested.
In contrast, five of eight SF STEC O157:NM strains were
positive for cdt-V (Table 3). The association of cdt-V with
SF STEC O157:NM confirms the data of Janka et al. (2003).
However, in contrast to these authors, who found cdt-V in
six out of 100 NSF E. coli O157:H7 (Janka et al., 2003), we
found neither cdt-V nor any of the other cdt alleles in any of
the 72 NSF E. coli O157:H7/NM strains. In addition to cdt,
SF E. coli O157:NM also possess additional loci which are
Table 1. Distribution of serotypes of the 202 investigated STEC strains
ONT, O non-typeable.
SerotypeNumber of strains
SF O157:NM, O125:H8/H7/H4, O103:H21/H7/H2/NM
O177:NM, O111:H8/NM, O114:H18, O113:H4
O175:H40/H8, O127:H40, O116:NM, O91:H21/NM, O76:H21/H19
O174:H21/H2, O163:H19/NM, O158:H40/H18, O128:H8/H2,
O100:NM, O75:H8, O8:H25, O6:H10, O5:NM
O185:H28, O182:H16, O143:H11, O139:H25, O119:H4, O118:H11,
O112:H18, O84:H2, O82:H11, O55:H7, O22:NM, O20:NM,
1488 Journal of Medical Microbiology 55
D. Orth and others
absent from NSF E. coli O157:H7/NM. These include efa1,
locus (Friedrich et al., 2004; Janka et al., 2002, 2005).
However, SF STEC O157:NM lack the gene clusters which
encode urease and tellurite resistance in E. coli O157:H7
Table 2. PCR primers and conditions used to detect eae, stx and cdt genes
Primer Sequence (5§–3§) Target PCR conditions*PCR
Den.D Ann.d Ext.§
CDT-IVas TTG CTC CAG AAT CTA TAC CT
c338f AGC ATT AAA TAA AAG CAC GA
c2135r TAC TTG CTG TGG TCT GCT AT
c1309f AGC ACC CGC AGT ATC TTT GA
c2166rAGC CTC TTT TAT CGT CTG GA
P105 GTC AAC GAA CAT TAG ATT AT
c2767r ATG GTC ATG CTT TGT TAT AT
SK1CCC GAA TTC GGC ACA AGC ATA AGC
SK2CCC GGA TCC GTC TCG CCA GTA TTC G
KS7 CCC GGA TCC ATG AAA AAA ACA TTA TTA
KS8CCC GAA TTC AGC TAT TCT GAG TCA ACG
LP43 ATC CTA TTC CCG GGA GTT TAC G
LP44 GCG TCA TCG TAT ACA CAG GAG C
TGG TGA GAA TCG GAA CTG
CAT TCC ATC AGG TTT GTC
AAT CCC TAT CCC TGA ACC
GTT CTA TTG GCT GTG GTG
AAA CAG GAC GGT AAT AAT GAC TAA TA
GTG ATC TCC TTC CAT GAA AAT ATA GT
cdt-IA 30 s 51uC 60 s 418Bielaszewska et al. (2004)
cdt-IIA30 s 52uC 60 s 542 Bielaszewska et al. (2004)
cdt-III|| 30 s 54uC 180 s 2230Clark et al. (2002)
cdt-IVB 60 s 55uC 60 s350 Toth et al. (2003)
cdt-VA 30 s 52uC 60 s 1329Janka et al. (2003)
cdt-VB30 s 52uC 60 s 1363Janka et al. (2003)
cdt-VC30 s 49uC 60 s 748Janka et al. (2003)
eae 30 s 52uC 60 s 863Friedrich et al. (2002)
stx1B 30 s 52uC 40 s 285 Friedrich et al. (2002)
stx2A 30 s 57uC 60 s584 Friedrich et al. (2002)
*All PCRs were performed in 30 cycles with final extension of 5 min at 72uC.
DDen., denaturing (all reactions at 94uC).
dAnn., annealing (all reactions 60 s).
§Ext., extension (all reactions at 72uC).
Table 3. Distribution of cdt-I, cdt-II, cdt-III, cdt-IV and cdt-V alleles among STEC strains, and CDT production
Serotype Strain no. OriginClinical diagnosis stx1
eae cdt-Icdt-II cdt-IIIcdt-IVcdt-V CDT titre (CHO cells)
Cytolethal distending toxin in STEC
(Bielaszewska et al., 2005b; Friedrich et al., 2005), which
Frequency of cdt genes among STEC non-O157
serotypes, nine (7?4%) tested positive for cdt genes.
Serotypes of the cdt-positive strains are shown in Table 3.
