ResearchPad - bacillus-anthracis https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Serological evidence for human exposure to <i>Bacillus cereus</i> biovar <i>anthracis</i> in the villages around Taï National Park, Côte d’Ivoire]]> https://www.researchpad.co/article/elastic_article_14539 Anthrax is a zoonotic disease transmitted from animals to humans and normally caused by B. anthracis mainly in savanna regions. However, untypical bacteria named Bacillus cereus biovar anthracis (Bcbva) were detected in a variety of wild animals in the rain forest region of the Taï National Park (TNP) in Côte d’Ivoire. No anthrax infections in humans living in the region around TNP were reported until now. Therefore, we assessed exposure to the pathogen by analysis of sera from human volunteers for the presence of antibodies against the protective antigen (PA), which is produced by B. anthracis and Bcbva, and against the Bcbva-specific protein pXO2-60. We found antibodies against PA in more than 20% of sera from humans living in the TNP region, and around 10% possessed also antibodies against pXO2-60, confirming exposure to Bcbva. As only Bcbva, but not classic B. anthracis was found in TNP, we assume that the majority of humans had contact with Bcbva and that pXO2-60 is less immunogenic than PA. Although most people reported animal contacts, there was no statistically significant correlation with the presence of antibodies against Bcbva. Nevertheless, our study confirmed that Bcbva represents a danger for humans living in the affected area.

]]>
<![CDATA[Toxin-neutralizing antibodies elicited by naturally acquired cutaneous anthrax are elevated following severe disease and appear to target conformational epitopes]]> https://www.researchpad.co/article/N0733fdcc-4c39-44e4-82cd-032e69d54dbc

Understanding immune responses to native antigens in response to natural infections can lead to improved approaches to vaccination. This study sought to characterize the humoral immune response to anthrax toxin components, capsule and spore antigens in individuals (n = 46) from the Kayseri and Malatya regions of Turkey who had recovered from mild or severe forms of cutaneous anthrax infection, compared to regional healthy controls (n = 20). IgG antibodies to each toxin component, the poly-γ-D-glutamic acid capsule, the Bacillus collagen-like protein of anthracis (BclA) spore antigen, and the spore carbohydrate anthrose, were detected in the cases, with anthrax toxin neutralization and responses to Protective Antigen (PA) and Lethal Factor (LF) being higher following severe forms of the disease. Significant correlative relationships among responses to PA, LF, Edema Factor (EF) and capsule were observed among the cases. Though some regional control sera exhibited binding to a subset of the tested antigens, these samples did not neutralize anthrax toxins and lacked correlative relationships among antigen binding specificities observed in the cases. Comparison of serum binding to overlapping decapeptides covering the entire length of PA, LF and EF proteins in 26 cases compared to 8 regional controls revealed that anthrax toxin-neutralizing antibody responses elicited following natural cutaneous anthrax infection are directed to conformational epitopes. These studies support the concept of vaccination approaches that preserve conformational epitopes.

]]>
<![CDATA[Evaluation of liposomal ciprofloxacin formulations in a murine model of anthrax]]> https://www.researchpad.co/article/Ne17111d7-5152-4c88-81b1-0e84a1b58e42

The in vivo efficacy of liposomal encapsulated ciprofloxacin in two formulations, lipoquin and apulmiq, were evaluated against the causative agent of anthrax, Bacillus anthracis. Liposomal encapsulated ciprofloxacin is attractive as a therapy since it allows for once daily dosing and achieves higher concentrations of the antibiotic at the site of initial mucosal entry but lower systemic drug concentrations. The in vivo efficacy of lipoquin and apulmiq delivered by intranasal instillation was studied at different doses and schedules in both a post exposure prophylaxis (PEP) therapy model and in a delayed treatment model of murine inhalational anthrax. In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control. Ciprofloxacin, lipoquin and apulmiq provided a high level of protection (87–90%) after 7 days of therapy when administered within 24 hours of exposure. Reducing therapy to only three days still provided protection of 60–87%, if therapy was provided within 24 hours of exposure. If treatment was initiated 48 hours after exposure the survival rate was reduced to 46–65%. These studies suggest that lipoquin and apulmiq may be attractive therapies as PEP and as part of a treatment cocktail for B. anthracis.

