Anthracis Isolates Research Articles

Anthrax in the Russian Federation in 2023 or in other words, «the same old story»

Introduction. The current anthrax situation in Russia is characterized by instability. In 2023, there was an increase in the number of infection outbreaks compared to the long-term average (for five years). The aim of the study is to assess the epizootological and epidemiological situation regarding anthrax in the Russian Federation in 2023 and the reasons for its deterioration, and to analyze data from genomic epidemiological surveillance of this infection. Materials and methods. The information of the territorial bodies of Rospotrebnadzor on the investigation of anthrax outbreaks, reference materials about anthrax stationary hazardous areas and anthrax burials were used. The phylogenetic position of the identified Bacíllus anthracis strains and genomes structure were determined based on whole-genome sequencing data. Results. In 2023 anthrax outbreaks were registered in the Chuvash Republic — Chuvashia (1), the Tyva Republic (1), Tambov (1), Ryazan (1) and Voronezh (3) regions. 14 farm animals and 19 people fell ill. The infection of animals not vaccinated against anthrax, as well as vaccinated long before contact with the source of infection, occurred mainly during grazing in the territories of old (unregistered) anthrax soil foci. Human disease is caused by contact with sick animals during care, forced slaughter, cutting, transportation of carcasses and meat, cooking processing of contaminated meat and offal, and consumption of insufficiently heat-treated liver. 17 patients were diagnosed with a cutaneous form of anthrax, while 2 had an oropharyngeal form combined with a cutaneous form of the disease. In all cases, the genome structure typical of the B. anthracis species has been established. The phylogenetic relationship of B. anthracis isolates with B. anthracis strains previously isolated in Russia is shown. Conclusion. The reason for the trouble in anthrax in 2023 was a number of violations of veterinary and sanitary-epidemiological regulations against the background of the presence of soil foci of infection. Stabilization of the situation can be achieved only in full range of regulated preventive measures are constantly implementated. The results of molecular genetic typing of B. anthracis strains isolated during the epidemiologic investigation of seven anthrax outbreaks in the Russian Federation in 2023 allow us to conclude that they are of local origin and have a genome structure typical of the species. Genetic analysis of the isolated strains demonstrated the effectiveness of the developed wgSNP typing system in the epidemiologic investigation of outbreaks.

Antimicrobial Resistance Profile, Whole-Genome Sequencing and Core Genome Multilocus Sequence Typing of B. anthracis Isolates in Croatia from 2001 to 2022.

Bacillus anthracis, the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. It is also considered one of the most important pathogens of bioterrorism. Rapid and reliable diagnosis and administration of antimicrobials are essential for effective anthrax treatment. In this study, we determined the in vitro susceptibilities of 40 isolates of B. anthracis isolated in Croatia over the recent two decades to 18 antimicrobials. Whole-genome sequencing was performed, and bioinformatics tools were used to determine virulence factors and antimicrobial resistance genes. Core genome-based multilocus sequence typing was used for isolate comparison and phylogenetic analysis. All isolates were susceptible to all antimicrobials recommended for post-exposure prophylaxis or anthrax therapy. Susceptibility was found to all other tested antimicrobials that are an alternative for primary therapy. We found two beta-lactamase genes, but their expression is not sufficient to confer resistance. In all isolates used in this study, we found 21 virulence genes, 8 of which are responsible for toxin and capsule production. As far as phylogenetic analysis is concerned, the B. anthracis isolates from Croatia are categorised into two clades. The first is clade A, subclade Trans Eurasia, and the other is clade B, subclade B2.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 μg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Spatial and phylogenetic patterns reveal hidden infection sources of Bacillus anthracis in an anthrax outbreak in Son La province, Vietnam.

