First PCR Confirmed anthrax outbreaks in Ethiopia-Amhara region, 2018-2019.

BACKGROUND: Anthrax is a disease that affects humans and animals. In Ethiopia, anthrax is a reportable disease and assumed to be endemic, although laboratory confirmation has not been routinely performed until recently. We describe the findings from the investigation of two outbreaks in Amhara region. METHODS: Following reports of suspected outbreaks in Wag Hamra zone (Outbreak 1) and South Gondar zone (Outbreak 2), multi-sectoral teams involving both animal and public health officials were deployed to investigate and establish control programs. A suspect case was defined as: sudden death with rapid bloating or bleeding from orifice(s) with unclotted blood (animals); and signs compatible with cutaneous, ingestion, or inhalation anthrax ≤7 days after exposure to a suspect animal (humans). Suspect human cases were interviewed using a standard questionnaire. Samples were collected from humans with suspected anthrax (Outbreak 1 and Outbreak 2) as well as dried meat of suspect animal cases (Outbreak 2). A case was confirmed if a positive test was returned using real-time polymerase chain reaction (qPCR). RESULTS: In Outbreak 1, a total of 49 cows died due to suspected anthrax and 22 humans developed symptoms consistent with cutaneous anthrax (40% attack rate), two of whom died due to suspected ingestion anthrax. Three people were confirmed to have anthrax by qPCR. In Outbreak 2, anthrax was suspected to have caused the deaths of two livestock animals and one human. Subsequent investigation revealed 18 suspected cases of cutaneous anthrax in humans (27% attack rate). None of the 12 human samples collected tested positive, however, a swab taken from the dried meat of one animal case (goat) was positive by qPCR. CONCLUSION: We report the first qPCR-confirmed outbreaks of anthrax in Ethiopia. Both outbreaks were controlled through active case finding, carcass management, ring vaccination of livestock, training of health professionals and outreach with livestock owners. Human and animal health authorities should work together using a One Health approach to improve case reporting and vaccine coverage.

Phylogenetics of Bacillus anthracis isolates from Russia and bordering countries.

Anthrax is a concern for public health and veterinary medicine in Russia. The available phylogenetic data on isolates from Russia and neighboring CIS countries are clearly not enough to gain a better understanding of their position in the global phylogenetic population structure of this pathogen. In this study, we analyzed the genomes of 66 Bacillus anthracis strains, which were isolated between 1935 and 2019 from different sources in Russia, as well as in Ukraine, Azerbaijan, Georgia, Armenia and Moldova. Whole genome SNP analysis of genomes of 66 strains obtained in this study along with 222 B. anthracis genomes available in the GenBank database revealed 7242 SNPs used to construct a phylogenetic reconstruction with the method of Maximum Likelihood. Studied strains belong to 6 different genetic groups: A.Br.008(A.Br.008/009), A.Br.081(Ames), A.Br.014(A.Br.Aust94), A.Br.082(A.Br.001/002), A.Br.034(A.Br.005/006, Ancient A) and B.Br.002 (B.Br.001/002). Within the group A.Br.014(A.Br.Aust94) a subcluster A.Br.029 of strains isolated in Georgia, Armenia, Azerbaijan, Russia (Republic of Dagestan) and Turkey, named Caucasus-East Anatolia (CEA), was identified. In the subgroup A.Br.105(Tsiankovskii) the cluster A.Br.117 of strains from Russia, Ukraine and Slovakia are assigned, in the subgroup A.Br118 (STI) - cluster A.Br.123 with strains from Russia and Georgia and cluster A.Br.125 with strains from Republic of Dagestan. New subclusters B.Br.017("EUROPE") were identified in the B.Br.002(B.Br.001/002) cluster, represented by strains from the European part of Russia, as well as from South Korea and Finland. For 8 clusters and subclusters, the SNP markers were identified. The study confirmed a significant genetic diversity of the strains isolated in Russia and border countries and clarified their position in the phylogenetic structure of the global B. anthracis population. New genetic clusters A.Br.029 (CEA), A.Br.117, A.Br.123, A.Br.125, and B.Br.017 («EUROPE¼) were defined. 96 marker SNPs specific for these clusters were identified.

Molecular genotyping of 15 B. anthracis strains isolated in Eastern Siberia and Far East.

