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18 pages, 2015 KiB

Open AccessArticle

C3 Opsonization of Anthrax Bacterium and Peptidoglycan Supports Recognition and Activation of Neutrophils

by Narcis I. Popescu, Ravi S. Keshari, Jackie Cochran, K. Mark Coggeshall and Florea Lupu

Microorganisms 2020, 8(7), 1039; https://doi.org/10.3390/microorganisms8071039 - 13 Jul 2020

Cited by 6 | Viewed by 2670

Abstract

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis, the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils [...] Read more.

Neutrophils are the most abundant innate cell population and a key immune player against invading pathogens. Neutrophils can kill both bacterium and spores of Bacillus anthracis, the causative anthrax pathogen. Unlike interactions with professional phagocytes, the molecular recognition of anthrax by neutrophils is largely unknown. In this study, we investigated the role of complement C3 deposition on anthrax particles for neutrophil recognition of bacterium and/or its cell wall peptidoglycan, an abundant pathogen-associated molecular pattern that supports anthrax sepsis. C3 opsonization and recognition by complement receptors accounted for 70–80% of the affinity interactions between neutrophils and anthrax particles at subphysiologic temperatures. In contrast, C3 supported up to 50% of the anthrax particle ingestion under thermophysiologic conditions. Opsonin-dependent low affinity interactions and, to a lower extent, opsonin-independent mechanisms, provide alternative entry routes. Similarly, C3 supported 58% of peptidoglycan-induced degranulation and, to a lower extent, 23% of bacterium-induced degranulation. Interestingly, an opsonin independent mechanism mediated by complement C5, likely through C5a anaphylatoxin, primes azurophilic granules in response to anthrax particles. Overall, we show that C3 deposition supports anthrax recognition by neutrophils but is dispensable for pathogen ingestion and neutrophil degranulation, highlighting immune recognition redundancies that minimize the risk of pathogen evasion. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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21 pages, 4008 KiB

Open AccessArticle

Rapid Microscopic Detection of Bacillus anthracis by Fluorescent Receptor Binding Proteins of Bacteriophages

by Peter Braun, Immanuel Wolfschläger, Leonie Reetz, Lilia Bachstein, Ana Clara Jacinto, Carolina Tocantins, Johannes Poppe and Gregor Grass

Microorganisms 2020, 8(6), 934; https://doi.org/10.3390/microorganisms8060934 - 21 Jun 2020

Cited by 12 | Viewed by 5871

Abstract

Bacillus anthracis, the etiological agent of anthrax disease, is typically diagnosed by immunological and molecular methods such as polymerase chain reaction (PCR). Alternatively, mass spectrometry techniques may aid in confirming the presence of the pathogen or its toxins. However, because of the [...] Read more.

Bacillus anthracis, the etiological agent of anthrax disease, is typically diagnosed by immunological and molecular methods such as polymerase chain reaction (PCR). Alternatively, mass spectrometry techniques may aid in confirming the presence of the pathogen or its toxins. However, because of the close genetic relationship between B. anthracis and other members of the Bacillus cereus sensu lato group (such as Bacillus cereus or Bacillus thuringiensis) mis- or questionable identification occurs frequently. Also, bacteriophages such as phage gamma (which is highly specific for B. anthracis) have been in use for anthrax diagnostics for many decades. Here we employed host cell-specific receptor binding proteins (RBP) of (pro)-phages, also known as tail or head fibers, to develop a microscopy-based approach for the facile, rapid and unambiguous detection of B. anthracis cells. For this, the genes of (putative) RBP from Bacillus phages gamma, Wip1, AP50c and from lambdoid prophage 03 located on the chromosome of B. anthracis were selected. Respective phage genes were heterologously expressed in Escherichia coli and purified as fusions with fluorescent proteins. B. anthracis cells incubated with either of the reporter fusion proteins were successfully surface-labeled. Binding specificity was confirmed as RBP fusion proteins did not bind to most isolates of a panel of other B. cereus s.l. species or to more distantly related bacteria. Remarkably, RBP fusions detected encapsulated B. anthracis cells, thus RBP were able to penetrate the poly-γ-d-glutamate capsule of B. anthracis. From these results we anticipate this RBP-reporter assay may be useful for rapid confirmative identification of B. anthracis. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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29 pages, 4366 KiB

Open AccessArticle

A Unique Isolation of a Lytic Bacteriophage Infected Bacillus anthracis Isolate from Pafuri, South Africa

by Ayesha Hassim, Kgaugelo Edward Lekota, David Schalk van Dyk, Edgar Henry Dekker and Henriette van Heerden

Microorganisms 2020, 8(6), 932; https://doi.org/10.3390/microorganisms8060932 - 20 Jun 2020

