Plasmid pXO1 Research Articles

Anthrax toxins-producing Bacillus spp. isolated from handwashing stations during COVID-19 pandemic in Lagos, Nigeria.

The virulence binding factor, protective antigen (pag) and poly-D-γ-glutamate capsular (cap) genes, peculiar to Bacillus anthracis are located in the pXO1 and pXO2 plasmids which are transferable horizontally to related species called "cereus group". The cereus group are usually isolated from the environmental/food samples and have been implicated in debilitating human and animal anthrax-like diseases. This study was designed to investigate the presence of the anthrax virulence genes in different Bacillus spp. isolated from handwashing facilities during COVID-19 pandemic in Lagos, Nigeria. The Bacillus anthracis (OK316847), B. thuringiensis (OK316855), B. amyloliquefaciens (OK316857), B. cereus (OK316858) and B. thuringiensis (OK316859) previously isolated from rinsates and bowl water in two local government areas (LGAs) of Lagos state were further investigated by the polymerase chain reaction (PCR) amplification of the pag and cap genes using specific primers. Bacillus anthracis and B. cereus co-harboured the two 578 bp cap and 364 bp pag genes while B. thuringiensis only harboured the cap gene. Similarly, the non-cereus B. amyloliquefaciens was found to habour the pag gene. The two anthrax toxin genes were amplified in the Bacillus spp isolated from rinsates and bowl water used in hand washing in the two study LGAs. Given that these virulence genes have a global consequence and are a potential threat to life, this study calls for an extensive surveillance, and reassessment of gene regulators and plasmid distribution among these strains in our environment.

Development and application of DETECTR-based rapid detection for pathogenic Bacillusanthracis.

Bacillus anthracis (B. anthracis) is a gram-positive bacterium responsible for the acute disease anthrax. Rapid and reliable identification of pathogenic B. anthracis is important in the detection of natural infectious disease cases or bio-threats. Herein, a DNA endonuclease targeted CRISPR trans reporter (DETECTR) detection platform based on recombinase polymerase amplification (RPA) was studied. The DETECTR system targeted three sequences from B. anthracis (the BA_5345 chromosomal specific marker, the protective antigen gene pag A from pXO1 plasmid and the capsule-biosynthesis-related gene cap A from pXO2 plasmid). We developed a rapid (<40min), easy-to-implement and accurate identification method for of B. anthracis nucleic acid with near two-copies sensitivity. The combination of tripartite primer sets is effective for the reliable identification of B. anthracis but also for fast screening of pathogenic strains. More importantly, DETECTR correctly detected simulated clinical blood samples and firstly detected positive samples collected from the location of world War-II site, preserved at north-east China (45°36'55.940″ N, 126°38'33.738″ E) with high sensitivity and specificity. Our study provides insight into the DETECTR-based detection of B. anthracis. We present a novel screening and diagnostic option for pathogenic B. anthracis that can be performed on a user-friendly portable device. Based on its proven reliability, sensitivity, specificity and simplicity, our proposed method can be readily adapted to detect pathogenic B. anthracis, anthrax and biothreats.

Modeling gastrointestinal anthrax disease

Bacillus anthracis is a spore-forming microbe that persists in soil and causes anthrax disease. The most natural route of infection is ingestion by grazing animals. Gastrointestinal (GI) anthrax also occurs in their monogastric predators, including humans. Exposure of carcasses to oxygen triggers sporulation and contamination of the surrounding soil completing the unusual life cycle of this microbe. The pathogenesis of GI anthrax is poorly characterized. Here, we use B. anthracis carrying the virulence plasmids pXO1 and pXO2, to model gastrointestinal disease in Guinea pigs and mice. We find that spores germinate in the GI tract and precipitate disease in a dose-dependent manner. Inoculation of vegetative bacilli also results in GI anthrax. Virulence is impacted severely by the loss of capsule (pXO2-encoded) but only moderately in absence of toxins (pXO1-encoded). Nonetheless, the lack of toxins leads to reduced bacterial replication in infected hosts. B. cereus Elc4, a strain isolated from a fatal case of inhalational anthrax-like disease, was also found to cause GI anthrax. Because transmission to new hosts depends on the release of large numbers of spores in the environment, we propose that the acquisition of pXO1- and pXO2-like plasmids may promote the successful expansion of members of the Bacillus cereus sensu lato group able to cause anthrax-like disease.

