Bull Vet Inst Pulawy 48, 219-223, 2004
RELATIONSHIPS BETWEEN PHENOTYPE AND GENOTYPE
OF ESCHERICHIA COLI O149:K91, F4 STRAINS
ISOLATED FROM PIGS WITH DIARRHOEA
KINGA WIECZOREK, ANDRZEJ KOWALCZYK AND JACEK OSEK
Department of Hygiene of Food of Animal Origin,
National Veterinary Research Institute,
24-100 Pulawy, Poland
e-mail: [email protected]
Received for publication February 26, 2004.
Abstract
Toxic and fimbrial virulence factors as well as
genetic diversity of 11 Escherichia coli O149:K91, F4 strains
isolated from pigs with diarrhoea in Germany were examined.
The ability of enterobacterial repetitive intergenic consensus –
polymerase chain reaction (ERIC-PCR) to discriminate E. coli
strains was evaluated. Two different virulence marker profiles:
F4/EAST1/LTI/STII and F4/EAST1/LTI/STI/STII were
obtained. Comparison of ERIC-PCR fingerprinting revealed a
relatively low percentage of genotypic similarity between the
tested isolates in spite of the presence almost the same
virulence marker genes. The obtained results showed that the
ERIC-PCR method could be very useful for study of genetic
difference between bacterial isolates especially when the tested
strains showed the same virulence markers.
Key words: swine, Escherichia coli, ERICPCR, virulence marker genes, genetic relatedness.
Enterotoxigenic Escherichia coli (ETEC)
strains are important cause of diarrhoea in the newborn
and weaned pigs. Although a large number of
serogroups of E. coli have been described, O149 group
is the predominant one in both suckling (one week old)
and weaned (4-6 weeks old) pigs (22). The reason for
dominance of this serogroup may be the virulence factor
composition which makes them better adapted for
colonization and multiplication in the small intestine of
affected pigs (5). The main virulence attributes of ETEC
are fimbriae and enterotoxins which allow the bacterial
cell to adhere to the intestinal epithelium and to induce
diarrhoea, respectively. ETEC may produce several
fimbrial colonization factors such as F4, F5, F6, F17,
F18, F41. However, the most predominant kind of
fimbriae among porcine E. coli are F4, F18 or F5 (5,
11). Three types of enterotoxins have been described in
ETEC recovered from pigs with diarrhoea, i.e. heatlabile enterotoxin LTI(encoded by the eltI gene) and two
variants of heat-stable enterotoxin: STI and STII
encoded by the estI and estII genes, respectively.
Recently, a new kind of heat-stable enterotoxin,
designated as enteroaggregative E. coli heat-stable
enterotoxin 1 (EAST1) has been described in human and
animal E. coli isolated from diarrhoea (11). This toxin
has also been found among ETEC strains responsible for
postweaning colibacillosis (15).
There are many discriminatory molecular
methods which enable to study relationships among
bacterial species and strains recovered from the same or
different sources as well as isolates belonging to the
same serotype or with identical biochemical profile (23).
One of them is enterobacterial repetitive intergenic
consensus – polymerase chain reaction (ERIC-PCR),
otherwise known as intergenic repeat units (IRUs).
These elements contain a highly conserved central
inverted repeat and located in extragenic regions. ERICPCR enables rapid discrimination of bacterial species
and strains and is a powerful tool for the analysis of
prokaryotic genomes (6, 21, 25).
The aim of our study was to reveal genetic
differences among E. coli O149:K91, F4 strains
recovered from piglets with diarrhoea from different pig
farms in Germany by using ERIC-PCR and to compare
the clonal structure of the tested strains with the
presence of fimbrial and toxin virulence marker genes.
Material and Methods
Bacterial strains. E. coli strains (n=167)
were isolated from sucking piglets with diarrhoea as
described previously (13). The strains were obtained
from 18 geographically separated pig farms located in
the eastern part of Germany. Rectal swabs or faecal
samples were taken from 167 piglets and plated on
MacConkey’s agar (Oxoid). Those identified as E. coli
using the API 20E biochemical system (bioMerieux)
were tested by the PCR test with primers specific for the
E. coli universal stress protein gene (uspA) as described
220
previously (2, 14). After isolation, the bacterial strains
were stored in agar stabs at room temperature and were
not subcultured more than twice before examination.
