Genomic sequence data and single nucleotide polymorphism genotyping of Bacillus anthracis strains isolated from animal anthrax outbreaks in Northern Cape Province, South Africa

This report presents genomic data on sequence reads and draft genomes of Bacillus anthracis isolates from anthrax outbreaks in animals in an endemic region of South Africa as well as genotyping of the strains using canonical single nucleotide polymorphisms (canSNPs). It is derived from an article entitle “Phylogenomic structure of B. anthracis strains in the Northern Cape Province, South Africa revealed novel single nucleotide polymorphisms”. Whole genome sequencing (WGS) of twenty-three B. anthracis strains isolated during 1998 and 2009 anthrax outbreaks in the Northern Cape Province (NCP), as well as a strain from Botswana (6102_6B) and one from Namibia-South Africa transfrontier conservation area (Sendlingsdrift, 6461_SP2) were obtained using both the HiSeq 2500 and MiSeq Illumina platforms. Mismatch amplification mutation assay (melt-MAMA) qPCR were used to identify the canSNP genotypes within the global population of B. anthracis. DNA sequencing data is available at NCBI Sequence Read Archive and GenBank database under accession N0. PRJNA580142 and PRJNA510736 respectively. A phylogenetic tree and CanSNP typing profiles of the isolates are presented within this article.The Institute of Tropical Medicine (ITM), Belgium, National Research Foundation and AgriSETA (Agriculture Sector Education Training Authority) in South Africa.http://www.elsevier.com/locate/dibam2020Veterinary Tropical Disease

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

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.The National Research Foundation (NRF) and NRF-THRIP.http://www.jidc.orgam2017Veterinary Tropical Disease

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

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.Dissertation (MSc)--University of Pretoria, 2013.Veterinary Tropical DiseasesMScUnrestricte

Comparative Genomics Revealed a Potential Threat of <i>Aeromonas rivipollensis</i> G87 Strain and Its Antibiotic Resistance

Aeromonas rivipollensis is an emerging pathogen linked to a broad range of infections in humans. Due to the inability to accurately differentiate Aeromonas species using conventional techniques, in-depth comparative genomics analysis is imperative to identify them. This study characterized 4 A. rivipollensis strains that were isolated from river water in Johannesburg, South Africa, by whole-genome sequencing (WGS). WGS was carried out, and taxonomic classification was employed to profile virulence and antibiotic resistance (AR). The AR profiles of the A. rivipollensis genomes consisted of betalactams and cephalosporin-resistance genes, while the tetracycline-resistance gene (tetE) was only determined to be in the G87 strain. A mobile genetic element (MGE), transposons TnC, was determined to be in this strain that mediates tetracycline resistance MFS efflux tetE. A pangenomic investigation revealed the G87 strain’s unique characteristic, which included immunoglobulin A-binding proteins, extracellular polysialic acid, and exogenous sialic acid as virulence factors. The identified polysialic acid and sialic acid genes can be associated with antiphagocytic and antibactericidal properties, respectively. MGEs such as transposases introduce virulence and AR genes in the A. rivipollensis G87 genome. This study showed that A. rivipollensis is generally resistant to a class of beta-lactams and cephalosporins. MGEs pose a challenge in some of the Aeromonas species strains and are subjected to antibiotics resistance and the acquisition of virulence genes in the ecosystem

Phylogenomic structure of Bacillus anthracis isolates in the Northern Cape Province, South Africa revealed novel single nucleotide polymorphisms

Bacillus anthracis, the aetiological agent of anthrax, is regarded as a highly monomorphic pathogen that presents a low genetic diversity using standard molecular techniques. Whole genome sequencing and single nucleotide polymorphisms (SNPs) are definitive signatures for subtyping of B. anthracis. Here we employed whole genome single nucleotide polymorphism (wgSNP) analysis to investigate the genetic diversity of B. anthracis in the historically endemic region of Northern Cape Province (NCP), South Africa. Twenty-six isolates from anthrax outbreaks that occurred between 1998 and 2008/9 in NCP as well as from Namibia-South Africa Transfontier Conservation area and Botswana were compared to global B. anthracis genomes. Most NCP B. anthracis strains (n = 22) clustered in the A.Br.003/004 (A.Br.101) branch and are closely related to the Zimbabwe and Mozambique strains (A.Br.102 branch). A total of 4923 parsimony informative-SNPs accurately established the A.Br.003/004 phylogenetic relationships of the NCP isolates into two distinct sub-clades and SNP markers designated as A.Br.172 and A.Br.173 were developed. Other NCP strains (n = 2) grouped in the A.Br.001/002 (Sterne) branch while strains (n = 2) from the Namibia-South Africa Transfontier Conservation area and Botswana clustered in A.Br.005/006 (Ancient A) branch. The sequenced B. anthracis strains (A0094, A0096 and A0097) that clustered in the A.Br.064 (V770) clade were isolated from Vaalbos National Park and similar strains have not been isolated. The B. anthracis A0088 strain cluster with the NCP strains in the A.Br.003/004 (A.Br.172) SNP branch which has been isolated in NCP, South Africa. This study highlights the phylogenetic structure of NCP B. anthracis strains with distinctive SNP branches important for forensic tracing and novel SNP discovery purposes. The sequenced strains will serve as a means to further trace the dissemination of B. anthracis outbreaks in NCP, South Africa, and on the continent, as well as for forensic tracking on a global scale.The National Research Foundation (NRF), Institute of Tropical Medicine (ITM), Belgium, and Agriculture Sector Education Training Authority (AgriSETA).http://www.elsevier.com/locate/meegidhj2021BiochemistryGeneticsMicrobiology and Plant PathologyVeterinary Tropical Disease