Cytokine profile in &lt;i&gt;in vitro&lt;/i&gt; mouse macrophage culture infected with &lt;i&gt;Bacillus anthracis&lt;/i&gt; spores with varying plasmid composition

Bacillus anthracis, the causative agent of anthrax, is able to exist both in environmental conditions (soil) and in the macroorganism. The manifestation of pathogenic properties of B. anthracis strains is determined by relevant plasmid composition, because the main toxin and the capsule-related virulence factors are located in bacterial plasmid. Modeling anthrax infection in vitro in macrophage culture might reveal an influence of individual B. anthracis strain characteristics on infection and development of infectious process. The aim of this study was to analyze cytokine level during infection of in vitro macrophage cell cultures with spores of anthrax microbe strains bearing varying plasmid composition. The dependence of the macrophage cell cytokine profile on the plasmid composition of B. anthracis strains was revealed while modeling anthrax infection in vitro. The presence of the toxin-producing plasmid pXO1 in anthrax microbe strains has a powerful stimulating effect on the production of macrophages J774A cell line cytokines. B. anthracis strains lacking the pXO1 plasmid virtually stimulated no production of IL-1, caused very low secretion of IL-1, IL-6, MCP-1, MIP-1, MIP-1, IL-12 (p70) and active G-CSF products. The low cytokine response of macrophage cells infected with monoplasmid strains bearing only the capsule-forming plasmid was due not only to the absence of a binary toxin, but also to disturbed regulation of capsule production associated with the absence of the atxA gene. The capsule, along with lethal and edematous toxins, belongs to the main virulence factors of B. anthracis, but strains lacking the pXO1 virulence plasmid, had its production impaired, because the main regulator of capsule synthesis is the atxA gene localized on the pXO1 plasmid being positively regulated by the acpA and acpB genes, so that strains lacking the toxin-forming plasmid, even in the presence of the encapsulation plasmid, elicit a weak cytokine response in infected cells. Diplasmid strains of B. anthracis, due to produced main virulence factors a two-component toxin and a capsule, enforce macrophages (in the experiment) to actively produce IL-1, IL-6, MCP-1, G-CSF, MIP-1; MIP-1, IL-12 (p70). Strains with moderate virulence and capable of capsulation in air virtually did not differ from highly virulent strains in terms of their effect on in vitro macrophage culture.

Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

What Is Anthrax?

Anthrax has been feared for its high mortality in animals and humans for centuries. The etiologic agent is considered a potentially devastating bioweapon, and since 1876―when Robert Koch demonstrated that Bacillus anthracis caused anthrax―it has been considered the sole cause of the disease. Anthrax is, however, a toxin-mediated disease. The toxins edema toxin and lethal toxin are formed from protein components encoded for by the pXO1 virulence plasmid present in pathogenic B. anthracis strains. However, other members of the Bacillus cereus group, to which B. anthracis belongs, have recently been shown to harbor the pXO1 plasmid and produce anthrax toxins. Infection with these Bacillus cereus group organisms produces a disease clinically similar to anthrax. This suggests that anthrax should be defined by the exotoxins encoded for by the pXO1 plasmid rather than the bacterial species it has historically been associated with, and that the definition of anthrax should be expanded to include disease caused by any member of the B. cereus group containing the toxin-producing pXO1 plasmid or anthrax toxin genes specifically.

Detection of B. anthracis from Environmental Samples during Outbreak in Tamilnadu by Molecular Methods

The study was undertaken during 2013−2017 at Zoonoses Research Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Chennai. Blood smears, soil and environmental samples were collected from 23 sporadic incidences of anthrax reported in different parts of Tamil Nadu from 2013−2017. 11 incidences were presumptively diagnosed as anthrax by detecting capsulated bacilli on polychrome methylene blue staining and microscopy. The spores in soil samples (62) were extracted, cultured and DNA was extracted for molecular method of diagnosis. The molecular method detected twelve incidences as anthrax by amplifying the virulence factors like lef gene (385bp) and capsular gene (264bp) encoded in pXO1 and pXO2 plasmids respectively and chromosomal marker (Ba813) gene (152bp) using multiplex PCR. The partial sequence of cap gene of isolates (Vellore and Nolambur strain) revealed a close relationship with B. anthracis species. The relatedness of the isolated strains to virulent strains such as Ames and Vollum strain on phylogenetic analysis showed the nature of the outbreak. Since the multiplex PCR detects the outbreak with short turn-around time by detecting both chromosomal and Plasmid DNA, considered as a confirmative diagnosis. Further the molecular method of diagnosis precludes the need for culturing thereby avoiding unnecessary occupational risks and environmental contamination.

Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

ABSTRACTThe zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Efficient Genome Editing by a Miniature CRISPR-AsCas12f1 Nuclease in Bacillus anthracis.

A miniature CRISPR-Cas12f has been demonstrated to serve as an effective genome editing tool in gram negative bacteria as well as human cells. Here, we developed an alternative method to edit the genome of Bacillus anthracis based on the AsCas12f1 nuclease from Acidibacillus sulfuroxidans. When the htrA gene on the chromosome and the lef gene on the plasmid pXO1 were selected as targets, the CRISPR-AsCas12f1 system showed very high efficiency (100%). At the same time, a high efficiency was observed for large-fragment deletion. Our results also indicated that the length of the homologous arms of the donor DNA had a close relationship with the editing efficiency. Furthermore, a two-plasmid CRISPR-AsCas12f1 system was also constructed and combined with the endonuclease I-SceI for potential multi-gene modification. This represents a novel tool for mutant strain construction and gene function analyses in B. anthracis and other Bacillus cereus group bacteria.

A comparative analysis of short tandem repeats in PXO1 and PXO2 plasmids in the human and bovine isolates of Bacillus anthracis

A comparative analysis of short tandem repeats in PXO1 and PXO2 plasmids in the human and bovine isolates of Bacillus anthracis

Sequence Variability of pXO1-Located Pathogenicity Genes of Bacillus anthracis Natural Strains of Different Geographic Origin.

The main pathogenic factor of Bacillus anthracis is a three-component toxin encoded by the pagA, lef, and cya genes, which are located on the pXO1 plasmid. The atxA gene, which encodes the primary regulator of pathogenicity factor expression, is located on the same plasmid. In this work, we evaluated the polymorphism of the pagA, lef, cya, and atxA genes for 85 B. anthracis strains from different evolutionary lineages and canSNP groups. We have found a strong correlation of 19 genotypes with the main evolutionary lineages, but the correlation with the canSNP group of the strain was not as strong. We have detected several genetic markers indicating the geographical origin of the strains, for example, their source from the steppe zone of the former USSR. We also found that strains of the B.Br.001/002 group caused an anthrax epidemic in Russia in 2016 and strains isolated during paleontological excavations in the Russian Arctic have the same genotype as the strains of the B.Br.CNEVA group circulating in Central Europe. This data could testify in favor of the genetic relationship of these two groups of strains and hypothesize the ways of distribution of their ancestral forms between Europe and the Arctic.

Development of ELISA based on Bacillus anthracis capsule biosynthesis protein CapA for naturally acquired antibodies against anthrax.

Anthrax is a zoonotic disease caused by the gram-positive spore-forming bacterium Bacillus anthracis. Detecting naturally acquired antibodies against anthrax sublethal exposure in animals is essential for anthrax surveillance and effective control measures. Serological assays based on protective antigen (PA) of B. anthracis are mainly used for anthrax surveillance and vaccine evaluation. Although the assay is reliable, it is challenging to distinguish the naturally acquired antibodies from vaccine-induced immunity in animals because PA is cross-reactive to both antibodies. Although additional data on the vaccination history of animals could bypass this problem, such data are not readily accessible in many cases. In this study, we established a new enzyme-linked immunosorbent assay (ELISA) specific to antibodies against capsule biosynthesis protein CapA antigen of B. anthracis, which is non-cross-reactive to vaccine-induced antibodies in horses. Using in silico analyses, we screened coding sequences encoded on pXO2 plasmid, which is absent in the veterinary vaccine strain Sterne 34F2 but present in virulent strains of B. anthracis. Among the 8 selected antigen candidates, capsule biosynthesis protein CapA (GBAA_RS28240) and peptide ABC transporter substrate-binding protein (GBAA_RS28340) were detected by antibodies in infected horse sera. Of these, CapA has not yet been identified as immunoreactive in other studies to the best of our knowledge. Considering the protein solubility and specificity of B. anthracis, we prepared the C-terminus region of CapA, named CapA322, and developed CapA322-ELISA based on a horse model. Comparative analysis of the CapA322-ELISA and PAD1-ELISA (ELISA uses domain one of the PA) showed that CapA322-ELISA could detect anti-CapA antibodies in sera from infected horses but was non-reactive to sera from vaccinated horses. The CapA322-ELISA could contribute to the anthrax surveillance in endemic areas, and two immunoreactive proteins identified in this study could be additives to the improvement of current or future vaccine development.

