Comparative genomic profiling of Dutch clinical Bordetella pertussis isolates using DNA microarrays: identification of genes absent from epidemic strains

Abstract

Background: Whooping cough caused by Bordetella pertussis in humans, is re-emerging in many countries despite vaccination. Several studies have shown that significant shifts have occurred in the B. pertussis population resulting in antigenic divergence between vaccine strains and circulating strains and suggesting pathogen adaptation. In the Netherlands, the resurgence of pertussis is associated with the rise of B. pertussis strains with an altered promoter region for pertussis toxin (ptxP3).

Results: We used Multi-Locus Sequence Typing (MLST), Multiple-Locus Variable Number of Tandem Repeat Analysis (MLVA) and microarray-based comparative genomic hybridization (CGH) to characterize the ptxP3 strains associated with the Dutch epidemic. For CGH analysis, we developed an oligonucleotide (70-mers) microarray consisting of 3,581 oligonucleotides representing 94% of the gene repertoire of the B. pertussis strain Tohama I. Nine different MLST profiles and 38 different MLVA types were found in the period 1993 to 2004. Forty-three Dutch clinical isolates were analyzed with CGH, 98 genes were found to be absent in at least one of the B. pertussis strains tested, these genes were clustered in 8 distinct regions of difference.

Conclusion: The presented MLST, MLVA and CGH-analysis identified distinctive characteristics of ptxP3 B. pertussis strains -the most prominent of which was a genomic deletion removing about 23,000 bp. We propose a model for the emergence of ptxP3 strains.

Figure 1

Temporal trends in MLST frequencies of Dutch B. pertussis strains from 1993 to 2004. The MLST designation was based on the allele number in the order ptxP, fim3 and prn. E.g. MLST113 represents strains with ptxP1, fim3-1 and prn3. MLSTs found in frequencies less then 1.5% in the whole period were combined into a single group designated R.

Figure 2

Temporal trends in MLVA frequencies of Dutch B. pertussis strains from 1993 to 2004. Frequencies of different MLVA-types are shown. MLVA types found in frequencies lower than 1.6% in the whole period were combined into a single group designated R.

Figure 3

Genetic diversity of the Dutch B. pertussis population in the period 1993 to 2004. The genetic diversity was calculated for each year using MLVA frequencies.

Figure 4

Minimum spanning tree based on MLVA of Dutch B. pertussis strains isolated from 1993 to 2004. Each circle represents a different MLVA-type, indicated by the number in the middle of the circle. The size of the circle is related to the number of isolates within the particular MLVA-type. Green and red colors indicate, respectively the ptxP1 and ptxP3 allele frequencies within MLVA. The distribution of the MLST types is shown in boxes for the most commonly found MLVA types. In some cases the MLST types are not 100% due to the fact that not all strains were types by MLST.

Figure 5

CGH data for Dutch clinical isolates. CGH analysis of 25 different B. pertussis isolates. A. Each column represents one strain. Strain designation, ptxP type, year of isolation, MLST type and MLVA type are indicated above the column. Each row represents one ORF (in B. pertussis Tohama I gene order). Text on the right indicates prominent RD's. B. Gene content of the locus containing BP1948-BP1966 genes (RD-10) as determined by CGH. Each column represents one strain, strains are sorted by the ptxP type in order to visualize the difference found in this region between ptxP1 and ptxP3 type strains and each row represents one ORF (in B. pertussis Tohama I gene order).

Figure 6

Model for the evolution of the Bordetella pertussis ptxP1 and ptxP3 lineages. The tree is mainly based on two point mutations found in ptxP and fim3 resulting in three lineages, ptxP1-fim3-1, ptxP3-fim3-1 and ptxP3-fim3-2. A single strain carrying the ptxP1-fim3-4 alleles was not included. The deleted regions are indicated in boxes and the frequency in the population is shown above the boxes. The frequencies of prn and MLVA types are given in the right part of the figure. See text for further details.