Bacillus anthracis, the bacterial cause of anthrax, is a zoonosis affecting livestock and wildlife often spilling over into humans. In Vietnam, anthrax has been nationally reportable since 2015 with cases occurring annually, mostly in the northern provinces. In April 2022, an outbreak was reported in Son La province following the butchering of a water buffalo, Bubalus bubalis. A total of 137 humans from three villages were likely exposed to contaminated meat from the animal. Early epidemiological investigations suggested a single animal was involved in all exposures. Five B. anthracis isolates were recovered from human clinical cases along with one from the buffalo hide, another from associated maggots, and one from soil at the carcass site. The isolates were whole genome sequenced, allowing global, regional, and local molecular epidemiological analyses of the outbreak strains. All recovered B. anthracis belong to the A.Br.001/002 lineage based on canonical single nucleotide polymorphism analysis (canSNP). Although not previously identified in Vietnam, this lineage has been identified in the nearby countries of China, India, Indonesia, Thailand, as well as Australia. A twenty-five marker multi-locus variable number tandem repeat analysis (MLVA-25) was used to investigate the relationship between human, soil, and buffalo strains. Locally, four MLVA-25 genotypes were identified from the eight isolates. This level of genetic diversity is unusual for the limited geography and timing of cases and differs from past literature using MLVA-25. The coupled spatial and phylogenetic data suggest this outbreak originated from multiple, likely undetected, animal sources. These findings were further supported by local news reports that identified at least two additional buffalo deaths beyond the initial animal sampled in response to the human cases. Future outbreak response should include intensive surveillance for additional animal cases and additional molecular epidemiological traceback to identify pathogen sources.

Resistance finding of Bacillus anthracis towards penicillin in East Java, Central Java, and Yogyakarta Provinces, Indonesia

Anthrax is a worldwide distributing zoonotic disease, caused by Bacillus anthracis, which occurs sporadically in Indonesia, particularly in the provinces of East Java, Central Java, and Yogyakarta, which are the working areas of the Disease Investigation Center (DIC) Wates. Penicillin has been the primary antimicrobial treatment recommended for anthrax since there has never been a report of resistance to this antibiotic in Indonesia. The objective of this research was to assess the sensitivity of B. anthracis isolates from Central Java, East Java, and Yogyakarta to penicillin and tetracycline. Sixteen B. anthracis isolates from DIC Wates collected between 1990-2021 recovered from environmental samples were used in this study. All isolates were identified by phenotype, then tested for sensitivity to penicillin and tetracycline by agar diffusion (Kirby-Bauer) and broth dilution method. The data obtained were compared with the standard and analyzed descriptively. The results showed that all isolates were B. anthracis. One of 16 isolates (6,25%) consistently showed resistance to penicillin, but was sensitive to tetracycline, while 15 isolates (93,75%) showed sensitive to both antibiotics. A penicillin-resistant isolate was soil sample from anthrax endemic area. In conclusion, there was B. anthracis isolate that was found resistance to penicillin. Therefore, tetracycline can be used as an alternative for anthrax treatment.

Spanish Outbreak Isolates Bridge Phylogenies of European and American Bacillus anthracis.

The geographical origin of a major present-day phylogenetic group (A branch WNA; A.Br.WNA) of American Bacillus anthracis is controversial. One hypothesis postulated that the anthrax pathogen reached North America via a then-existing land bridge from northeastern Asia thousands of years ago. A competing hypothesis suggested that B. anthracis was introduced to America a couple of hundred years ago, related to European colonization. The latter view is strongly supported by genomic analysis of a group of French B. anthracis isolates that are phylogenetically closely related to the North American strains of the A branch A.Br.WNA clade. In addition, three West African strains also belong to this relationship group. Recently, we have added a Spanish strain to these close relatives of the WNA lineage of American B. anthracis. Nevertheless, the diversity of Spanish B. anthracis remains largely unexplored, and phylogenetic links to European or American relatives are not well resolved. Here, we genome sequenced and characterized 29 new B. anthracis isolates (yielding 18 unique genotypes) from outbreaks in west central and central Spain in 2021. Applying comparative chromosomal analysis, we placed the chromosomes of these isolates within the established phylogeny of the A.Br.008/009 (A.Br.TEA) canonical SNP group. From this analysis, a new sub-clade, named A.Br.11/ESPc, emerged that constitutes a sister group of American A.Br.WNA.

From cereus to anthrax and back again: Assessment of the temperature-dependent phenotypic switching in the "cross-over" strain Bacillus cereus G9241.

Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other in Bacillus anthracis: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all B. anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and hemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25°C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37°C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25°C compared to 37°C. Furthermore, results suggest that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37°C, giving rise to the temperature-dependent hemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to "switch" between B. cereus and B. anthracis-like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA.

Systematic Review of In Vitro Antimicrobial Susceptibility Testing for Bacillus anthracis, 1947-2019.

Bacillus anthracis, the causative agent of anthrax, is a high-consequence bacterial pathogen that occurs naturally in many parts of the world and is considered an agent of biowarfare or bioterrorism. Understanding antimicrobial susceptibility profiles of B. anthracis isolates is foundational to treating naturally occurring outbreaks and to public health preparedness in the event of an intentional release. In this systematic review, we searched the peer-reviewed literature for all publications detailing antimicrobial susceptibility testing of B. anthracis. Within the set of discovered articles, we collated a subset of publications detailing susceptibility testing that followed standardized protocols for Food and Drug Administration-approved, commercially available antimicrobials. We analyzed the findings from the discovered articles, including the reported minimal inhibitory concentrations. Across the literature, most B. anthracis isolates were reported as susceptible to current first-line antimicrobials recommended for postexposure prophylaxis and treatment. The data presented for potential alternative antimicrobials will be of use if significant resistance to first-line antimicrobials arises, the strain is bioengineered, or first-line antimicrobials are not tolerated or available.

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.

Novel Strategy for Rapidly and Safely Distinguishing Bacillus anthracis and Bacillus cereus by Use of Peptide Mass Fingerprints Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

The objective of this study was to construct a rapid, high-throughput, and biosafety-compatible screening method for Bacillus anthracis and Bacillus cereus based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS coupled to ClinProTools was used to discover MALDI-TOF MS biomarker peaks and generate a classification model based on a genetic algorithm (GA) to differentiate between different Bacillus anthracis and Bacillus cereus isolates. Thirty Bacillus anthracis and 19 Bacillus cereus strains were used to construct and analyze the model, and 40 Bacillus strains were used for validation. For the GA screening model, the cross-validation values, which reflect the ability of the model to handle variability among the test spectra, and the recognition capability values, which reflect the model's ability to correctly identify its component spectra, were all 100%. This model contained 10 biomarker peaks (m/z 3,339.9, 3,396.3, 3,682.4, 5,476.7, 6,610.6, 6,680.1, 7,365.3, 7,792.4, 9,475.8, and 10,934.1) used to correctly identify 28 Bacillus anthracis and 12 Bacillus cereus isolates from 40 Bacillus isolates, with a sensitivity and specificity of 100%. With the obvious advantages of being rapid, highly accurate, and highly sensitive and having a low cost and high throughput, MALDI-TOF MS ClinProTools is a powerful and reliable tool for screening Bacillus anthracis and Bacillus cereus strains.

An atypical localization of Bacillusanthracis in a cow’s milk and udder

A 6-year-old Simental-Swiss Brown cross-bred cow, which aborted in the first trimester of the gestation and involuntarily presented anthrax bacilli around the udder, was subjected to this study. The cow was vaccinated with an attenuated anthrax vaccine (Bacillus anthracis 34 F2) 6 months ago and had normal physical examination findings. Samples taken 10 days after the abortion were representative of all possible spaces that could carry the bacteria such as milk, blood, environment, and swabs from rectum, udder and tits of an infected cow. Microbiological examination was carried out by staining the smears with Azure and in vitro culture of the samples on some enriched and selective media such as 7% sheep blood agar (SBA), Brain Heart Infusion (BHI) Broth media with 7% sheep blood, and polymyxin-lysozyme-ethylenediaminetetraacetic acid-thallous acetate (PLET) agar. On a molecular basis, the virulence plasmid-targeted PCR analysis of the isolates was performed. In addition, MLVA-8 and MLVA-25 typing of B. anthracis isolates were conducted. Upon the isolation of B. anthracis with a concentration of 6 × 102 cfu/mL in milk sample and different subtypes obtained from the isolates by MLVA-8 and MLVA-25 typing, the present study enables us to instantiate the presence and unusual spread of bacteria around the udder of cows.