Cited by 4 | Viewed by 5001

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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10 pages, 568 KiB

Open AccessArticle

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

by Janine Muller, Ilhan Mohammad, Simone Warner, Roger Paskin, Fiona Constable and Mark Fegan

Microorganisms 2020, 8(6), 886; https://doi.org/10.3390/microorganisms8060886 - 11 Jun 2020

Cited by 3 | Viewed by 2511

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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18 pages, 11318 KiB

Open AccessArticle

Evaluation of a Highly Efficient DNA Extraction Method for Bacillus anthracis Endospores

by Mandy Knüpfer, Peter Braun, Kathrin Baumann, Alexandra Rehn, Markus Antwerpen, Gregor Grass and and Roman Wölfel

Microorganisms 2020, 8(5), 763; https://doi.org/10.3390/microorganisms8050763 - 20 May 2020

Cited by 13 | Viewed by 7561

Abstract

A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. [...] Read more.

A variety of methods have been established in order to optimize the accessibility of DNA originating from Bacillus anthracis cells and endospores to facilitate highly sensitive molecular diagnostics. However, most endospore lysis techniques have not been evaluated in respect to their quantitative proficiencies. Here, we started by systematically assessing the efficiencies of 20 DNA extraction kits for vegetative B. anthracis cells. Of these, the Epicentre MasterPure kit gave the best DNA yields and quality suitable for further genomic analysis. Yet, none of the kits tested were able to extract reasonable quantities of DNA from cores of the endospores. Thus, we developed a mechanical endospore lysis protocol, facilitating the extraction of high-quality DNA. Transmission electron microscopy or the labelling of spores with the indicator dye propidium monoazide was utilized to assess lysis efficiency. Finally, the yield and quality of genomic spore DNA were quantified by PCR and they were found to be dependent on lysis matrix composition, instrumental parameters, and the method used for subsequent DNA purification. Our final standardized lysis and DNA extraction protocol allows for the quantitative detection of low levels (<50 CFU/mL) of B. anthracis endospores and it is suitable for direct quantification, even under resource-limited field conditions, where culturing is not an option. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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7 pages, 1272 KiB

Open AccessArticle

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

by Peter Braun, Mandy Knüpfer, Markus Antwerpen, Dagmar Triebel and Gregor Grass

Microorganisms 2020, 8(2), 298; https://doi.org/10.3390/microorganisms8020298 - 21 Feb 2020

Cited by 2 | Viewed by 3965

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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10 pages, 3325 KiB

Open AccessArticle

Whole Genome Sequencing for Studying Bacillus anthracis from an Outbreak in the Abruzzo Region of Italy

by Alexandra Chiaverini, Mostafa Y. Abdel-Glil, Jörg Linde, Domenico Galante, Valeria Rondinone, Antonio Fasanella, Cesare Cammà, Nicola D’Alterio, Giuliano Garofolo and Herbert Tomaso

Microorganisms 2020, 8(1), 87; https://doi.org/10.3390/microorganisms8010087 - 08 Jan 2020

Cited by 8 | Viewed by 3699

Abstract

Anthrax is a serious infectious disease caused by the gram-positive and spore-forming bacterium Bacillus anthracis. In Italy, anthrax is an endemic disease with sporadic cases each year and few outbreaks, especially in Southern Italy. However, new foci have been discovered in zones [...] Read more.

Anthrax is a serious infectious disease caused by the gram-positive and spore-forming bacterium Bacillus anthracis. In Italy, anthrax is an endemic disease with sporadic cases each year and few outbreaks, especially in Southern Italy. However, new foci have been discovered in zones without previous history of anthrax. During summer 2016, an outbreak of anthrax caused the death of four goats in the Abruzzo region, where the disease had not been reported before. In order to investigate the outbreak, we sequenced one strain and compared it to 19 Italian B. anthracis genomes. Furthermore, we downloaded 71 whole genome sequences representing the global distribution of canonical SNP lineages and used them to verify the phylogenetic positioning. To this end, we analyzed and compared the genome sequences using canonical SNPs and the whole genome SNP-based analysis. Our results demonstrate that the outbreak strain belonged to the Trans-Eurasian (TEA) group A.Br.011/009, which is the predominant clade in Central-Southern Italy. In conclusion, the high genomic relatedness of the Italian TEA strains suggests their evolution from a common ancestor, while the spread is supposedly driven by trade as well as human and transhumance activities. Here, we demonstrated the capabilities of whole genome sequencing (WGS), which can be used as a tool for outbreak analyses and surveillance activities. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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12 pages, 1627 KiB

Open AccessArticle

Phylogenetic Placement of Isolates Within the Trans-Eurasian Clade A.Br.008/009 of Bacillus anthracis

by Markus Antwerpen, Wolfgang Beyer, Olga Bassy, María Victoria Ortega-García, Juan Carlos Cabria-Ramos, Gregor Grass and Roman Wölfel

Microorganisms 2019, 7(12), 689; https://doi.org/10.3390/microorganisms7120689 - 12 Dec 2019

Cited by 10 | Viewed by 3141

Abstract

The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis [...] Read more.

