Polyphasic characterization of Bacillus species from anthrax outbreaks in animals from South Africa and Lesotho.

Kgaugelo Edward Lekota,Jasper Rees,Joseph Mafofo,Farai Catherine Muchadeyi,Evelyn Madoroba,Henriette Van Heerden,Ayesha Hassim

doi:10.3855/jidc.7798

Abstract

Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Accurate identification of virulent B. anthracis is essential but challenging due to its close relationship with other members of B. cereus group. This study characterized B. anthracis and Bacillus species that were recovered from animals and the environment where animals died of anthrax symptoms in southern Africa using a polyphasic approach. For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. The Bacillus isolates were non-hemolytic, non-motile, and susceptible to penicillin, which is typical of B. anthracis, but resistant to gamma phage, unlike typical B. anthracis. The Biolog system and 16S rRNA gene sequence analysis identified most of the Bacillus isolates as B. endophyticus (7 of 10). Conventional PCR revealed that most of the Bacillus isolates contained capBCA gene regions. This highlights the limitation of the specificity of conventional PCR and the fact that the real-time PCR is more specific and reliable for anthrax diagnosis. Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Identification of B. anthracis should be done using a polyphasic approach.

Highlights

Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa
The colonies of Bacillus isolates were typically Gram-positive rods showing slightly variable thickness, non-motile, sensitive to penicillin like classic B. anthracis, but in contrast to the classic anthrax bacteria, the Bacillus bacteria were not lysed by gamma phage
The BiologOmnilog system and 16S ribosomal RNA (rRNA) gene sequencing could identify most of the isolates as B. endophyticus

Summary

Introduction

Bacillus anthracis is the causative agent of anthrax, a disease endemic in regions of Northern Cape Province and Kruger National Park of South Africa. Methodology: For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, BiologOmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, polymerase chain reaction (PCR) detection of protective antigen (pag) and capsule (cap) regions, and real-time PCR using hybridization probes targeting chromosomal, pag, and capC genes. Conclusions: Real-time PCR, 16S rRNA sequencing, and confirmatory microbiology tests including phage resistance distinguished Bacillus isolates from B. anthracis in this study. Bacillus cereus sensu lato group comprises six members, namely B. cereus, B. anthracis, B. thuringiensis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis. The plasmid-encoding toxin and capsule proteins are encoded by pXO1 and pXO2, which have been found in other members of B. cereus sensu lato and are restricted to B. anthracis as previously thought [5,6]. The capsule encoded by pXO2 consists of a five-gene operon (capBCADE) that synthesizes the poly-γ-D-glutamic acid capsule of Bacillus species, which enable host

Results

Discussion

Conclusion