A unique isolation of a lytic bacteriophage infected Bacillus anthracisIsolate from Pafuri, South Africa

Bacillus anthracis is a soil-borne, Gram-positive endospore-forming bacterium and the causative agent of anthrax. It is enzootic in Pafuri, Kruger National Park in South Africa. The bacterium is amplified in a wild ungulate host, which then becomes a source of infection to the next host upon its death. The exact mechanisms involving the onset (index case) and termination of an outbreak are poorly understood, in part due to a paucity of information about the soil-based component of the bacterium’s lifecycle. In this study, we present the unique isolation of a dsDNA bacteriophage from a wildebeest carcass site suspected of having succumbed to anthrax. The aggressively lytic bacteriophage hampered the initial isolation of B. anthracis from samples collected at the carcass site. Classic bacteriologic methods were used to test the isolated phage on B. anthracis under different conditions to simulate deteriorating carcass conditions. Whole genome sequencing was employed to determine the relationship between the bacterium isolated on site and the bacteriophage-dubbed Bacillus phage Crookii. The 154,012 bp phage belongs to Myoviridae and groups closely with another African anthrax carcass-associated Bacillus phage WPh. Bacillus phage Crookii was lytic against B. cereus sensu lato group members but demonstrated a greater affinity for encapsulated B. anthracis at lower concentrations (<1 × 108 pfu) of bacteriophage. The unusual isolation of this bacteriophage demonstrates the phage’s role in decreasing the inoculum in the environment and impact on the life cycle of B. anthracis at a carcass sit

Molecular characterization Brucella species from Zimbabwe

Brucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world.S1 Table. Reference strains and Zimbabwean Brucella spp. isolates identified by Bruce-ladder and repeat copy number of the indicated loci. https://doi.org/10.1371/journal.pntd.0007311.s001S2 Table. Brucella abortus and B. suis genome sequences retrieved from GenBank, used in the study for comparison of whole genome single nucleotide polymorphisms (WGS-SNPS) phylogenetic analysis. https://doi.org/10.1371/journal.pntd.0007311.s002S1 Fig. Suis-ladder multiplex PCR assay of Brucella DNA from Zimbabwe and reference strains. https://doi.org/10.1371/journal.pntd.0007311.s003S2 Fig. Minimum spanning tree analysis of published data and Zimbabwean Brucella isolates using the MLVA8 data (Panel 1 genotypes). https://doi.org/10.1371/journal.pntd.0007311.s004The ITM Belgium and National Research Foundation (NRF South Africa). Work by PLF and GV is supported by the ANR project Microtype-14-ASMA-0002-02.http://www.plosntds.orghj2020Veterinary Tropical Disease

Molecular characterization of Brucella species from Zimbabwe

International audienceBrucella abortus and B. melitensis have been reported in several studies in animals in Zimbabwe but the extent of the disease remains poorly known. Thus, characterizing the circulating strains is a critical first step in understanding brucellosis in the country. In this study we used an array of molecular assays including AMOS-PCR, Bruce-ladder, multiple locus variable number tandem repeats analysis (MLVA) and single nucleotide polymorphisms from whole genome sequencing (WGS-SNP) to characterize Brucella isolates to the species, biovar, and individual strain level. Sixteen Brucella strains isolated in Zimbabwe at the Central Veterinary laboratory from various hosts were characterized using all or some of these assays. The strains were identified as B. ovis, B. abortus, B. canis and B. suis, with B. canis being the first report of this species in Zimbabwe. Zimbabwean strains identified as B. suis and B. abortus were further characterized with whole genome sequencing and were closely related to reference strains 1330 and 86/8/59, respectively. We demonstrate the range of different tests that can be performed from simple assays that can be run in laboratories lacking sophisticated instrumentation to whole genome analyses that currently require substantial expertise and infrastructure often not available in the developing world