Serotyping. Somatic O and capsular K
antigens were identified in all E. coli strains by the tube
and slide agglutination tests, respectively, as described
previously (12). The set of antisera with the specificity
for the following O antigens was used: 1, 8, 9, 20, 26,
45, 66, 72, 101, 111, 127, 138, 139, 141, 147, 149 and
157. The anti-K specific antisera were directed against
the following K antigens: 1, 6, 30, 72, 81, 82, 85, 87, 89,
91, 92 (14). For the present study, 11 E. coli strains
belonging to O149:K91 serogroup were randomly
selected and subsequently used for further phenotypic
and genotypic analyses.
Preparation of genomic DNA. Isolation and
purification of bacterial DNA from E. coli bacteria
grown in Luria Bertani (LB) broth were performed using
a genomic DNA isolation kit (Bio-Rad). The purity and
concentration of the DNA preparations were estimated
spectrophotometrically at 260 and 280 nm.
Toxic and fimbrial virulence factors. The
genes encoding heat-labile (LTI), heat-stable I and II
(STI and STII), cytolethal distending toxins I, II and III
(CDTI, CDTII and CDTIII), as well as heat-stable
enterotoxin 1 (EAST 1) were analysed by PCR as
described previously (15, 16, 18). Fimbrial antigens F4,
F5, F6, F17 and F41 were determined by the slide
agglutination test as described before (17).
ERIC PCR amplification conditions. ERIC
PCR was performed in a mixture consisting of 5 µl of
the PCR buffer (10-times concentrated), 5 µl of dNTPs
(Fermentas, Vilnius, Lithuania, final concentration 200
µM), 10 µl of MgCl2 (final concentration 5 mM), 1 µl of
each
primer:
ERIC
1-R
(5’ATGTAAGCTCCTGGGATC AC3’) and ERIC 2
(5’AAGTAAGTGACTGGG GTGAGCG3’) - final
concentration of 0.2 µM, 2 µl (2 U) of the Taq
thermostable DNA polymerase (Fermentas), 5 µl of the
template DNA (10 ng, concentration 2 ng/µl) and
DNase-, RNase-free deionized water (Biomedicals) to a
final volume of 50 µl. Amplification was carried out in a
thermal cycler (PTC-100, MJ Research, Watertown,
USA) using the following programme: one cycle of five
minutes at 94oC, followed by 35 cycles of one minute at
52oC, five minutes at 70oC, one minute at 92oC, and one
final extension step was done for ten minutes at 70oC.
The analysis of the amplified products was performed in
2% agarose (Sigma) in Tris-Acetated–EDTA (TAE)
buffer at 100 V. DNA bands were visualized by staining
with ethidium bromide with distilled water and analysed
under UV light (300 nm) and photographed using the
GEL Doc 2000 documentation system (Bio-Rad).
ERIC PCR profiles of the analysed E. coli
isolates were compared with one another using the
Molecular Analyst Fingerprinting Plus software (BioRad). Cluster analysis was done using the Dice
coefficient and the unweighted pair group method with
arithmetic mean (UPGMA) and a dendrogram was
constructed. The discriminatory ability of the test used
was determined by Simpson’s index of diversity (D) as
described previously (7, 23).
Results
Analysis of virulence markers of E. coli
O149 strains. The analysis of the virulence marker
genes revealed that the isolates obtained from different
pig farms showed almost identical pathogenic gene
profiles. All of them were positive for the EAST1, LTI
and STII toxin genetic markers as well as for the
fimbrial antigen F4 (Table 1). On the other hand, all the
strains were negative for the CDTI, CDTII, CDTIII and
STI toxin genes except one isolate (293) which
harboured the STI gene.
Arbitrary Primer PCR analyses. In the
ERIC-PCR analysis, the strains were represented by 10
different fingerprints (FPs) and generated informative
arrays of bands composed of a minimum of 4 and
maximum of 12 bands (Fig. 1). According to the
UPGMA analysis the strains could be classified into 7
different groups. The ERIC-PCR method had the
discriminatory index of D=0.96 as calculated by the
Simpson’s diversity. Comparison of these fingerprints
showed a relatively low percentage of genotypic
similarity between tested isolates in spite of the presence
almost the same virulence marker genes. However,
100% genetic relatedness among 367 and 371 was
found. Furthermore, these two isolates generated a
separate cluster with a very low percentage of similarity
(40%) as compared to the other strains analysed. High
genetic relatedness among 289 and 293, 296 and 286 as
well as 269 and 360 strains was observed (94%, 92%
and 91%, respectively) (Fig. 2).
