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

Abstract

Bacillus anthracis, B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B. weihenstephanensis belong to the B. cereus group (B. cereus sensu lato). Bacillus anthracis is a causative agent of anthrax that primarily affects herbivores. In South Africa, anthrax occurs sporadic and is endemic or epidemic in the Northern Cape Province (NCP) and Kruger National Park (KNP). The Agricultural Research Council- Onderstepoort Veterinary (ARC-OVI) Institute is a national reference laboratory for diagnosis of anthrax. The ARC-OVI reference laboratory received isolates that were reported as anthrax cases and other Bacillus strains that were found to have caused anthrax-like symptoms, but showed inconclusive results with the standard B. anthracis biochemical tests. It was hypothesis that these isolates were unique strains of the B. cereus group, which previously could have been distinguished from B. cereus group using a few of the standard biochemical tests. However, the biochemical tests did not seem to be sensitive enough to give unequivocal results for these atypical B. anthracis strains that caused anthrax-like symptoms. Molecular based methods were therefore employed to further investigate the strains. The study therefore characterized B. anthracis and Bacillus species that were isolated during anthrax outbreaks in the Northern Cape, Limpopo, Mpumalanga Provinces and Lesotho, using a polyphasic approach that included phenotypic and molecular techniques. For this purpose, 3 B. anthracis and 10 Bacillus isolates were subjected to microbiology tests, Biolog OmniLog identification system (Biolog), 16S ribosomal RNA (rRNA) sequence analysis, PCR detection of protective antigen (pag) and capsule (cap) regions, real-time PCR using hybridization probes targeting the chromosome, pag and capC genes, and multi-loci variable number of tandem repeat (VNTR) analysis (MLVA). The classical microbiological tests indicated that the Bacillus species could be differentiated from the typical B. anthracis only by the use of gamma phage resistant and the morphology of B. anthracis. Phenotypic and genotypic, some of the strains showed the presence of capsules that might be important for virulence or environmental survival. Some of the Bacillus species produced weak PCR amplicons of the pagA (pXO1) and different capBCA (pXO2) regions. The Biolog OmniLog system and 16S rRNA gene sequencing identified the isolates as B. endophyticus, B. thuringiensis and Brevibacterium frigotoleransis with 16S rRNA sequencing being more reliable than Biolog Omnilog. The 16S rRNA sequencing identified the B. anthracis strains that clustered separately from the Bacillus species. The API 50 CHB system was rendered irrelevant for diagnosis of Bacillus species (B. anthracis-like) isolates in this study as it lacked correlation with the 16S rRNA sequences and Biolog Omnilog. The real-time PCR identified the B. anthracis strains. MLVA-26 differentiated the Bacillus species isolates from the typical B. anthracis. Some of the VNTR markers produced non-specific amplicons, which indicated differences in the genomes as the markers are specifically designed for B. anthracis. In this study we determined that B. anthracis can be differentiated from other Bacillus species using some of the confirmatory microbiological that includes gamma-phage sensitivity, 16S rRNA sequencing, real-time PCR and MLVA. Most of the Bacillus species isolates were identified by 16S rRNA sequencing and Biolog OmniLog as the endospore-forming B. endophyticus, which was first isolated from the inner tissue of healthy cotton plants and their role in anthrax-associated animal deaths could not be established. This was the first time that virulence genes of B. anthracis were detected in B. endophyticus bacteria that do not belong to the B. cereus group. However PCR analysis provided limited information about the genetic basis of these observations since it relies on predetermined, known genetic sequences. Whole genome sequencing was therefore considered to further investigate and resolve the variability within species and sub-species groups that are closely related amongst B. cereus/subtilis group. It was decided, as a first step, to analyse typical South African Bacillus anthracis strains using whole genomes sequence data and build an understanding of the genomic structure and level of diversity in these isolates. Bacillus anthracis strains (20SD and 3631-1C) were characterized by whole genome sequencing. Sequencing of the B. anthracis strains showed the presence of the chromosome and plasmids pXO1 and pXO2 replicons on strain 20SD, whereas isolate 3631-1C lacked plasmid pXO2. Genome comparison of the B. anthracis strains showed similar and highly conserved regions relative to B. anthracis Ames ancestor, with differences in the number of single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs). High proportion of SNPs were observed in the chromosome with few genetic rearrangements. Approximately 2% of the SNPs in both strains were found on the pXO1 and pXO2 plasmids when compared to B. anthracis Ames ancestor. Four-lambda prophages (the latent form of a bacteriophage in which the viral genes are incorporated into the bacterial chromosome) has been reported to be unique to B. anthracis. The presence of the four putative prophage regions in B. anthracis was investigated as these regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. In the study the prophages identified on the two strains were common to the B. anthracis Ames ancestor with slight variations in the sizes and number of prophages. B. anthracis 20SD and 3631-1C had phage minor proteins regions that are similar to the B. anthracis H901 and CDC strains. Sequence data was also compared to the only available South African B anthracis whole genome sequences of B. anthracis KrugerB and A0442 that also exhibited high number of prophages. The four-lambda prophages indicated to be characteristic of B. anthracis were shared amongst the B. anthracis strains i.e Ames ancestor, KrugerB, A0442, 3631-1C and 20SD. Results from this study suggested that regions that have capsid or mobile phage elements were likely to change the structural genomic complex of the strain resulting in inverted regions. The genomic complexity of strain 3631-1C was highly similar to B. anthracis Ames ancestor, while 20SD has a few genetic rearrangements of inversions. The results from this study agreed with and explained why prophages like conjugative transposons, insertion sequences, introns and other elements that make up a mobile portion of bacterial genomes often accounts for large-scale genomic rearrangements, insertion and deletions in bacterial chromosome. Overall the study showed the importance of using polyphasic approach to characterize the Bacillus isolates from B. anthracis and better diagnostic method should be developed to identify B. anthracis and Bacillus sepcies containing anthrax virulence genes. The study also provided the importance of using whole genome wide characterization for comparative genomics.