Bacillus anthracis is a pathogenic bacterium, which causes anthrax disease. The ability of this bacterium to form spores, which can be preserved in soil for decades and cause outbreaks later on, makes this pathogen a serious problem for veterinary and health services of many countries. Siberia is one of the most anthrax-influenced regions of Russia. In this research we report on the results of genotyping based on whole genome SNP analysis of 15 strains, isolated on the territory of Eastern Siberia and the Far East in 1956-2018. In this research, we sequenced 15 genomes of B. anthracis strains isolated from infected humans and animals, and from soil samples from the territory of Eastern Siberia and the Far East in the period from 1956 to 2018. We used genomic sequences obtained in this study and 219 B. anthracis genomes available in the international GenBank database to perform a comparative analysis. As a result we detected 6400 chromosomal SNPs which allowed to differentiate the studied strains. We built phylogenetic reconstruction of the global B. anthracis population based on the detected SNPs using the Maximum Likelihood Method and described genetic diversity of the strains isolated on the territory of Eastern Siberia and the Far East. Strains, isolated on this territory from 1956 to 2018 belong to 5 different genetic groups: "Ames", "STI", "Tsiankovskii", "Siberia" and "Asia". The greatest diversity of the strains is registered for two regions of the southern part of Eastern Siberia - Tyva and Buryatia. This research expands current understanding of genetic diversity of B. anthracis strains circulating on the territory of Russia.

Molecular characterization of B. anthracis isolates from the anthrax outbreak among cattle in Karnataka, India.

BACKGROUND: Anthrax, a zoonotic disease is caused by the Gram positive bacterium Bacillus anthracis. During January 2013, an anthrax outbreak among cattle was reported in Gundlupet Taluk, neighboring Bandipur National Park and tiger reserve, India. The present study aims at the molecular identification and characterization of 12 B. anthracis isolates from this outbreak by 16S rRNA gene sequencing, screening B. anthracis specific prophages and chromosomal markers, protective antigen (pag) gene and canonical single nucleotide polymorphism (canSNP) analysis to subtype the isolates into one of the twelve globally identified clonal sub-lineages of B. anthracis. RESULTS: These isolates had identical 16S rDNA nucleotide sequences with B. anthracis specific dual peaks showing mixed base pair R (G/A) at position 1139 with visual inspection while the automated basecaller software indicated a G. Alternatively the nucleotide A at 1146 position was indicative of the 16S rDNA type 7. Multiple sequence alignment with additional 170 (16S rDNA) sequences of B. cereus sensu lato group from GenBank database revealed 28 new 16S types in addition to eleven 16S types reported earlier. The twelve B. anthracis isolates were found to harbor the four B. anthracis specific prophages (lambdaBa01, lambdaBa02, lambdaBa03, and lambdaBa04) along with its four specific loci markers (dhp 61.183, dhp 77.002, dhp 73.019, and dhp 73.017). The pag gene sequencing identified the isolates as protective antigen (PA) genotype I with phenylalanine-proline-alanine phenotype (FPA phenotype). However, sequence clustering with additional 34 pag sequences from GenBank revealed two additional missense mutations at nucleotide positions 196 bp and 869 bp of the 2294 bp pag sequence among the 5 B. cereus strains with pXO1 like plasmids. The canSNP analysis showed that the isolates belong to A.Br.Aust94 sub-lineage that is distributed geographically in countries of Asia, Africa, Europe and Australia. CONCLUSIONS: The analysis of 16S rDNA sequences reiterated the earlier findings that visual inspection of electropherogram for position 1139 having nucleotide R could be used for B. anthracis identification and not the consensus sequence from base caller. The canSNP results indicated that the anthrax outbreak among cattle was caused by B. anthracis of A.Br.Aust94 sub-lineage.

Characterization of Ba813 harbouring Bacillus cereus in patients with haematological malignancy and hospital environments at a medical centre in Japan.

