Kgaugelo E. Lekota | Geology Reports

USGS2025

Comparative genomics of Bacillus anthracis A and B-clades reveals genetic variation in genes responsible for spore germination

Bacillus anthracis , the causative agent of anthrax, is composed of three genetic clades (A, B, and C). Clade-A is the most common and distributed worldwide, B-clade has a narrow geographic distribution, and C-clade is rare. South Africa's Kruger National Park (KNP) has high diversity of B. anthracis , with strains from A and B clades described from its northernmost region, Pafuri. We employed whole genome sequencing to investigate the genomic diversity of B. anthracis strains isolated from animal carcasses ( n = 34) during the 2012–2015 outbreaks. Whole-genome single-nucleotide polymorphism (wgSNP) analysis assigned the 2012–2015 B. anthracis genomes to the A-clade branch, distributed across the branch's two minor sub-clades A.Br.005/006. Additionally, pan-genomic analysis distinguished the A- and B-clade genomes, identifying unique accessory genes. Notable genetic differences include the biosynthetic spore cell wall genes; long-chain fatty acid CoA ligases ( FaD13 ), Bacillus collagen-like protein of anthracis (BclA) involved in the exosporium germination, as well as a truncated murein DD-endopeptidase ( mepH ) found in the pXO2 plasmid of the B-clade strains. The tryptophan synthase subunit alpha gene ( trpA ), which results in a pseudogene in B-clade genomes separates the A- and B-clade genomes. These differences in biosynthetic cell wall genes suggest variation in adaptability or cell growth of the B-clade strains in the environment, further influenced by the truncation of the trpA gene involved in spore germination. The A.Br.005/006-clade strains in KNP exhibit higher genetic diversity, which may enhance their resilience to environmental stressors. In contrast, the KNP B-clade (B.Br.001/002) strains show limited genetic variation, potentially reducing their adaptability. This pattern is evident through whole-genome SNP analysis and pan-genomics investigating the evolution of B. anthracis .

Kruger National Park↗

USGS2024

Comparing microbiological and molecular diagnostic tools for the surveillance of anthrax

The diagnosis of anthrax, a zoonotic disease caused by Bacillus anthracis can be complicated by detection of closely related species. Conventional diagnosis of anthrax involves microscopy, culture identification of bacterial colonies and molecular detection. Genetic markers used are often virulence gene targets such as B. anthracis protective antigen ( pagA , also called BAPA, occurring on plasmid pXO1), lethal factor ( lef , on pXO1), capsule-encoding capB/C (located on pXO2) as well as chromosomal Ba-1. Combinations of genetic markers using real-time/quantitative polymerase chain reaction (qPCR) are used to confirm B . anthracis from culture but can also be used directly on diagnostic samples to avoid propagation and its associated biorisks and for faster identification. We investigated how the presence of closely related species could complicate anthrax diagnoses with and without culture to standardise the use of genetic markers using qPCR for accurate anthrax diagnosis. Using blood smears from 2012–2020 from wildlife mortalities (n = 1708) in Kruger National Park in South Africa where anthrax is endemic, we contrasted anthrax diagnostic results based on qPCR, microscopy, and culture. From smears, 113/1708 grew bacteria in culture, from which 506 isolates were obtained. Of these isolates, only 24.7% (125 isolates) were positive for B . anthracis based on genetic markers or microscopy. However, among these, merely 4/125 (3.2%) were confirmed B . anthracis isolates (based on morphology, microscopy, and sensitivity testing to penicillin and gamma-phage) from the blood smear, likely due to poor survival of spores on stored smears. This study identified B . cereus sensu lato , which included B . cereus and B . anthracis , Peribacillus spp., and Priestia spp. clusters using gyrB gene in selected bacterial isolates positive for pagA region using BAPA probe. Using qPCR on blood smears, 52.1% (890 samples) tested positive for B . anthracis based on one or a combination of genetic markers which included the 25 positive controls. Notably, the standard lef primer set displayed the lowest specificity and accuracy. The Ba-1+BAPA+ lef combination showed 100% specificity, sensitivity, and accuracy. Various marker combinations, such as Ba-1+ capB , BAPA+ capB , Ba-1+BAPA+ capB + lef , and BAPA+ lef + capB , all demonstrated 100.0% specificity and 98.7% accuracy, while maintaining a sensitivity of 96.6%. Using Ba-1+BAPA+ lef + capB , as well as Ba-1+BAPA+ lef with molecular diagnosis accurately detects B . anthracis in the absence of bacterial culture. Systematically combining microscopy and molecular markers holds promise for notably reducing false positives. This significantly enhances the detection and surveillance of diseases like anthrax in southern Africa and beyond and reduces the need for propagation of the bacteria in culture.

PLoS Neglected Tropical Diseases↗