Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates.
BACKGROUND
Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR. However, we have identified 23 Bacillus spp. isolates that gave discrepant results when assayed by standard microbiological methods and PCR. We used multiple-locus variable-number tandem repeat analysis (MLVA), multiple-locus sequence typing (MLST), and phenotypic analysis to characterize these isolates, determine if they cluster phylogenetically and establish whether standard microbiological identification or PCR were associated with false positive/negative results.


RESULTS
Six isolates were LRN real-time PCR-positive but resistant to gamma phage; MLVA data supported the identification of these isolates as gamma phage-resistant B. anthracis. Seventeen isolates were LRN real-time PCR-negative but susceptible to gamma phage lysis; these isolates appear to be a group of unusual gamma phage-susceptible B. cereus isolates that are closely related to each other and to B. anthracis. All six B. anthracis MLVA chromosomal loci were amplified from one unusual gamma phage-susceptible, motile, B. cereus isolate (although the amplicons were atypical sizes), and when analyzed phylogenetically, clustered with B. anthracis by MLST.


CONCLUSIONS
MLVA and MLST aided in the identification of these isolates when standard microbiological methods and PCR could not definitely identify or rule out B. anthracis. This study emphasized the need to perform multiple tests when attempting to identify B. anthracis since relying on a single assay remains problematic due to the diverse nature of bacteria.
Marston
Chung K
CK
Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd,, MS G34, Atlanta, Georgia 30333, USA. cdk5@cdc.gov
Gee
Jay E
JE
Popovic
Tanja
T
Hoffmaster
Alex R
AR
D001407:Bacillus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017103:Bacillus Phages / Q000472:pathogenicity; D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015373:Bacterial Typing Techniques / Q000379:methods*; D014644:Genetic Variation; D006461:Hemolysis; D018598:Minisatellite Repeats / Q000235:genetics; D010406:Penicillins / Q000494:pharmacology; D010641:Phenotype; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods
D010406:Penicillins