Abstract
BackgroundAn isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-D-glutamic acid capsule. These phenotypes prompted Ezzell et al., (J. Clin. Microbiol. 28:223) to reclassify this isolate to Bacillus anthracis in 1990.ResultsWe demonstrate that despite these B. anthracis features, the isolate is severely attenuated in a guinea pig model. This prompted whole genome sequencing and closure. The comparative analysis of CDC 684 to other sequenced B. anthracis isolates and further analysis reveals: a) CDC 684 is a close relative of a virulent strain, Vollum A0488; b) CDC 684 defines a new B. anthracis lineage (at least 51 SNPs) that includes 15 other isolates; c) the genome of CDC 684 contains a large chromosomal inversion that spans 3.3 Mbp; d) this inversion has caused a displacement of the usual spatial orientation of the origin of replication (ori) to the termination of replication (ter) from 180° in wild-type B. anthracis to 120° in CDC 684 and e) this isolate also has altered growth kinetics in liquid media.ConclusionsWe propose two alternative hypotheses explaining the attenuated phenotype of this isolate. Hypothesis 1 suggests that the skewed ori/ter relationship in CDC 684 has altered its DNA replication and/or transcriptome processes resulting in altered growth kinetics and virulence capacity. Hypothesis 2 suggests that one or more of the single nucleotide polymorphisms in CDC 684 has altered the expression of a regulatory element or other genes necessary for virulence.
Highlights
An isolate originally labeled Bacillus megaterium Centers for Disease Control (CDC) 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-Dglutamic acid capsule
In a second experiment to confirm attenuation, 10 guinea pigs injected i.m. with 1 × 108 cfu/mL CDC 684 spores all survived. These results confirm that CDC 684 is highly attenuated with an LD50 of >1 × 108 spores in the guinea pig model
We address two hypotheses that could have a role for the attenuated phenotype in B. anthracis CDC 684
Summary
An isolate originally labeled Bacillus megaterium CDC 684 was found to contain both pXO1 and pXO2, was non-hemolytic, sensitive to gamma-phage, and produced both the protective antigen and the poly-Dglutamic acid capsule. Work by Pasteur and Greenfield [1,2] capitalized upon strains missing one of the mega-plasmids (pXO1), which resulted in attenuation This enabled the development of the first bacterial disease to be prevented through the use of an attenuated live vaccine. An isolate from the Centers for Disease Control (CDC) originally identified as B. megaterium, CDC 684/ NRRL-349S/NRS 234 ( called CDC 684), was being used as an avirulent outgroup control in experiments with B. anthracis [15] This particular isolate shares key phenotypic traits with B. anthracis such as non-hemolytic on blood agar, production of protective antigen and the poly-D-glutamic acid capsule, and sensitivity to gamma bacteriophage. The advent of massively parallel whole genome sequencing (WGS) provides an opportunity to examine the complete genetic component of CDC 684 for clues that might bear on this problem
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