Application of CRISPR/Cas9 System for Plasmid Elimination and Bacterial Killing of Bacillus cereus Group Strains.

The CRISPR-Cas system has been widely applied in prokaryotic genome editing with its high efficiency and easy operation. We constructed some “scissors plasmids” via using the temperature-sensitive pJOE8999 shuttle plasmid, which carry the different 20nt (N20) guiding the Cas9 nuclease as a scissors to break the target DNA. We successfully used scissors plasmids to eliminate native plasmids from Bacillus anthracis and Bacillus cereus, and specifically killed B. anthracis. When curing pXO1 and pXO2 virulence plasmids from B. anthracis A16PI2 and A16Q1, respectively, we found that the plasmid elimination percentage was slightly higher when the sgRNA targeted the replication initiation region (96–100%), rather than the non-replication initiation region (88–92%). We also tried using a mixture of two scissors plasmids to simultaneously eliminate pXO1 and pXO2 plasmids from B. anthracis, and the single and double plasmid-cured rates were 29 and 14%, respectively. To our surprise, when we used the scissor plasmid containing two tandem sgRNAs to cure the target plasmids pXO1 and pXO2 from wild strain B. anthracis A16 simultaneously, only the second sgRNA could guide Cas9 to cleave the target plasmid with high efficiency, while the first sgRNA didn't work in all the experiments we designed. When we used the CRISPR/cas9 system to eliminate the pCE1 mega-virulence plasmid from B. cereus BC307 by simply changing the sgRNA, we also obtained a plasmid-cured isogenic strain at a very high elimination rate (69%). The sterilization efficiency of B. anthracis was about 93%, which is similar to the efficiency of plasmid curing, and there was no significant difference in the efficiency of among the scissors plasmids containing single sgRNA, targeting multi-sites, or single-site targeting and the two tandem sgRNA. This simple and effective curing method, which is applicable to B. cereus group strains, provides a new way to study these bacteria and their virulence profiles.

Development of a PCR Lateral Flow Assay for Rapid Detection of Bacillus anthracis, the Causative Agent of Anthrax.

Bacillus anthracis, the causative agent of anthrax is one of the most potent listed biological warfare agents. The conventional microbiological methods of its detection are labor intensive and time consuming, whereas molecular assays are fast, sensitive and specific. PCR is one of the most reliable diagnostic tools in molecular biology. The combination of PCR with lateral flow strips can reduce the diagnostic/detection time. It gives an alternative to gel electrophoresis and offers easy and clear interpretation of results. In the present study, a PCR Lateral flow (PCR-LF) assay targeting cya gene present on pXO1 plasmid of B. anthracis has been developed. The forward and reverse primers were tagged with 6-carboxyflourescein (6-FAM) and biotin, respectively, at 5' end. The dual labeled PCR products were detected using lateral flow (LF) strips developed in this study. The PCR-LF assay could detect ≥ 5 pg of genomic DNA and ≥ 500 copies of target DNA harboured in a recombinant plasmid. The assay was able to detect as few as 103 and 10 CFU/mL of B. anthracis Sterne cells spiked in human blood after 6 and 24 h of enrichment, respectively.

Development of a set of three real-time loop-mediated isothermal amplification (LAMP) assays for detection of Bacillus anthracis, the causative agent of anthrax.