In contrast to SF STEC O157:NM, which contained only
cdt-V, seven of nine non-O157 STEC contained cdt-III, and
two of these harboured cdt-V (Table 3). We demonstrated
the presence of cdt-III in three STEC strains belonging to
serotype O127:H40. Interestingly, E. coli isolates of serotype
O127:NM have been the most prevalent CDT+strains
found among enteropathogenic E. coli (EPEC) in several
studies (Albert et al., 1996; Ansaruzzaman et al., 2000). The
presence of only cdt-III and cdt-V in this study is in
agreement with earlier studies from Germany (Bielaszewska
et al., 2004) and North America (Pickett et al., 2004), in
which only these two alleles have been identified. However,
cdt-I was the only cdt allele found in STEC strains isolated in
differences may exist in cdt alleles occurring in STEC strains
in earlier studies from other countries (Bielaszewska et al.,
2004, 2005c; Pickett et al., 2004; Prager et al., 2005) were cdt
genes identified in STEC of the major non-O157 serogroups
O145, which, however, are mostly eae positive (Bielaszewska
et al., 2005c; Friedrich et al., 2002).
Distribution of cdt-positive STEC among human
and environmental isolates
cdt genes, including cdt-III and cdt-V, were found in nine
(6?0%) of 150 human isolates, in five (13?2%) of 38 animal
isolates, and in none of 14 food isolates. The origins of the
STEC isolates harbouring cdt-III and cdt-V are shown in
Table 3. Eight of the nine human cdt-positive isolates
originated from diseased persons, including two patients
The presence of cdt-V in STEC strains isolated from patients
with severe disease, including HUS, supports reports by
other investigators (Bielaszewska et al., 2004; Janka et al.,
from cattle is in agreement with the report of Clark et al.
(2002). Moreover, the absence of cdt from the goat STEC
isolate investigated in our study is in accordance with
previous studies by other authors (Morabito et al., 2001;
Sonntag et al., 2005a) which suggest species-specific
differences in the prevalence of cdt among STEC of
animal origin. Specifically, whereas cdt genes are regularly
found in STEC strains isolated from pigeons (Morabito
et al., 2001; Sonntag et al., 2005a), STEC isolated from pigs
with oedema disease or diarrhoea lack cdt (Sonntag et al.,
2005b). Since cdt in STEC occurs in a variety of serotypes
from a number of different origins, it is possible that these
genes spread horizontally. Janka and colleagues have shown
that the cdt-V gene in SF STEC O157:NM is flanked
by sequences of bacteriophage P2 (Janka et al., 2003),
suggesting that the gene might have been acquired by phage
cluster in STEC has been shown to be plasmid-
borne (Bielaszewska et al., 2004). Thus, conjugation could
represent a mechanism by which cdt-III is spread among
Association of cdt genes with the eae gene
Ten (7?6%) of 132 eae-positive and four (5?7%) of 70 eae-
negative STEC possessed cdt-III or cdt-V, demonstrating
that there was no significant difference in the prevalence of
after 4 days’ incubation with culture filtrates
control strain 493/89 (a),
CDT-V (b), and STEC strain 33 (O128:H8)
producing CDT-III (c). (d) CHO cells incu-
bated for 4 days in Ham’s F12 medium only,
in the absence of any CDTs. Bars, 75 mm.
1490 Journal of Medical Microbiology 55
D. Orth and others
cdt between eae-positive and eae-negative STEC from our
collection. Moreover, eae was found in the same frequency
among cdt-III-positive and cdt-V-positive STEC strains
(Table 3). These data demonstrate that there is no
association between a particular cdt allele and the presence
of eae in STEC. Our finding of cdt in non-O157 STEC
carrying eae extends the findings of previous studies in
which cdt in non-O157 STEC was restricted to eae-negative
STEC strains (Bielaszewska et al., 2004; Prager et al., 2005).
CDT-III and CDT-V expression in STEC
Of the 14 STEC strains harbouring cdt-III or cdt-V genes, all
produced active CDT according to the CHO cell assay. This
was evidenced by a progressive distension in CHO cells for
up to 4 days after exposure to culture filtrates of the STEC
strains. The CDT titres ranged from 1:2 to 1:64 (Table 3).
There was no difference in CDT titre between strains
harbouring cdt-III and those harbouring cdt-V (Table 3).
The typical distending effects of STEC isolates producing
CDT-III or CDT-V are demonstrated in Fig. 1.
In conclusion, we demonstrate that two different cdt alleles
encode biologically active CDT in STEC of particular
serotypes, some of which are associated with severe human
diseases, including HUS and bloody diarrhoea. Further
investigation is necessary to clarify the mechanisms that
govern the cdt genetic coding and spread among STEC
strains, and to determine the role of CDT in the
pathogenesis of STEC-mediated diseases.
We thank Professor H. Karch (University of Mu ¨nster) for providing us
with cdt-positive control strains and for stimulating discussions. We
thank C. Ortner, A. Rief and G. Hechenblaikner for excellent technical
assistance. This study was supported by the Network of Excellence
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