Introduction. Bacillus cereus harbouring Ba813, a specific chromosomal marker of Bacillus anthtacis, is found in patients with severe manifestations and causes nosocomial outbreaks.Aim. We assessed the genetic characteristics and virulence of Ba813(+) B. cereus in a hospital setting.Methodology. Three neutropenic patients with haematological malignancy developed B. cereus bacteraemia within a short period. Fifteen B. cereus were isolated from different sites in a haematology ward. A total of 18 isolates were evaluated for Ba813- and B. anthracis-related virulence, food poisoning-related virulence, genetic diversity, bacteria motility and biofilm formation.Results. Ba813(+) B. cereus was detected in 33 % (1/3) of patients and 66 % (9/15) of the hospital environment. The 18 strains were divided into 2 major clusters (clade 1 and clade 2), and 14 strains were classified into clade 1. All Ba813(+) strains, including four sequence types, were classified into clade 1/the cereus III lineage, which is most closely related to the anthracis lineage. Two strains belonging to clade 1/non-cereus III carried the B. anthracis-associated cap gene, but not Ba813. B. cereus, including Ba813(+) strains, had significantly lower prevalence of enterotoxin genes than clade 2 strains. In clade 1, B. cereus, Ba813(+) strains showed significantly higher swimming motility and biofilm formation ability than Ba813(-) strains.Conclusion. Ba813(+) B. cereus, which are genetically closely related to B. anthracis, were abundant in a haematological ward. Ba813(+) B. cereus with high motility and biofilm formation abilities may spread easily in hospital environments, and could become a hospital-acquired infection.

Genotyping and population diversity of Bacillus anthracis in China based on MLVA and canSNP analysis.

In pastoral parts of China, anthrax still presents a major risk to livestock and threatens the health of local human populations. Currently, whole-genome-based molecular markers, such as single-nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs), are the most effective tools for genotyping Bacillus anthracis. In this study, 191 isolates were selected to assess the diversity of B. anthracis in China. Five isolates were confirmed not to be B. anthracis by clustered regularly interspaced short palindromic repeat analysis, while the remaining 186 isolates were typed using canonical SNP (canSNP) and VNTR analyses. Five sublineages/subgroups, A.Br.001/002, A.Br.Vollum, A.Br.Aust.94, A.Br.Ames, and A.Br.008/009, were detected based on 13 canSNP sites. The 186 isolates were further assigned 114 sequence types based on 27 VNTR loci, with major branches correlating with the canSNP analysis. We then used a simplified multiple-locus variable number tandem repeat analysis (MLVA) protocol (MLVAmin) based on eight high-resolution VNTR sites to analyze the Chinese isolates, with the resulting phylogeny again agreeing with the canSNP analysis. We also developed two schemes, MLVAc and MLVAp, using various numbers of VNTRs to analyze different canSNP sublineages to increase the typing resolution of the canSNP protocol. The results showed a highly imbalanced geographical distribution of the B. anthracis population, with four different sublineages observed in Xinjiang Province, while only one sublineage, A.Br.001/002, was found in the other six provinces, except for three A.Br.Ames strains isolated from Inner Mongolia. Based on the MLVA and canSNP analysis, the spread of B. anthracis appears to have occurred from west to east via three independent routes.

Genetic characterization of Bacillus anthracis strains circulating in Italy from 1972 to 2018.

In Italy anthrax is an endemic disease, with a few outbreaks occurring almost every year. We surveyed 234 B. anthracis strains from animals (n = 196), humans (n = 3) and the environment (n = 35) isolated during Italian outbreaks in the years 1972-2018. Despite the considerable genetic homogeneity of B. anthracis, the strains were effectively differentiated using canonical single nucleotide polymorphisms (CanSNPs) assay and multiple-locus variable-number tandem repeat analysis (MLVA). The phylogenetic identity was determined through the characterization of 14 CanSNPs. In addition, a subsequent 31-loci MLVA assay was also used to further discriminate B. anthracis genotypes into subgroups. The analysis of 14 CanSNPs allowed for the identification of four main lineages: A.Br.011/009, A.Br.008/011 (respectively belonging to A.Br.008/009 sublineage, also known Trans-Eurasian or TEA group), A.Br.005/006 and B.Br.CNEVA. A.Br.011/009, the most common subgroup of lineage A, is the major genotype of B. anthracis in Italy. The MLVA analysis revealed the presence of 55 different genotypes in Italy. Most of the genotypes are genetically very similar, supporting the hypothesis that all strains evolved from a local common ancestral strain, except for two genotypes representing the branch A.Br.005/006 and B.Br.CNEVA. The genotyping analysis applied in this study remains a very valuable tool for studying the diversity, evolution, and molecular epidemiology of B. anthracis.

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.

