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Journal of Clinical Microbiology, February 2001, p. 670-674, Vol. 39, No. 2
Unité des Entérobactéries,
INSERM U389, Institut Pasteur, Paris, France
Received 11 July 2000/Returned for modification 11 September
2000/Accepted 4 November 2000
The presence of cryptic fliC alleles in the genomes of
120 strains representative of the four Shigella species was
investigated. One fragment was obtained by PCR amplification of
fliC, with a size varying from 1.2 to 3.2 kbp, depending on
the species or serotype. After digestion with endonuclease
HhaI, the number of fragments in patterns varied from three
to nine, with sizes of between 115 and 1,020 bp. Patterns sharing most
of their bands were grouped to constitute an F type. A total of 17 different F types were obtained from all strains included in this
study. A unique pattern was observed for each the following serotypes: Shigella dysenteriae 1, 2, 8, and 10 and S. boydii 7, 13, 15, 16, and 17. On the contrary, S. dysenteriae serotype 13 and S. sonnei biotype e were
each subdivided into two different F types. S. flexneri
serotypes 3a and X could be distinguished from the cluster containing
S. flexneri serotypes 1 to 5 and Y. S. flexneri serotype 6 clustered with S. boydii serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and S. dysenteriae serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising
S. dysenteriae serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining S. boydii serotypes 5 and
9. None of the 17 fliC patterns was found in the fliC
HhaI pattern database previously described for Escherichia
coli. Overall, this work supports the hypothesis that
Shigella evolved from different ancestral strains of
E. coli. Moreover, the method outlined here is a promising tool for the identification of some clinically important
Shigella strains as well as for confirmation of atypical
isolates as Shigella spp.
Shigella species and
serotypes, except for Shigella boydii serotype 13, show more
than 73% DNA relatedness to Escherichia coli K-12
(4). Because of this close genetic relationship, Shigella strains can be considered E. coli clones
which are much less biochemically active, are host restricted, and
carry a plasmid encoding invasiveness. However, for historical reasons,
nonmotile, anaerogenic Enterobacteriaceae causing dysentery
are classified in the genus Shigella, which comprises four
species: S. dysenteriae, S. flexneri, S. boydii, and
S. sonnei (11).
Since Shigella strains produce neither flagella nor capsular
antigens, their antigenic characterization relies exclusively on the
properties of their somatic antigens (O antigens) (3, 17).
S. dysenteriae, S. boydii, and S. flexneri have
been subdivided into 15, 18, and 6 serotypes, respectively. S. flexneri contains two additional variants (X and Y), and serotypes
1 to 5 are further subdivided into subserotypes. Only one serotype has
been described for S. sonnei, but strains of this species
can be divided into five biotypes.
In contrast to Shigella strains, E. coli strains
are often motile and produce a structural complex flagellum consisting
of three main structural regions: the basal body, the hook, and the filament (22, 23). The flagellar filament is composed of
many thousands of copies of a single protein subunit, flagellin
(18), which carries antigenic determinants of the H
(flagellar) antigen. The variability of the H antigen is associated
with the flagellin amino acid sequence and its structural gene
(fliC). The N- and C-terminal portions of flagellin are
important for the structure of flagella and are highly conserved
(25). The middle region can be quite variable and contains
portions of the protein that are surface exposed and H type specific.
Several sequences of the gene encoding flagellin (fliC) are
now available (33), allowing restriction of amplified
fliC genes to be used for the identification of H types in
different bacteria (1, 2, 9, 20, 36, 37).
Recently, Machado et al. (21) demonstrated that
HhaI restriction of the amplified fliC gene could
be used for a flagellar identification system fitting all E. coli serovars. The correlation between F types
(fliC-RFLP types) and H types allowed the deduction of H
types from F types. Furthermore, F types of nonmotile isolates could be
identified. Actually, nonmotile strains generally possess the
structural gene but are unable to build up a functional flagellum (23). In another study, it was found that all E. coli O157:NM strains producing Shiga toxin carried the gene
encoding H7 (12, 13).
Although Shigella has long been described as a
nonflagellated organism, intact, but cryptic, flagellin genes have been
detected for S. flexneri and S. sonnei strains
(35). Furthermore, the production of flagella by
prototypic strains of all four Shigella species
has been demonstrated by electron microscopy (14).
The purposes of this work were to (i) detect the cryptic flagellar gene
in all Shigella serotypes, (ii) report on the distribution of flagellar restriction patterns among serotypes, and (iii) compare these patterns with those of E. coli serotypes.
Bacterial strains.
A total of 120 reference, collection, or
clinical strains representing all recognized Shigella
serotypes and biotypes were included in this study (Table
1).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.670-674.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Clonal Relationships among Shigella
Serotypes Suggested by Cryptic Flagellin Gene
Polymorphism
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
List of strains and their F types
Amplification of the fliC gene and restriction of PCR products. Cell lysis and DNA extraction were done as previously described (16). The amplification of flagellin genes by PCR, digestion of the amplified product with endonuclease HhaI, and electrophoresis for fragment separation were performed following the protocol of Machado et al. (21). Three DNA fragments (101, 210, and 701 bp) obtained by HhaI restriction of the amplified fliC gene from E. coli O4:H5:K3 (strain U4/41) were added to all gel lanes and used as internal fragment size standards (21). Gels were stained with ethidium bromide, and images of band patterns were electronically captured using a charge-coupled device video camera interfaced to a microcomputer (Genomic, Collonges-sous-Saleve, France). Tagged image file format (TIFF) images were transferred to a Macintosh computer (Apple Computers, Cupertino, Calif.) for further analysis.
