Evolution, ecology and the role of bacteriophages in horizontal gene transfer:
The working hypothesis is that the organism evolved from a common B. cereus ancestor through the acquisition of two virulence plasmids which transformed the bacterium from a ubiquitous environmental organism to an obligate pathogen unable to replicate outside of an infected host. In collaboration with colleagues at Universities of Oxford and Herriot Watts in the UK we employed Multi-Locus Sequence Typing (MLST) to characterize the genetic interrelatedness of the members of the B. cereus group (Priest et al., 2005). Interestingly B. anthracis represents a distinct, closely defined group with geographically distinct isolates showing little if any intergroup diversity suggesting perhaps that the organism has lost the ability to replicate outside of a susceptible host. In addition to MLST screening we have also investigated the role of B. anthracis specific bacteriophages as a vehicles for gene transfer. Analysis of the recently completed B. anthracis genome sequence (Read et al., 2003) revealed the presence of four prophages integrated into the chromosome. Subsequent screening of over 200 geographically distinct isolates of B. anthracis confirmed the presence of all four phages in every isolate examined to date reinforcing the clonal nature of the species (Sozhamannan et al., 2006). While we are yet to detect the presence of all four phages in any non-anthracis isolate we have seen homologs to phages 3, in some strains of B. cereus suggesting that phages which infect B.anthracis and other members of the cereus group exist and as such are capable of transferring genetic material between strains. Indeed the recent isolation of a phage from soil collected in Maryland capable of infecting isolates of B.anthracis, B.cereus and B.thuringiensis lends further support to this supposition.
Aims of Project
The transfer of virulence genes between different members of the B.cereus family was recently highlighted by the isolation of a strain of B.cereus, G9241, from humans with an infection clinically indistinguishable from that of inhalation anthrax. Analysis of the organism revealed the presence of a homolog of the anthrax virulence plasmid pXO1 which encodes one of the two major virulence factors of B.anthracis, a tripartite toxin (Baillie, 2001). The ability of what was previously though to be B.anthracis specific virulence factors to spread to other, non virulent close relatives such as B.cereus has major implications for the evolution of new pathogens and agent specific detection. The emergence of human pathogenic strains of B.cereus, an organism with considerably greater antibiotic resistance than B.anthracis, raises concerns over our ability to treat infections with these bacteria. The high false positive rate of environmental bio-detectors deployed across the US designed to detect B.anthracis, in some cases up to 20% depending on the time of year, speaks to the problem of identifying organism specific targets.
In contrast to the controlled environment of the laboratory, the mechanisms by which gene transfer occur in nature are unclear. In the case of the bacillus spp the organism must be in its vegetative state. The conditions required outside the test tube for the germination of the anthracis spore, its subsequent replication and the transfer of genetic material are unknown. Indeed there is conflicting data concerning the ability of B. anthracis to persist in the soil as anything other than a spore. This is an important issue with regards to designing post exposure decontamination strategies, if the bug cannot replicate then it is unlikely to reach a level which poses a threat to health. To address these questions studies are in progress in my laboratory to determine the soil conditions, using microcosms, which enable spores of the vaccine strain of B. anthracis to under go germination, replication and exchange genetic material. In collaboration with Professor Fergus Priest of Herriot Watt University in the UK we are investigating the ability of phages to infect B.anthracis and its close relatives as defined by MLST, and to transfer genetic markers in soil. Studies are also in progress to isolate a range of B.anthracis specific lytic phages which could be used, in combination with a suitable germinant mixture, as environmentally friendly decontaminants.