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

BackgroundAnthrax, 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.ResultsThese 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.ConclusionsThe 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.

A Unique Isolation of a Lytic Bacteriophage Infected Bacillus anthracis Isolate 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 site.

Genetic Diversity of Australian Bacillus anthracis Isolates Revealed by Multiple-Locus Variable-Number Tandem Repeat Analysis.

Outbreaks of anthrax occur sporadically in Australia and most commonly in the “anthrax belt”, a region which extends from southern Queensland through the centre of New South Wales and into northern Victoria. Little is known about the epidemiological links between Bacillus anthracis isolates taken from different outbreaks and the diversity of strains within Australia. We used multiple-locus variable-number tandem repeat analysis employing 25 markers (MLVA25) to genotype 99 B. anthracis isolates from an archival collection of Australian isolates. MLVA25 genotyping revealed eight unique genotypes which clustered within the previously defined A3 genotype of B. anthracis. Genotyping of B. anthracis strains from outbreaks of disease in Victoria identified the presence of multiple genotypes associated with these outbreaks. The geographical distribution of genotypes within Australia suggests that a single genotype was introduced into the eastern states of Australia, followed by the spread and localised differentiation of the pathogen (MLVA25 genotypes MG1-MG6) throughout the anthrax belt. In contrast, unexplained occurrences of disease in areas outside of this anthrax belt which are associated with different genotypes, (MLVA25 genotypes MG7 and MG8) indicate separate introductions of B. anthracis into Australia.

Epidemiological Investigation and Etiological Analysis of a Cutaneous Anthrax Epidemic Caused by Butchering Sick Cattle in Guizhou, China.

A suspected human cutaneous anthrax epidemic caused by butchering sick cattle occurred in Zhijin County of Guizhou Province, Southwest of China, in 2016. Epidemiological investigation and etiological analysis were performed to provide a scientific basis for the source tracking of the epidemic. The epidemic was epidemiologically investigated, and skin blister samples collected from patients and soil samples collected from the butchering spots were used for Bacillus anthracis isolation. The suspicious B. anthracis isolates were identified using conventional methods and PCR, followed by genotyping using multiple-locus variable-number tandem repeats (VNTRs) analysis (MLVA-15) and canonical single-nucleotide polymorphism (canSNP). The genetic relationship of epidemic strains and isolates collected from other regions was analyzed. Epidemiological investigation results showed that the patients may be infected by B. anthracis during butchering sick cattle. Two suspected B. anthracis strains were isolated from blood samples and blister fluids, respectively. Conventional methods identified the two suspected isolates as B. anthracis, while PCR results showed that anti-protective antigen (PA) and capsule (CAP) gene were positive in the two isolates. MLVA-15 showed that the MLVA profiles of the two isolates were 9-20-12-53-16-2-8-8-8-4-4-4-4-10-4, which is different from the MLVA profiles of representative strains from other regions. CanSNP analysis showed that the two strains belonged to cluster A.Br.001/002. Clustering analysis and minimum spanning tree (MST) demonstrated that the two isolates were clustered with strains previously isolated from Guizhou Province. The results indicated that B. anthracis was the pathogen for this epidemic, and the patients were infected during butchering the sick. The genetic characteristics and the relationship of the B. anthracis isolates to strains from other regions indicated that the epidemic was a local occurrence.

A Rare Glimpse into the Past of the Anthrax Pathogen Bacillus anthracis.

The bacterium Bacillus anthracis is the causative agent of the zoonotic disease anthrax. While genomics of extant B. anthracis isolates established in-depth phylogenomic relationships, there is scarce information on the historic genomics of the pathogen. Here, we characterized the oldest documented B. anthracis specimen. The inactive 142-year-old material originated from a bovine diseased in Chemnitz (Germany) in 1878 and is contemporary with the seminal studies of Robert Koch on B. anthracis. A specifically developed isolation method yielded high-quality DNA from this specimen for genomic sequencing. The bacterial chromosome featuring 242 unique base-characters placed it into a major phylogenetic clade of B. anthracis (B.Branch CNEVA), which is typical for central Europe today. Our results support the notion that the CNEVA-clade represents part of the indigenous genetic lineage of B. anthracis in this part of Europe. This work emphasizes the value of historic specimens as precious resources for reconstructing the past phylogeny of the anthrax pathogen.