Bacillus anthracis, the causative agent of anthrax is a Gram-positive, non-motile, spore forming bacterium. Its spores can persist in soil and water for years and can also be aerosolized. A rapid, sensitive and specific method to detect B. anthracis is important for clinical management and preventing spread of anthrax. Loop-mediated isothermal amplification (LAMP) assay is a rapid technique that amplifies target DNA in isothermal conditions with high sensitivity and specificity. In this study, a LAMP assay set targeting a chromosomal and two plasmid markers was developed. The individual assays of the LAMP set targeting pXO1 plasmid (lef), pXO2 plasmid (capB), and chromosome (BA5345) sequences could detect 10, 250, and 100fg of genomic DNA and 10, 100, and 50 copies of the DNA targets harboured in recombinant plasmids, respectively. The lef and capB LAMP assays could detect ≥ 1 × 103CFU per mL of bacteria in spiked human blood samples, while BA5345 LAMP assay could detect ≥ 1 × 104CFU of bacteria per mL of spiked blood. The amplification was monitored in real-time by turbidimeter, and visual detection was also accomplished under normal and UV light after adding SYBR Green 1 dye on completion of the reaction. The assay set was found to be highly sensitive and did not cross-react with the closely related Bacillus spp. and other bacterial strains used in the study.

AtxA-Controlled Small RNAs of Bacillus anthracis Virulence Plasmid pXO1 Regulate Gene Expression in trans.

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram-negative bacteria; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for pXO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA-null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis.

Screening of possibly anthrax-contaminated burial sites in eastern and southern Ukraine

Aim. The aim of this study was to screen soil samples of 17 anthrax burial sites in Eastern and Southern Ukraine for the presence of B. anthracis. Methods. Soil samples were collected from anthrax grave sites located in Kharkiv, Sumy and Mykolaiv regions (diseased animals dated from 1946 to 2003). Isolation of B. anthracis from collected soil samples was performed with the GABRI method. From single colonies without hemolysis, that were inactivated with peracetic acid- containing 2 % Terralin PAA solution, DNA was extracted and analyzed by qPCR for the presence of chromosomal marker dhp61, as well as the markers pagA and capC located on virulence plasmids pXO1 and pXO2, respectively. Results. Eleven fi eld trips were conducted from July, 2016 to October, 2018 in which 369 soil samples from 17 burial sites in Kharkiv, Sumy and Mykolaiv oblasts were collected from different depths of presumed anthrax carcass sites. In most cases (12 out of 17 cases), the current status of these burial sites was deteriorated and not prop- erly accounted for. It was possible to obrain viable B. anthracis isolate was obtained from 50 cm depth at the grave site near Koviagy village, Valky district, Kharkiv region (49.92373°N, 35.48951°E). This isolate was named KhR/ VD/Kov2-2-05-3 and deposited in the Collection of Animal Infectious Pathogens of the National Scientifi c Center “Institute of Experimental and Clinical Veterinary Medicine”, Kharkiv, Ukraine. The contamination level of soil at the isolation site reached about 10 4 CFU per g as determined by plate counting. qPCR analysis of this isolate identi- fi ed both the dhp61 B. anthracis chromosomal and the pagA virulence plasmid marker. However, the plasmid pXO2 marker, required for capsule-formation could not be detected. Conclusions. The anthrax burial sites were created between the 1920s and 1960s, however, only approximate locations could be found and demarcated. In most cases the status of the sites was unsuitable for sampling. Nevertheless, isolation of B. anthracis in one case in the Valky district shows that old anthrax burial sites (13.500 exist in Ukraine) still pose a risk as potential source of the infection and therefore require more attention and surveillance, for which a surveillance plan will be developed.

Complete Genome Sequence of an Environmental Bacillus cereus Isolate Belonging to the Bacillus anthracis Clade.

We report here the complete genome sequence of a Bacillus cereus isolate identified in a soil sample from Namibia. This isolate is closely related to the B. anthracis clade. While the plasmids (500 and 12 kb) carry no detectable B. anthracis virulence gene, the large plasmid shares a 50-kb continuous region similar to plasmid pXO1.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil

BACKGROUND Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil.OBJECTIVES This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil.METHODS Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics.FINDINGS We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand.MAIN CONCLUSIONSThis work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.