M
259 269 286 289 293 296 305 360 365 367 371
Fig. 1. Agarose gel electrophoresis showing ERIC-PCR
fingerprintings generated by eleven E. coli O149:K91,
F4 strains isolated from pigs. Lane M: 100 bp DNA
ladder.
Table 1
Characteristics of E. coli O149:K91, F4 strains isolated from pigs with diarrhoea used in the study
E. coli
40
50
Virulence marker
strain
EAST
CDT I
CDT II
CDT III
LT I
ST I
ST II
F4
F5
F6
F17
F41
259
+
-
-
-
+
-
+
+
-
-
-
-
269
+
-
-
-
+
-
+
+
-
-
-
-
286
+
-
-
-
+
-
+
+
-
-
-
-
289
+
-
-
-
+
-
+
+
-
-
-
-
293
+
-
-
-
+
+
+
+
-
-
-
-
296
+
-
-
-
+
-
+
+
-
-
-
-
305
+
-
-
-
+
-
+
+
-
-
-
-
360
+
-
-
-
+
-
+
+
-
-
-
-
365
+
-
-
-
+
-
+
+
-
-
-
-
367
+
-
-
-
+
-
+
+
-
-
-
-
371
+
-
-
-
+
-
+
+
-
-
-
-
60
70
80
90
100
286
296
305
289
293
259
269
360
365
367
371
Fig. 2. The dendrogram outlining the clonal relationship of the bacterial isolates, performed with UPGMA and Dice’s coefficient.
221
222
Discussion
In this study the presence of virulence marker
genes and genetic differences among E. coli O149:K91,
F4 were investigated. Two pathotypes appeared among
the all isolates examined: F4/EAST1/LTI/STII (ten
strains) and F4/EAST1/LTI/STI/STII (one isolate).
ETEC of O149 serogroup is frequently defined as STInegative, but some reports identified this serotype as
STI-positive (1, 5, 11). The presence of STI gene would
enhance the capacity of strains to cause diarrhoea
because of different mechanisms act on intestinal
epithelial cells from STII and LTI enterotoxins. None of
the tested strains harboured the CDTI, CDTII or CDTII
genes although Lortie et. al. (9) described CDT-positive
strains belonging to O149 serogroup. All the analysed
strains produced F4 fimbriae, which are the major
adhesin of ETEC of porcine origin. Moreover, they
possessed the genetic virulence marker encoding EAST1
toxin. This confirms a close association of the astA gene
(EAST1 toxin) with the presence of porcine fimbrial
colonization factor F4, which was also described in
another studies (10, 13, 15).
Analyses based on the distribution of ERIC
sequences were previously done for environmental
strains of several bacterial species (25). In ERIC-PCR
used for toxigenic strains of Vibrio cholerae O1 and
O139 isolated from different sources showed high
resemblance between FPs and distantly relation to the
non-toxigenic strains (20). For Shigella sonnei, the
ability of ERIC-PCR to discriminate bacterial clones
was equivalent to that of pulsed-field gel electrophoresis
(8). ERIC-PCR based analysis were also used in
comparative test to discriminate strains of the
Acinetobacter calcoaceticus-A. baumannii complex
(26). Thus, ERIC-PCR seems to be relevant for the
genetic discrimination of enteric bacteria. As
demonstrated in many previous studies the ERIC primer
set seems to be also useful for the differentiation
between E. coli strains (3, 4, 19, 25, 26). This statement
was also proved in our analysis. The PCR–based method
as ERIC–PCR allows the simple and rapid genetic
characterization of bacteria tested. Therefore, such tool
could be widely used for epidemiological studies,
especially when the investigated strains are
phenotypically similar or identical (19).
In this study we used purified genomic DNA,
although Woods et. al. (24) described the ERIC-PCR
method which allow the use of the whole bacterial cells.
The authors obtained identical band positions and
fingerprints derived from the whole-cell ERIC-PCR as
well as from purified genomic DNA.
In summary, the relationship between similar
phenotypic traits and genetic diversity which was
described in the present study shows high usefulness of
ERIC-PCR fingerprinting method for epidemiological
purposes for E. coli O149 strains.
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