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Journal of Clinical Microbiology, June 2008, p. 2125-2128, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.02484-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Is the Sequenced Bordetella pertussis Strain Tohama I Representative of the Species?

Valérie Caro,^* Valérie Bouchez, and Nicole Guiso

Institut Pasteur, Molecular Prevention and Therapy of Human Diseases Unit, URA-CNRS 3012, National Center of Reference of Whooping Cough and other Bordetelloses, Paris, France

Received 28 December 2007/ Returned for modification 10 March 2008/ Accepted 24 March 2008

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Subtractive hybridization was carried out to identify differences between the sequenced genome of Bordetella pertussis Tohama I and those of two recently collected isolates. We identified genetic regions specific to recent isolates, old isolates, and isolates of B. parapertussis and B. bronchiseptica species. We conclude that Tohama I strain is not representative of the B. pertussis species.

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Bordetella pertussis is the bacterial agent of whooping cough in humans, which is a serious public health problem worldwide. Whooping cough is endemic in certain regions, and its reemergence in adolescents and adults has been reported in several countries, despite extensive vaccination programs (7, 13).

B. pertussis, an obligate pathogen of humans, is a model for analyzing bacterial speciation, host restriction, and differences in genotype that may account for pathogenesis. Great progress in bacterial genomics has made it possible to determine the structures of the whole genomes of many bacteria, including the 4.08-Mb genome of B. pertussis. One remarkable trait of this pathogen is its limited genetic variability, which indicates that the species derived from a common ancestor in the recent past, perhaps only a few thousand years ago (11). The B. pertussis strain chosen for genome sequencing was Tohama I, which was originally isolated from a case of whooping cough in Japan in the 1950s. This strain is widely used for genetic studies and as a vaccine strain.

However, it is unclear whether this strain is representative of the species. The B. pertussis species is clonal, but differences have been found (by various techniques) between vaccine strains and currently circulating isolates (9). These differences have been confirmed recently with DNA microarrays (3, 5). For example, we have previously developed a whole-genome DNA microarray, representing more than 91% of predicted coding sequences of Tohama I, to analyze clinical isolates with various pulsed-field gel electrophoresis (PFGE) profile patterns. This spatiotemporal study of the B. pertussis population in France revealed a decrease in genetic diversity over time in this highly vaccinated country. The decrease in diversity is mainly due to loss of genes and pseudogenes driven by homologous recombination between insertion sequences (3). Regions of difference (RD-1 to RD-10) harbored by the Tohama I strain are absent in the genome of isolates of the prevaccine era or of currently circulating B. pertussis isolates.

Nevertheless, this powerful approach cannot be used to identify genes in an isolate that are absent in the chosen sequenced strain. An alternative and efficient approach is subtractive hybridization, which allows direct identification of genomic differences between isolates without requiring a reference genomic strain. This method has already been successfully developed and used to identify genetic differences among plant-pathogenic Xylella fastidiosa strains (10) and between two Yersinia species, pestis and pseudotuberculosis (12). A recent study used suppression subtractive hybridization to search for potential additional DNA harbored by the genome of B. pertussis isolates (1a). The recovered fragments share high (or complete) nucleotide identity with sequences from the driver pool of DNA from the sequenced genomes of B. pertussis, B. parapertussis, and B. bronchiseptica strains. However, no precise sequence information for these fragments was given.

In the present study, we used a new subtractive hybridization approach to identify genomic differences between the reference genome sequence for B. pertussis Tohama I (classified as PFGE group II), the PFGE group IV reference strain FR0743 (isolated in 1999), and the PFGE group V reference strain FR0287 (isolated in 1996) (3, 4). These other two strains were chosen because they are representative of isolates recently circulating in Europe (4), North America (1), and South America (2).

Genomic DNA differences between the three B. pertussis PFGE reference strains were investigated with the collaboration of Cogenics-GENOME Express with a technology based on a subtractive hybridization method (8). This technique allows investigation of genomic fragments specific to the strain of interest, as DNA fragments shared by both strains (tester and driver strains) are specifically removed from the sample. Moreover, prior knowledge of target sequences of interest is not required, and unknown genomic DNA sequences can be isolated and identified.

Subtractive hybridization was carried out with extracted genomic DNA digested with Sau3A or RsaI to increase the coverage of the region of interest, with a theoretical size of 256 bp. We amplified the DNA to avoid PCR saturation, thus preserving representativeness of genomic DNA fragments in each sample.

Tester DNA was mixed with a four times molar excess of biotinylated driver DNA. After an initial denaturation step, the sample was allowed to renature for 20 h. After annealing, the DNA was incubated with streptavidin magnetic beads for driver removal (driver-driver and driver-tester hybrids corresponding to sequences commonly shared by both samples). Specific double-stranded tester DNA was then isolated by incubation of the sample with a single-stranded DNA-binding protein which binds specifically to single-stranded DNA molecules and allows the removal of single-stranded DNA, avoiding normalized library construction. An excess of driver DNA was added to the sample, and the renaturation/subtraction process was repeated two more times as described above. After the third round of subtraction, DNA was used as a template for PCR with the tester-specific primers to allow amplification of a specific DNA target from the tester.

Purified subtracted DNA fragments were ligated to pUC18 and then used to transform Escherichia coli. For each subtraction, plasmids with insert were sequenced with an M13rev universal primer with a BigDye terminator cycle sequencing kit and an ABI Prism 3730 sequencer.

Each analysis step described below was applied independently to sequencing data from each subtraction. DNA sequences were analyzed through an analysis pipeline written in the Perl language (version 5.00502) and using dedicated software for each analysis step.