]]>
<![CDATA[Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct]]> https://www.researchpad.co/article/5c8c1951d5eed0c484b4d3e6

A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion.

]]>
<![CDATA[Spatio-temporal epidemiology of anthrax in Hippopotamus amphibious in Queen Elizabeth Protected Area, Uganda]]> https://www.researchpad.co/article/5c0841a3d5eed0c484fca4e8

Background

Anthrax is a zoonotic disease primarily of herbivores, caused by Bacillus anthracis, a bacterium with diverse geographical and global distribution. Globally, livestock outbreaks have declined but in Africa significant outbreaks continue to occur with most countries still categorized as enzootic, hyper endemic or sporadic. Uganda experiences sporadic human and livestock cases. Severe large-scale outbreaks occur periodically in hippos (Hippopotamus amphibious) at Queen Elizabeth Protected Area, where in 2004/2005 and 2010 anthrax killed 437 hippos. Ecological drivers of these outbreaks and potential of hippos to maintain anthrax in the ecosystem remain unknown. This study aimed to describe spatio-temporal patterns of anthrax among hippos; examine significant trends associated with case distributions; and generate hypotheses for investigation of ecological drivers of anthrax.

Methods

Spatio-temporal patterns of 317 hippo cases in 2004/5 and 137 in 2010 were analyzed. QGIS was used to examine case distributions; Spearman’s nonparametric tests to determine correlations between cases and at-risk hippo populations; permutation models of the spatial scan statistics to examine spatio-temporal clustering of cases; directional tests to determine directionality in epidemic movements; and standard epidemic curves to determine patterns of epidemic propagation.

Key findings

Results showed hippopotamus cases extensively distributed along water shorelines with strong positive correlations (p<0.01) between cases and at-risk populations. Significant (p<0.001) spatio-temporal clustering of cases occurred throughout the epidemics, pointing towards a defined source. Significant directional epidemic spread was detected along water flow gradient (206.6°) in 2004/5 and against flow gradient (20.4°) in 2010. Temporal distributions showed clustered pulsed epidemic waves.

Conclusion

These findings suggest mixed point-source propagated pattern of epidemic spread amongst hippos and points to likelihood of indirect spread of anthrax spores between hippos mediated by their social behaviour, forces of water flow, and persistent presence of infectious carcasses amidst schools. This information sheds light on the epidemiology of anthrax in highly social wildlife, can help drive insight into disease control, wildlife conservation, and tourism management, but highlights the need for analytical and longitudinal studies aimed at clarifying the hypotheses.

]]>
<![CDATA[Fused Regression for Multi-source Gene Regulatory Network Inference]]> https://www.researchpad.co/article/5989d9e5ab0ee8fa60b6b09f

Understanding gene regulatory networks is critical to understanding cellular differentiation and response to external stimuli. Methods for global network inference have been developed and applied to a variety of species. Most approaches consider the problem of network inference independently in each species, despite evidence that gene regulation can be conserved even in distantly related species. Further, network inference is often confined to single data-types (single platforms) and single cell types. We introduce a method for multi-source network inference that allows simultaneous estimation of gene regulatory networks in multiple species or biological processes through the introduction of priors based on known gene relationships such as orthology incorporated using fused regression. This approach improves network inference performance even when orthology mapping and conservation are incomplete. We refine this method by presenting an algorithm that extracts the true conserved subnetwork from a larger set of potentially conserved interactions and demonstrate the utility of our method in cross species network inference. Last, we demonstrate our method’s utility in learning from data collected on different experimental platforms.