The largest phylogenetic lineage known to date of the anthrax pathogen Bacillus anthracis is the wide-spread, so-called Trans-Eurasian clade systematically categorized as the A.Br.008/009 group sharing two defining canonical single-nucleotide polymorphisms (canSNP). In this study, we genome-sequenced a collection of 35 B. anthracis strains of this clade, derived from human infections, animal outbreaks or soil, mostly from European countries isolated between 1936 and 2008. The new data were subjected to comparative chromosomal analysis, together with 75 B. anthracis genomes available in public databases, and the relative placements of these isolates were determined within the global phylogeny of the A.Br.008/009 canSNP group. From this analysis, we have detected 3754 chromosomal SNPs, allowing the assignation of the new chromosomal sequences to established sub-clades, to define new sub-clades, such as two new Spanish, one Bulgarian or one German group(s), or to introduce orphan lineages. SNP-based results were compared with that of a multilocus variable number of tandem repeat analysis (MLVA). This analysis indicated that MLVA typing might provide additional information in cases when genomics yields identical genotypes or shows only minor differences. Introducing the delayed mismatch amplification assay (DMAA) PCR-analysis, we developed a cost-effective method to interrogate for a set of ten phylogenetically informative SNPs within genomes of A.Br.008/009 canSNP clade strains of B. anthracis. By this approach, additional 32 strains could be assigned to five of ten defined clades. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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Review

Jump to: Research, Other

25 pages, 2305 KiB

Open AccessReview

The Bacillus anthracis Cell Envelope: Composition, Physiological Role, and Clinical Relevance

by Alice Chateau, Sander E. Van der Verren, Han Remaut and Antonella Fioravanti

Microorganisms 2020, 8(12), 1864; https://doi.org/10.3390/microorganisms8121864 - 26 Nov 2020

Cited by 10 | Viewed by 6496

Abstract

Anthrax is a highly resilient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. The bacterium presents a complex and dynamic composition of its cell envelope, which changes in response to developmental and environmental conditions and host-dependent signals. Because of [...] Read more.

Anthrax is a highly resilient and deadly disease caused by the spore-forming bacterial pathogen Bacillus anthracis. The bacterium presents a complex and dynamic composition of its cell envelope, which changes in response to developmental and environmental conditions and host-dependent signals. Because of their easy to access extracellular locations, B. anthracis cell envelope components represent interesting targets for the identification and development of novel therapeutic and vaccine strategies. This review will focus on the novel insights regarding the composition, physiological role, and clinical relevance of B. anthracis cell envelope components. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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16 pages, 1101 KiB

Open AccessReview

Anthrax Toxin Detection: From In Vivo Studies to Diagnostic Applications

by Jean-Nicolas Tournier and Clémence Rougeaux

Microorganisms 2020, 8(8), 1103; https://doi.org/10.3390/microorganisms8081103 - 23 Jul 2020

Cited by 7 | Viewed by 3008

Abstract

Anthrax toxins are produced by Bacillus anthracis throughout infection and shape the physiopathogenesis of the disease. They are produced in low quantities but are highly efficient. They have thus been long ignored, but recent biochemical methods have improved our knowledge in animal models. [...] Read more.

Anthrax toxins are produced by Bacillus anthracis throughout infection and shape the physiopathogenesis of the disease. They are produced in low quantities but are highly efficient. They have thus been long ignored, but recent biochemical methods have improved our knowledge in animal models. This article reviews the various methods that have been used and how they could be applied to clinical diagnosis. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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8 pages, 257 KiB

Open AccessReview

A Review of Arguments for the Existence of Latent Infections of Bacillus anthracis, and Research Needed to Understand Their Role in the Outbreaks of Anthrax

by Robert S. Gainer, Gilles Vergnaud and Martin E. Hugh-Jones

Microorganisms 2020, 8(6), 800; https://doi.org/10.3390/microorganisms8060800 - 26 May 2020

Cited by 4 | Viewed by 2380

Abstract

Hugh-Jones and Blackburn and Turnbull’s collective World Health Organization (WHO) report did literature reviews of the theories and the bases for causes of anthrax outbreaks. Both comment on an often-mentioned suspicion that, even though unproven, latent infections are likely involved. Hugh-Jones suggested Gainer [...] Read more.