A classification framework for Bacillus anthracis defined by global genomic structure.

Bacillus anthracis, the causative agent of anthrax, is a considerable global health threat affecting wildlife, livestock, and the general public. In this study, whole‐genome sequence analysis of over 350 B. anthracis isolates was used to establish a new high‐resolution global genotyping framework that is both biogeographically informative and compatible with multiple genomic assays. The data presented in this study shed new light on the diverse global dissemination of this species and indicate that many lineages may be uniquely suited to the geographic regions in which they are found. In addition, we demonstrate that plasmid genomic structure for this species is largely consistent with chromosomal population structure, suggesting vertical inheritance in this bacterium has contributed to its evolutionary persistence. This classification methodology is the first based on population genomic structure for this species and has potential use for local and broader institutions seeking to understand both disease outbreak origins and recent introductions. In addition, we provide access to a newly developed genotyping script as well as the full whole‐genome sequence analyses output for this study, allowing future studies to rapidly employ and append their data in the context of this global collection. This framework may act as a powerful tool for public health agencies, wildlife disease laboratories, and researchers seeking to utilize and expand this classification scheme for further investigations into B. anthracis evolution.

Nucleotide polymorphism assay for the identification of west African group Bacillus anthracis: a lineage lacking anthrose

BackgroundThe exosporium of the anthrax-causing Bacillus anthracis endospores display a tetrasaccharide composed of three rhamnose residues and an unusual sugar termed anthrose. Anthrose is a proposed potential target for immunotherapy and for specific detection of B. anthracis. Although originally thought to be ubiquitous in B. anthracis, previous work identified an anthrose negative strain from a West African lineage isolated from cattle that could represent a vaccine escape mutant. These strains carry genes required for expression of the anthrose operon but premature stop codons resulting from an 8-bp insertion in BAS3320 (an amino-transferase) and a C/T substitution at position 892 of the BAS3321 (a glycosyltransferase) gene prevent anthrose expression. Various other single nucleotide polymorphisms (SNPs) have been identified throughout the operon and could be the basis for detection of anthrose-deficient strains.ResultsIn this study, we evaluated rhAmp genotypic assays based on SNPs at positions 892 and 1352 of BAS3321 for detection and differentiation of anthrose negative (Ant−) West African strains. Discrimination of anthrose negative West African isolates was achieved with as low as 100 fg of DNA, whereas consistent genotyping of Sterne necessitated at least 1 pg of DNA.ConclusionsScreening of a global panel of B. anthracis isolates showed anthrose-expressing alleles are prevalent worldwide whereas the anthrose-deficient phenotype is to date limited to West Africa. Our work also revealed a third, previously unreported anthrose genotype in which the operon is altogether missing from a Polish B. anthracis isolate.

Development of a rapid immunochromatographic assay for detection of surface array protein (Sap), a potent biomarker of Bacillus anthracis

Bacillus anthracis, the causative agent of anthrax poses serious threat to human health. Genetically, B. anthracis is very close to the other species of this group. Hence, it is important to develop a rapid screening test for B. anthracis. Surface array protein (Sap), a surface layer protein produced by B. anthracis is considered an important biomarker for its detection as it is excreted in the growth media. In the present study, we have developed an Immunochromatography test (ICT) strip for detection of Sap, for rapid screening of B. anthracis strains. The recombinant Sap was produced and used for production of mouse monoclonal antibodies. The antibodies were conjugated with colloidal gold for preparation of ICT. For determination of sensitivity and specificity, different Bacillus species were grown in culture broth, heat inactivated to kill the live bacteria and tested on ICT. The B. anthracis Sterne could be detected after 3 h of growth. Sample testing by ICT takes only 2 min in comparison to 4–5 h in sandwich plate ELISA. The results corroborated that the ICT can be used for rapid screening of B. anthracis isolates. The proposed method is rapid, safe and user friendly for detection of B. anthracis culture.