Factors affecting conventional and molecular detection of Bacillus anthracis in the environment and the stability of B. anthracis identification plasmids PX01 and PX02 in vitro.
Conventional and molecular methods for the improved detection of B. anthracis in
environmental material were studied. A system was developed which combines a
selective enrichment broth with two-phase concentration using polyethylene glycol and
potassium phosphate to form the two immiscible phases.
The enrichment broth alone, based on polymyxin B sulphate, lysozyme, EDTA
and thallous acetate, which comprise PLET agar (PLETA), allowed the selective
recovery of B. anthracis from a mixture of B. anihracis and closely related B. cereus.
When soil was added to the broth, however, B. anthracis was rapidly overgrown by
other naturally occurring Bacillus species. Recovery of B. anlhracis was improved by
using semi-solid PLET broth or by adding chelating agents or the monobactam
antibiotic aztreonam to the broth. The combination of chemicals required for optimal
recovery of B. anthracis varied according to the composition of the soil. Use of the
two-phase concentration system showed that in soil B. anthracis spores are generally
attached to soil particles and need to be separated before they can be concentrated.
Separation was achieved by pre-soaking the soil in water.
The sensitivity of standard PLETA is approximately 5- 50 spores per gram of
soil depending on the sample composition. The system finally recommended for the
mostr eliablea nd sensitived etectiono f sporesi n soil achieveda n average2 5 fold
greater sensitivity than PLETA.
Further enrichment of the B. anthrcicis concentrate obtained using the
optimised enrichment method allowed the PCR detection of B. cinthracis DNA. The
sensitivity of the PCR was affected by the composition of the soil. In the absenceinhibition the PCR detection limit was approximately 10 - 100 spores per gram.
A multiplex PCR was developed which targets DNA from pXO1, pX02 and the
B. anthracis chromosome. The PCR allowed the rapid identification of colonies
suspected of being B. anthracis. In addition to being essential for the definitive
identification of B. anthracis, the ability to determine the presence of virulence
plasmids in B. anthracis has reduced the need to use animals for virulence tests.
Attenuated pXO 1+/pX02- or pXO 1'/pX02- strains of B. anthraci. s are
occasionally found in the environment. Naturally occurring pXO 1-/pX02` derivatives
have not been isolated. No other plasmid DNA has been identified in
B. anthracis. To examine the nature of the stability of pXOI and pX02 in
B. wzlh1"acist,h e effect of selective pressuref or non-indigenous plasmid DNA that had
been introduced into B. nnthracis was studied. A plasmid based on the minimal
replicon of pAMß 1 (pAEX-5E, 5.8. kb) was found to be stable in pX01"/pXO2' and
pXO1-/pX02+ derivatives of B. anthracis for more than 100 generations of growth. In
the pXO1+/pX02` and pXO1+/pX02- derivatives of B. anthracis, pAEX-5E was
expelled within 105 culture generations. Loss of pAEX-5E was most rapid in the
pXO1+/pXO2' derivative. Plasmids pXOI and pX02 both remained stable under
selection pressure for pAEX-5E, and in the pX01''/pXO2- derivative retention of pX01
led to a reduction in growth rate. This indicates that, in the absence of pX02, B.
anlhracis will endure a significant metabolic compromise in order to retain pX01.
This study has provided extensive new information about the selective recovery
of B. anthr"acis. in environmental material and novel'data about the stability of identity
plasmids pXO1 and pX02 under selective pressure for non-indigenous plasmid DNA