Hugh-Jones and Blackburn and Turnbull’s collective World Health Organization (WHO) report did literature reviews of the theories and the bases for causes of anthrax outbreaks. Both comment on an often-mentioned suspicion that, even though unproven, latent infections are likely involved. Hugh-Jones suggested Gainer do an updated review of our present-day knowledge of latent infections, which was the basis for Gainer’s talk at the Biology of Anthrax Conference in Bari, Italy 2019. At the Conference Gainer met Vergnaud who presented anthrax genome studies that implied that the disease might have spread throughout Asia and from Europe to North America in a short time span of three or four centuries. Vergnaud wondered if latent infections might have played a role in the process. Several other presenters at the Conference also mentioned results that might suggest the existence of latent infections. Vergnaud subsequently looked into some of the old French literature about related observations, results, and discussions of early Pasteur vaccine usage (late 1800′s) and found mentions of suspected latent infections. The first part of the paper is a focused summary and interpretation of Hugh-Jones and Blackburn’s and Turnbull’s reviews specifically looking for suggestions of latent infections, a few additional studies with slightly different approaches, and several mentions made of presentations and posters at the Conference in Italy. In general, many different investigators in different areas and aspects of the anthrax study at the Conference found reasons to suspect the existence of latent infections. The authors conclude that the affected species most studied, including Homo sapiens, provide circumstantial evidence of latent infections and modified host resistance. The last part of the review explores the research needed to prove or disprove the existence of latent infections. Full article

(This article belongs to the Special Issue An Update on Anthrax)

7 pages, 207 KiB

Open AccessReview

Current State of Anthrax Vaccines and Key R&D Gaps Moving Forward

by Adam Clark and Daniel N. Wolfe

Microorganisms 2020, 8(5), 651; https://doi.org/10.3390/microorganisms8050651 - 29 Apr 2020

Cited by 14 | Viewed by 3370

Abstract

A licensed anthrax vaccine has been available for pre-exposure prophylaxis in the United States since 1970, and it was approved for use as a post-exposure prophylaxis, in combination with antibiotic treatment, in 2015. A variety of other vaccines are available in other nations, [...] Read more.

A licensed anthrax vaccine has been available for pre-exposure prophylaxis in the United States since 1970, and it was approved for use as a post-exposure prophylaxis, in combination with antibiotic treatment, in 2015. A variety of other vaccines are available in other nations, approved under various regulatory frameworks. However, investments in anthrax vaccines continue due to the severity of the threat posed by this bacterium, as both a naturally occurring pathogen and the potential for use as a bioweapon. In this review, we will capture the current landscape of anthrax vaccine development, focusing on those lead candidates in clinical development. Although approved products are available, a robust pipeline of candidate vaccines are still in development to try to address some of the key research gaps in the anthrax vaccine field. We will then highlight some of the most pressing needs in terms of anthrax vaccine research. Full article

(This article belongs to the Special Issue An Update on Anthrax)

Other

Jump to: Research, Review

6 pages, 1923 KiB

Open AccessCase Report

Case Report of an Injectional Anthrax in France, 2012

by Jean-Marc Thouret, Olivier Rogeaux, Emmanuel Beaudouin, Marion Levast, Vincent Ramisse, Fabrice V. Biot, Eric Valade, François Thibault, Olivier Gorgé and Jean-Nicolas Tournier

Microorganisms 2020, 8(7), 985; https://doi.org/10.3390/microorganisms8070985 - 30 Jun 2020

Cited by 4 | Viewed by 2280

Abstract

(1) Background: Bacillus anthracis is a spore-forming, Gram-positive bacterium causing anthrax, a zoonosis affecting mainly livestock. When occasionally infecting humans, B. anthracis provokes three different clinical forms: cutaneous, digestive and inhalational anthrax. More recently, an injectional anthrax form has been described in intravenous [...] Read more.

(1) Background: Bacillus anthracis is a spore-forming, Gram-positive bacterium causing anthrax, a zoonosis affecting mainly livestock. When occasionally infecting humans, B. anthracis provokes three different clinical forms: cutaneous, digestive and inhalational anthrax. More recently, an injectional anthrax form has been described in intravenous drug users. (2) Case presentation: We report here the clinical and microbiological features, as well as the strain phylogenetic analysis, of the only injectional anthrax case observed in France so far. A 27-year-old patient presented a massive dermohypodermatitis with an extensive edema of the right arm, and the development of drug-resistant shocks. After three weeks in an intensive care unit, the patient recovered, but the microbiological identification of B. anthracis was achieved after a long delay. (3) Conclusions: Anthrax diagnostic may be difficult clinically and microbiologically. The phylogenetic analysis of the Bacillus anthracis strain PF1 confirmed its relatedness to the injectional anthrax European outbreak group-II. Full article

(This article belongs to the Special Issue An Update on Anthrax)

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