DNA fragment size determination. The Taxotron package (Taxolab; Institut Pasteur, Paris, France) was used for searching lanes and bands in the TIFF images and for interpretation of patterns (6).
The algorithm of Schaffer and Sederoff (32) was used to derive an experimental function relating molecular size to electrophoretic migration distances of the standard fragments and to interpolate the sizes of the other restriction fragments. In pattern comparisons, the percentage of tolerated variation (allowed error) was set to 3.5% for fragment size, indicating that two fragments were considered identical when their sizes did not differ by more than this percentage. Fragments of less than 100 bp were discarded, since error was larger in this range.Comparison of Shigella restriction patterns against a previously published E. coli database. The HhaI restriction patterns of fliC (F types) obtained for Shigella in this study were compared with the 62 previously published patterns for E. coli (21).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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One fragment was obtained by PCR amplification of fliC from each strain tested, except for the reference strain S. boydii serotype 12 NCDC266-59, which failed to give a PCR product. The size of the amplified fliC fragment varied from 1.2 to 3.2 kbp (Table 1).
The number of bands in patterns varied from three to nine, with sizes
of between 115 and 1,020 bp (Fig. 1 and
2). Patterns sharing most of their bands
were grouped to constitute an F type. Seventeen different F types
were obtained from all strains. Some F types contained variable
bands that were not always present, depending on the strain (bands
represented as dotted lines in Fig. 2). Common bands in each F type
(thick lines in Fig. 2) allowed F type identification.
|
|
A unique F type was observed for each of the following serotypes: S. dysenteriae 1, 2, 8, and 10 and S. boydii 7, 13, 15, 16, and 17. On the contrary, S. dysenteriae serotype 13 and S. sonnei biotype e were each subdivided into two different F types. S. flexneri serotypes 3a and X could be distinguished from the cluster containing S. flexneri serotypes 1 to 5 and Y. S. flexneri serotype 6 clustered with S. boydii serotypes 1, 2, 3, 4, 6, 8, 10, 11, 14, and 18 and S. dysenteriae serotypes 4, 5, 6, 7, 9, 11, and 12. Two other clusters were outlined: one comprising S. dysenteriae serotypes 3, 12, 13 (strain CDC598-77), 14, and 15 and the other one joining S. boydii serotypes 5 and 9 (Table 1).
None of the patterns in the present study had been described in the previously published database containing 62 F types of E. coli (21).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The notion that flagellar antigens could be an indicator of ancient evolutionary divergences was first suggested by Ørskov et al. (27) to explain the two H types detected among strains of nine enteropathogenic serotypes of E. coli from different origins. In the present study, cryptic fliC was found to be highly conserved among Shigella species, revealing the clonal structure of this genus. Shigella clusters with the same F types are described here, and these data are in agreement with the results of previous studies.
A previous analysis of esterase electrophoretic polymorphisms distinguished five clusters among Shigella serotypes: (i) S. dysenteriae serotype 1, (ii) S. flexneri serotypes 1 to 5, (iii) S. flexneri serotype 6 and S. boydii serotypes 2 and 4, (iv) S. sonnei, and (v) S. boydii serotype 13; these clusters were closely related to those of E. coli (15). These data are in accordance with the results of the present study, except for the close relationship of these clusters to those of E. coli, which was not observed with the method used here.
Clusters of Shigella have been distinguished by ribotyping. In an earlier study, Rolland et al. (31), using a ribotyping system based on restriction with two endonucleases (EcoRI and HindIII), showed that S. sonnei and S. flexneri were easily distinguishable from S. boydii and S. dysenteriae, whereas distinction between S. boydii and S. dysenteriae was less clear. Strains of S. boydii serotype 13, which belong to a discrete DNA hybridization group (5), were clearly distinguished from the other Shigella strains. Rolland et al. (31) could not demonstrate the close taxonomic relationship between S. flexneri serotype 6 strains and S. boydii strains, but this association was proven on the basis of antigenic, chemical, and genetic data (10, 29, 30, 34). Another ribotyping system based on restriction with endonuclease MluI was proposed (8). Results were correlated to serotyping results and, in many cases, the serotypes of clinical strains could be predicted from their ribotypes. Again, some clusters of serotypes were observed within each species. Moreover, S. flexneri serotype 3 and S. boydii serotype 12 had the same ribotype.
Serotyping (28), biotyping (24), and isoenzyme analysis (26) suggested that S. sonnei is genetically homogeneous. However, distinct clones within S. sonnei have been revealed by more discriminating molecular biology techniques (19, 31). Strains UE 94-2558 and UE 55-87 of S. sonnei biotype e had the same MluI ribotype (8). That pattern did not correspond to the one expected for S. sonnei. These two strains were also determined to be different from other S. sonnei strains by MboII restriction of the amplified chromosomal region coding for the enzymes responsible for O-antigen synthesis (rfb-RFLP technique) (7).
The results of this work support the hypothesis that there was no single primordial Shigella ancestral strain (15). Groups of Shigella serotypes may represent different lineages.
However, some clinically important serotypes had unique F types (Table 1), e.g., S. dysenteriae serotype 1. In practical terms, analysis of the HhaI restriction patterns of fliC is a promising tool for the identification of some clinically important Shigella strains as well as for confirmation of atypical isolates as Shigella spp.
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FOOTNOTES |
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* Corresponding author. Mailing address: Unité des Entérobactéries, Institut Pasteur, 28, Rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33 145688340. Fax: 33 145688837. E-mail: pgrimont{at}pasteur.fr.
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