Phylogenetic analysis of Bacillus anthracis strains from Western Siberia reveals a new genetic cluster in the global population of the species.

BACKGROUND: Anthrax is a zoonotic disease caused by the gram-positive bacterium Bacillus anthracis. The most anthrax-endemic regions of Russia are Siberia and North Caucasus. Previously, genotyping of Russian B.anthracis isolates was carried out using canSNP and MLVA data; these methods yield lower resolution results compared to whole genome SNP analysis (wgSNP). In this research, we have used wgSNP method for genotyping of 10 B.anthracis isolates, obtained during 1961-2016 in Russia on territory of Western Siberia. RESULTS: We have analyzed 185 B.anthracis genomes available in GenBank database and genomes of 10 isolates obtained in this study to determine the place of Russian isolates in the global phylogeny of B.anthracis. For the studied genomes we have detected 7203 SNPs, which were used for building a phylogenetic reconstruction with Maximum Likelihood Method. Results of the phylogenetic analysis indicate that Russian strains belong to three different genetic groups. Three strains belong to genetic group "Ames", two strains - to "STI" group. Five strains belong to the main genetic line B, and four of them form a subcluster, described for the first time, which we have named "Siberia". CONCLUSIONS: In this study, the data on genetic diversity of B.anthracis strains on the territory of Western Siberia is presented for the first time. As a result of complex phylogenetic analysis, the place of these isolates was determined in the global phylogenetic structure of the B.anthracis population. We describe a new cluster in the main genetic line B for the first time.

An unusual case of bovine anthrax in the canton of Jura, Switzerland in 2017.

BACKGROUND: Anthrax caused by Bacillus anthracis is a zoonotic disease mainly affecting herbivores. The last Swiss outbreak was over 20 years ago. We describe a recent anthrax outbreak involving two cows from the same herd. One cow was designated as a peracute clinical case with sudden death and typical lung lesions, while the other cow presented with protracted fever and abortion. CASE PRESENTATION: On April 29th 2017, a 3.5-year-old Montbéliard dairy cow was found dead while out at pasture with haemorrhage from the nose. The veterinarian suspected pneumonia and performed a necropsy on site. Subsequently, a lung and liver sample were sent to the laboratory. Unexpectedly, Bacillus anthracis was isolated, a pathogen not found in Switzerland for decades. Several days later, a second cow from the same farm showed signs of abortion after protracted fever. Since these symptoms are not typical for anthrax, and the bacteria could not be demonstrated in blood samples from this animal, a necropsy was performed under appropriate biosafety measures. Subsequently, Bacillus anthracis could be isolated from the placenta and the sublumbal lymph nodes but not from the blood, liver, spleen and kidney. The outbreak strain (17OD930) was shown to belong to the lineage B.Br.CNEVA, the same as Swiss strains from previous outbreaks in the region. We speculate that the disease came from a temporarily opened cave system that is connected to an old carcass burial site and was flushed by heavy rainfall preceding the outbreak. CONCLUSION: Even in countries like Switzerland, where anthrax is very rare, new cases can occur after unusual weather conditions or ground disturbance. It is important for public officials to be aware of this risk to avoid possible spread.

Insights from Bacillus anthracis strains isolated from permafrost in the tundra zone of Russia.

This article describes Bacillus anthracis strains isolated during an outbreak of anthrax on the Yamal Peninsula in the summer of 2016 and independently in Yakutia in 2015. A common feature of these strains is their conservation in permafrost, from which they were extracted either due to the thawing of permafrost (Yamal strains) or as the result of paleontological excavations (Yakut strains). All strains isolated on the Yamal share an identical genotype belonging to lineage B.Br.001/002, pointing to a common source of infection in a territory over 250 km in length. In contrast, during the excavations in Yakutia, three genetically different strains were recovered from a single pit. One strain belongs to B.Br.001/002, and whole genome sequence analysis showed that it is most closely related to the Yamal strains in spite of the remoteness of Yamal from Yakutia. The two other strains contribute to two different branches of A.Br.008/011, one of the remarkable polytomies described so far in the B. anthracis species. The geographic distribution of the strains belonging to A.Br.008/011 is suggesting that the polytomy emerged in the thirteenth century, in combination with the constitution of a unified Mongol empire extending from China to Eastern Europe. We propose an evolutionary model for B. anthracis recent evolution in which the B lineage spread throughout Eurasia and was subsequently replaced by the A lineage except in some geographically isolated areas.

