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

Abstract

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. 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. 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. Results: 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. Conclusions: 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.

Whole genome sequencing and identification of Bacillus endophyticus and B. anthracis isolated from anthrax outbreaks in South Africa

“…Meanwhile the overproduction of mac A and mac B results in an increase resistance to the macrolides antibiotics [ 19 ]. B. endophyticus is regarded as plant-endophytic bacterium that survives high-salt concentration [ 1 , 13 ]. The sigma-M predicted to response to high concentration of salt [ 20 ] was found in the 8 compared B. endophytcicus genomes in this study.…”

“…The presence of the PGA subunits pg s/ cap A-C in the South African B. endophyticus strains isolated alongside B. anthracis strains from the 2009 anthrax outbreak initiated the comparative investigation of these two species. B. endophyticus and B. anthracis can be differentiated based on sensitivity to the γ-phage [ 13 ], which is not a reliable differentiating character as resistance to γ-phage had been reported amongst the normally γ-phage sensitive B. anthracis [ 13 , 22 ] . In this study a more comprehensive approach that included morphology, biochemical as well as WGS were used to compare these two species in order to identify differentiating characteristics for diagnostic purposes.…”

“…The South African B. endophyticus strains were differentiated from B. anthracis based on γ-phage microbiological characteristics and real-time PCR, whereas 16S rRNA sequences and Omnilog identified the B. endophyticus strains [ 13 ]. However identification of the B. cereus sensu lato group using 16S rRNA gene sequencing is often challenging, since it has been regarded as a single taxon based on similar 16Sr RNA sequences [ 23 ].…”

“…The B. endophyticus strains that were isolated alongside B. anthracis strains had some of the similar morphological, biochemical and some genetic characteristics compared to the anthrax causing bacteria. In our previous study, conventional PCR detected PGA gene regions in both B. anthracis and B. endophyticus isolates and attempts were made to distinguish and identify these strains using routine and non-routine diagnostic methods [ 13 ]. The B. endophyticus strains were identified using non-routine diagnostic Omnilog (Biolog) and 16S rRNA sequencing methods and differentiated based on routine diagnostic microbiological tests and real time PCR.…”

Polyphasic Identification of Amylolytic Bacteria Producing Bioplastic Poly-β-hydroxybutyrate (PHB)

“…Identification of the three local amylolytic bacteria producing PHB with polyphasic approach based on characters of phenotypic, chemical (whole-cell protein profiles) and molecular (16S rRNA sequence) showed the congruence results. Several studies report that identification with the approach of systematics polyphasic using phenotypic, chemical and molecular characters can be used up to the species level (Lekota et al 2016;Mainville et al 2005;Randazzo et al 2009) the suitability of the results (congruence) of these three approaches, which establishes the identity of the bacterial strains in the classification system (Vandamme et al 1996).…”