Investigating genome reduction of Bordetella pertussis using a multiplex PCR-based reverse line blot assay (mPCR/RLB)

The genetic composition of the bacterium causing whooping cough, Bordetella pertussis, has been investigated using microarray studies in order to examine potential genetic contributors to the disease re-emergence in the past decade. Regions of difference (RDs) have been previously identified as clusters of genes flanked by insertion sequences which are variably present in different sets of isolates, and have also been shown to be potential markers of B. pertussis evolution.This study used microarray data to identify and select a panel of RDs; primers and probes for these RDs were then designed to test for the presence or absence of these regions in a novel and less expensive multiplex PCR-based reverse line blot (mPCR/RLB) assay. By comparing the presence or absence of RDs, we aimed to determine the genomic variability of a diverse collection of B. pertussis strains and how they have changed over time.

A B. pertussis specific mPCR/RLB using 43 genes representing 30 RDs, was developed and used to characterise a set of 42 B. pertussis isolates. When mapped against the previously identified evolutionary relationships of the strains, the losses of two RDs--BP0910A--BP00930 and BP1948-BP1962--were found to be associated with significant events in B. pertussis history: the loss of BP0910A--BP00930 coincided with introduction of whole cell vaccines in the 1950s while that of BP1948-BP1962 occurred after the introduction of acellular vaccines. The loss of BP1948-BP1962 also coincided with expansion of the most recent B. pertussis strains.

The mPCR/RLB assay offers an inexpensive and fast method of determining the gene content of B. pertussis strains and also confirms that gene losses are an ongoing feature of B. pertussis evolution.