The Bacillus cereus Group: Bacillus Species with Pathogenic Potential.

The Bacillus cereus group includes several Bacillus species with closely related phylogeny. The most well-studied members of the group, B. anthracis, B. cereus, and B. thuringiensis, are known for their pathogenic potential. Here, we present the historical rationale for speciation and discuss shared and unique features of these bacteria. Aspects of cell morphology and physiology, and genome sequence similarity and gene synteny support close evolutionary relationships for these three species. For many strains, distinct differences in virulence factor synthesis provide facile means for species assignment. B. anthracis is the causative agent of anthrax. Some B. cereus strains are commonly recognized as food poisoning agents, but strains can also cause localized wound and eye infections as well as systemic disease. Certain B. thuringiensis strains are entomopathogens and have been commercialized for use as biopesticides, while some strains have been reported to cause infection in immunocompromised individuals. In this article we compare and contrast B. anthracis, B. cereus, and B. thuringiensis, including ecology, cell structure and development, virulence attributes, gene regulation and genetic exchange systems, and experimental models of disease.

Comparative genomics and phylogenetic analysis of Bacillus anthracis strains isolated from domestic animals in Japan.

Anthrax, caused by Bacillus anthracis, is a severe zoonosis with a great impact on both human and animal health. In the present study, we identified the phylogenetic relationships among 16 Japanese strains of B. anthracis, including eight bovine strains, two equine strains, five swine strains, and one former vaccine strain, using in silico canonical single nucleotide polymorphism (canSNP) and core genome SNP analyses. The results of our in silico canSNP analysis suggest that these 16 Japanese strains could be divided into four lineages: i) one equine strain in A.Br.Ames, ii) one equine and six bovine strains in A.Br.001/002, iii) five swine and one bovine strain in A.Br.Aust94, and iv) one bovine and one vaccine strain in A.Br.008/011. A comparison with non-Japanese B. anthracis strains revealed a total of 3787 SNPs identified from the whole genome sequences of the Japanese strains; these SNP data were subjected to a phylogenetic analysis using the maximum parsimony (MP) method. Our core genome SNP analysis was also able to detect differences of a few chromosomal SNPs across clonal strains from the same cases that had different storage and passage histories. Additionally, our whole genome SNP analysis clearly indicated that the Japanese swine anthrax cases of 1982 were caused by at least three independent strains; however, their phylogeny revealed no clear relationship with swine strains from other countries. The bovine strain belonging to the A.Br.008/011 lineage differed from a former Japanese vaccine strain by only 12 SNPs. Together with the phylogenic results and epidemiological circumstances, the diversity of strains reveals that the B. anthracis available in Japan probably resulted from multiple relatively recent import events, rather than reflecting the persistence of a more ancient ecologically established group.

Genetic characteristics of Bacillus anthracis isolated from northwestern China from 1990 to 2016.

Anthrax is a global re-emerging zoonotic disease and is an endemic disease in China, especially in rural regions. In this study, the general characteristics of human anthrax outbreaks that occurred in areas of northwestern China over the past decade have been described. Meanwhile, the genetic characteristics of Bacillus anthracis isolated from these areas from 1990 to 2016 were analyzed by means of canonical single-nucleotide polymorphism (canSNP) analysis and multilocus variable-number tandem repeat analysis (MLVA) with 15 markers. Five sublineages/subgroups, namely, A.Br.001/002, A.Br.Vollum, A.Br.Aust94, A.Br.Ames and A.Br.008/009, were detected by using 13 canSNP sites. All of the sublineages were found in Xinjiang province, while one sublineage was found in Shaanxi, two in Gansu, three in Qinghai and four in Inner Mongolia. However, the geographical distribution of the B. anthracis populations exhibited different canSNP characteristics from those of the strains isolated before 1990 in China. In contrast to previous data, the A.Br.Ames subgroup was also observed to be scattered from Inner Mongolia to other provinces. All 106 strains were assigned to 36 MLVA15 genotypes, and 21 of these types were first observed in this study. The strains collected from anthrax outbreaks in recent decade were classified as subgroups A.Br.001/002 and A.Br.Ames and identified as genotypes MLVA15-28, MLVA15-30, MLVA15-31, MLVA15-38, MLVA15-CHN3, and MLVA15-CHN18. By canSNP analysis and MLVA, we found that the diversification of MLVA genotypes and the geographical distribution of B. anthracis populations is gradually becoming balanced across northwestern China. This study also provides preliminary survey results regarding the population diversity of B. anthracis in China, which will help promote the prevention and control of this important disease.