Briefly, trace files were basecalled and quality trimmed by PHRED software (version 0.000925.c, parameter trim_cutoff 0.01). Vector bases were masked (Crossmatch software version 0.960731, parameters minmatch 10 and minscore 20) against the sequence of pUC18 and scanned for detection of exact motifs of partial digestion and chimerism. Trimmed sequences shorter than 50 nucleotides in length were discarded. PHRAP software (version 0.960731, default parameters) was then used to assemble filtered sequences that had no obvious partial digestion and/or chimera motifs. We defined "target" sequences as contigs and singletons delivered by the assembly process. They reflect restriction fragments selected by the DFHY approach.

Eight subtractive hybridization experiments were carried out, including subtraction of Tohama I (driver) and FR0743 or FR0287 (tester) and vice versa and subtraction of FR0743 (driver) and FR0287 (tester) and vice versa. This technique confirmed the detection of the previous RDs identified by our whole-genome DNA microarray study (3). However, RD-1 (deleted from the genomes of the two isolates FR0743 and FR0287 but present in the genome of Tohama I) and RD-6 (deleted only from the genome of FR0287), identified by microarrays, were not detected by subtractive hybridization, indicating the limits of the method.

However, among 768 clones screened from the subtraction between Tohama I and FR0743 or FR0287, 436 fragments appeared original and specific. There were 252 fragments obtained from subtraction between FR0743 and FR0287. After contig analysis, these fragments appeared to cluster into four RDs that contained Bordetella genes absent from the Tohama I genome but present in the genomes of FR0287 and FR0743 isolates. The new identified RDs, numbered according to the previously identified RDs (according to the sequence of the B. pertussis genome) (3) and according to the sequenced B. parapertussis and B. bronchiseptica genomes (11), are summarized in Table 1. These new RDs are not flanked by IS elements, whereas deleted RDs previously identified by microarrays were. They are also harbored by the genomes of the sequenced B. parapertussis and B. bronchiseptica species.

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TABLE 1. Characteristics of RDs described in this studya

PCR analysis was then used to determine the precise genomic limits of these RDs and to determine the presence of these RDs in other B. pertussis, B. parapertussis, and B. bronchiseptica isolates. We amplified 28 specific regions deduced from these four RDs to confirm our results. The four RDs were present in the genomes of 27 B. pertussis isolates classified into PFGE groups II, III, IV, and V (4) and in the genomes of 10 B. parapertussis and 10 B. bronchiseptica isolates. Our analysis with subtractive hybridization showed that B. pertussis isolates did not acquire any new genetic material, indicating that horizontal transfer of genetic material does not occur in this species. The kept material is also present in the genome of B. bronchiseptica, considered an ancestor of B. pertussis (6). The limits of the four regions were determined, two of which are illustrated in Fig. 1. RD-11 contains mostly open reading frames (ORFs) encoding hypothetical proteins, enzymes, and regulatory proteins (e.g., the LysR family). RD-12 includes ORFs encoding autotransporter, membrane or exported proteins, and transcriptional regulators (e.g., the TetR family). RD-13 is the longest newly identified region, with a length of 17 kb. It consists of ORFs encoding membrane proteins, transcriptional regulators (e.g., the GntR-family), and many transporters (e.g., ABC, a branched-chain amino acid). Finally, RD-14 includes ORFs encoding enzymes, lipoproteins, and transcriptional regulators (e.g., the IclR family) (Table 2).

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FIG. 1. Genomic details for two new identified RDs, RD11 and RD14.

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TABLE 2. Genomic details of RDs described in this study

Figure 2 summarizes the observations from our previous study (3) and the present study.

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FIG. 2. Representation of the presence or absence of the different identified RD in the genomes of the B. pertussis population, B. parapertussis strain 12822, and B. bronchiseptica strain RB50.

In conclusion, the genomes of B. pertussis isolates from the prevaccine and postvaccine eras harbor approximately 46 kb of additional genetic material compared to the Tohama I genome, indicating that a sequenced genome is not always representative of a whole species.

We confirmed the particular trait of this bacterial species by subtractive hybridization. However, our observations indicate the limitations of microarray-based comparative genomic hybridization, which only detects the absence of genes or differences between genes, but not additional genes, with respect to a reference strain genome sequence. Furthermore, subtractive hybridization is not sufficient for the detection of all genetic differences between two similar isolates. Thus, our findings indicate that a single technique cannot be used to analyze a microbial population. Subtractive hybridization and DNA microarrays, as well as comparative genome sequencing, are useful complementary tools to analyze microbial genomic patterns. However, since B. pertussis seems not to acquire any new genetic material and the genetic content is also found in B. bronchiseptica, a "pan-Bordetella" microarray could therefore provide some useful information for the analysis of B. pertussis isolates genome.

Finally, our observations demonstrate the importance of the analysis of clinical isolates and not only one representative of a species. This is especially of important for the development of new molecular diagnostics.

 
We thank Christophe Bozic, François Paillier, and Diliana Dimova from Cogenics, Genome Express, for technical and bioinformatic assistance.

This study was supported by Groupe Malakoff, GlaxoSmithKline, and the Fondation Institut Pasteur.

 
* Corresponding author. Mailing address: Institut Pasteur, Unité PTMMH, URA-CNRS 3012, National Center of Reference of Whooping Cough and other Bordetelloses, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France. Phone: 33 (0)1 45 68 80 05. Fax: 33 (0)1 40 61 35 33. E-mail: vcaro{at}pasteur.fr

Published ahead of print on 2 April 2008.

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Journal of Clinical Microbiology, June 2008, p. 2125-2128, Vol. 46, No. 6
0095-1137/08/$08.00+0     doi:10.1128/JCM.02484-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Caro, V. Articles by Guiso, N. Search for Related Content PubMed PubMed Citation Articles by Caro, V. Articles by Guiso, N.