Genotyping and phylogenetic placement of Bacillus anthracis isolates from Finland, a country with rare anthrax cases.

BACKGROUND: Anthrax, the zoonotic disease caused by the gram-positive bacterium Bacillus anthracis, is nowadays rare in northern parts of Europe including Finland and Scandinavia. Only two minor outbreaks of anthrax in 1988 and in 2004 and one sporadic infection in 2008 have been detected in animals in Finland since the 1970's. Here, we report on two Finnish B. anthracis strains that were isolated from spleen and liver of a diseased calf related to the outbreak in 1988 (strain HKI4363/88) and from a local scrotum and testicle infection of a bull in 2008 (strain BA2968). These infections occurred in two rural Finnish regions, i.e., Ostrobothnia in western Finland and Päijänne Tavastia in southern Finland, respectively. RESULTS: The isolates were genetically characterized by PCR-based methods such as multilocus variable number of tandem repeat analysis (MLVA) and whole genome-sequence analysis (WGS). Phylogenetic comparison of the two strains HKI4363/88 and BA2968 by chromosomal single nucleotide polymorphism (SNP) analysis grouped these organisms within their relatives of the minor canonical A-branch canSNP-group A.Br.003/004 (A.Br.V770) or canonical B-branch B.Br.001/002, respectively. Strain HKI4363/88 clustered relatively closely with other members of the A.Br.003/004 lineage from Europe, South Africa, and South America. In contrast, strain BA2968 clearly constituted a new sublineage within B.Br.001/002 with its closest relative being HYO01 from South Korea. CONCLUSIONS: Our results suggest that Finland harbors both unique (autochthonous) and more widely distributed, common clades of B. anthracis. We suspect that members of the common clades such as strains HKI4363/88 have been introduced only recently by anthropogenic activities involving importation of contaminated animal products. On the other hand, autochthonous strains such as isolate BA2968 probably have an older history of their introduction into Finland as evidenced by a high number of single nucleotide variant sites in their genomes.

The identification of novel single nucleotide polymorphisms to assist in mapping the spread of Bacillus anthracis across the Southern Caucasus.

Anthrax is common as a zoonotic disease in the southern Caucasus area including parts of Turkey and Georgia. In this region, population genetics of the etiological agent Bacillus anthracis comprises, where known, the major canonical single nucleotide polymorphism (canSNP) groups A.Br.Aust94 and A.Br.008/009 of the pathogen's global phylogeny, respectively. Previously, isolates of B. anthracis from Turkey have been genotyped predominantly by multi locus variable number of tandem repeat analysis (MLVA) or canSNP typing. While whole genome sequencing is the future gold standard, it is currently still costly. For that reason we were interested in identifying novel SNPs which could assist in further distinguishing closely related isolates using low cost assay platforms. In this study we sequenced the genomes of seven B. anthracis strains collected from the Kars province of Eastern Anatolia in Turkey and discovered new SNPs which allowed us to assign these and other geographically related strains to three novel branches of the major A-branch canSNP-group (A.Br.) Aust94. These new branches were named Kafkas-Geo 1-3 and comprised isolates from the Kars region and the neighboring republic of Georgia suggesting a common ancestry. The novel SNPs identified in this study connect the population genetics of B. anthracis in the South Caucasus and Turkey and will likely assist efforts to map the spread of the pathogen across this region.

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

BACKGROUND: Bacillus endophyticus is a soil plant-endophytic bacterium, while B. anthracis is the causative agent of anthrax. The virulence factors of B. anthracis are the plasmid encoded tripartite toxins (pXO1) and poly-γ-glutamic acid (PGA) capsule (pXO2). B. endophyticus isolated alongside B. anthracis from animals that died of anthrax in Northern Cape Province (NCP), South Africa, harbored polyglutamate genes. The study compared the characteristics of B. anthracis and B. endophyticus with other Bacillus species with a focus on the presence of the PGA capsule or/and unbound PGA. The morphology and whole genome sequence analysis of B. endophyticus strains and B. anthracis were compared. RESULTS: In conventional microbiology, B. endophyticus showed gram-positive round-shaped rods in single/short chains, which were endospore-forming, non-motile, non-haemolytic with white and dry colonies, and γ-phage resistant. B. anthracis was differentiated from B. endophyticus based on the latter's box-shaped rods in pairs/long chains, white-grey and slimy colonies, encapsulated and γ-phage susceptible. The study identified a PGA polyglutamate synthase operon that consisted of pgsBCA, γ-glutamyltranspeptidase (ggt) and pgsE in B. endophyticus genomes. CONCLUSIONS: PGA regions of B. anthracis contain capBCADE genes located in the pXO2 required for capsulation formation, while B. endophyticus contain the pgsBCAE genes in the chromosome. Whole genome and microbiology analysis identified B. endophyticus, as a non-capsuled endospore-forming bacterium that consists of PGA required for biosynthesis. B. endophyticus strains do not synthesize surface associated PGA, therefore capsule visualization of B. anthracis is a key diagnostic characteristic. The study highlights the significance of using whole genome shotgun sequencing to identify virulence and other important genes that might be present amongst unknown samples from natural outbreaks. None of the B. anthracis related plasmids or virulence genes were found in the B. endophyticus genomes.

Pathogenicity, population genetics and dissemination of Bacillus anthracis.

Bacillus anthracis, the etiological agent of anthrax, procures its particular virulence by a capsule and two AB type toxins: the lethal factor LF and the edema factor EF. These toxins primarily disable immune cells. Both toxins are translocated to the host cell by the adhesin-internalin subunit called protective antigen PA. PA enables LF to reach intra-luminal vesicles, where it remains active for long periods. Subsequently, LF translocates to non-infected cells, leading to inefficient late therapy of anthrax. B. anthracis undergoes slow evolution because it alternates between vegetative and long spore phases. Full genome sequence analysis of a large number of worldwide strains resulted in a robust evolutionary reconstruction of this bacterium, showing that B. anthracis is split in three main clades: A, B and C. Clade A efficiently disseminated worldwide underpinned by human activities including heavy intercontinental trade of goat and sheep hair. Subclade A.Br.WNA, which is widespread in the Northern American continent, is estimated to have split from clade A reaching the Northern American continent in the late Pleistocene epoch via the former Bering Land Bridge and further spread from Northwest southwards. An alternative hypothesis is that subclade A.Br.WNA. evolved from clade A.Br.TEA tracing it back to strains from Northern France that were assumingly dispatched by European explorers that settled along the St. Lawrence River. Clade B established mostly in Europe along the alpine axis where it evolved in association with local cattle breeds and hence displays specific geographic subclusters. Sequencing technologies are also used for forensic applications to trace unintended or criminal acts of release of B. anthracis. Under natural conditions, B. anthracis generally affects domesticated and wild ruminants in arid ecosystems. The more recently discovered B. cereus biovar anthracis spreads in tropical forests, where it threatens particularly endangered primate populations.

Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Discrimination of highly pathogenic bacteria, such as Bacillus anthracis, from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of B. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse Bacillus strains of the Bacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of B. anthracis not present in closely related Bacillus species. We could identify 7, 10, 18, and 14 B. anthracis-specific biomarker candidates that were absent in B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection of Bacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of B. anthracis and B. cereus yielded improved score values. The B. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

Genome sequencing of two Bacillus anthracis strains: a virulent strain and a vaccinal strain

ABSTRACT Bacillus anthracis strain SPV842_15 was isolated from bovine fetus, while B. anthracis strain Brazilian vaccinal was recovered from a commercial vaccine. We report here the genome sequences of both strains. The SPV842_15 genome is composed of a single circular chromosome with a length of 5,228,664 base pairs, and comprises 5911 coding sequences. In turn, the Brazilian vaccinal genome remains in 201 contigs with 5733 coding sequences. Both genomes have an overall C + G content of 35.4%, and 11 genes encoding the ribosomal RNAs (rRNAs) 5S, 16S and 23S. Only the plasmid pX01 sequence, which carries genes for toxins synthesis, was detected and completely assembled for both strains. These plasmids have a length of 181,684 base pairs and a C + G content of 32.5%. These genomic data generate insights about vaccinal B. anthracis virulence.