]]>
<![CDATA[High stringency evaluation of the inactivation / exclusion efficacy of a MALDI-TOF MS chemical extraction method, with filtration of extract through 0.1 µm filters, on Bacillus anthracis Vollum vegetative cells and spores]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf60f

A previous report indicated that a formic acid chemical extraction method for the preparation of protein extracts for matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF MS) identification, with filtration of extracts through 0.2 μm regenerated cellulose (RC) filters, would not reliably inactivate or exclude Bacillus anthracis Vollum cells or spores when tested under high stringency conditions. B. anthracis was recovered from 13/36 extracts (3/18 from vegetative cell extracts and 10/18 from bacterial spore extracts). In this paper we report the repetition of this study but with the substitution of the 0.2 μm, regenerated cellulose, filters with 0.1 μm polyvinylidene fluoride (PVDF) filters. Experiments were conducted under the same high stringency post-treatment viability test methods (100% of resulting protein content; 7 days Luria (L)-broth and a further 7 days L-agar plate incubation; or 7 days L-agar plate only incubation). B. anthracis was not recovered from any of 18 replicates generated from high concentrations of vegetative cells (107 to 108 cfu), but a single B. anthracis colony was recovered from one of 18 replicates generated from high concentrations of bacterial spores (108 cfu), using a post-treatment viability culture method of 7 days on L-agar plate only. We discuss our results in the context of other similar studies and also a requirement to develop standardised post-treatment viability test methods.

]]>
<![CDATA[The putative drug efflux systems of the Bacillus cereus group]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf5e1

The Bacillus cereus group of bacteria includes seven closely related species, three of which, B. anthracis, B. cereus and B. thuringiensis, are pathogens of humans, animals and/or insects. Preliminary investigations into the transport capabilities of different bacterial lineages suggested that genes encoding putative efflux systems were unusually abundant in the B. cereus group compared to other bacteria. To explore the drug efflux potential of the B. cereus group all putative efflux systems were identified in the genomes of prototypical strains of B. cereus, B. anthracis and B. thuringiensis using our Transporter Automated Annotation Pipeline. More than 90 putative drug efflux systems were found within each of these strains, accounting for up to 2.7% of their protein coding potential. Comparative analyses demonstrated that the efflux systems are highly conserved between these species; 70–80% of the putative efflux pumps were shared between all three strains studied. Furthermore, 82% of the putative efflux system proteins encoded by the prototypical B. cereus strain ATCC 14579 (type strain) were found to be conserved in at least 80% of 169 B. cereus group strains that have high quality genome sequences available. However, only a handful of these efflux pumps have been functionally characterized. Deletion of individual efflux pump genes from B. cereus typically had little impact to drug resistance phenotypes or the general fitness of the strains, possibly because of the large numbers of alternative efflux systems that may have overlapping substrate specificities. Therefore, to gain insight into the possible transport functions of efflux systems in B. cereus, we undertook large-scale qRT-PCR analyses of efflux pump gene expression following drug shocks and other stress treatments. Clustering of gene expression changes identified several groups of similarly regulated systems that may have overlapping drug resistance functions. In this article we review current knowledge of the small molecule efflux pumps encoded by the B. cereus group and suggest the likely functions of numerous uncharacterised pumps.

]]>
<![CDATA[Multigeneration Cross-Contamination of Mail with Bacillus anthracis Spores]]> https://www.researchpad.co/article/5989da22ab0ee8fa60b7f4a7

The release of biological agents, including those which could be used in biowarfare or bioterrorism in large urban areas, has been a concern for governments for nearly three decades. Previous incidents from Sverdlosk and the postal anthrax attack of 2001 have raised questions on the mechanism of spread of Bacillus anthracis spores as an aerosol or contaminant. Prior studies have demonstrated that Bacillus atrophaeus is easily transferred through simulated mail handing, but no reports have demonstrated this ability with Bacillus anthracis spores, which have morphological differences that may affect adhesion properties between spore and formite. In this study, equipment developed to simulate interactions across three generations of envelopes subjected to tumbling and mixing was used to evaluate the potential for cross-contamination of B. anthracis spores in simulated mail handling. In these experiments, we found that the potential for cross-contamination through letter tumbling from one generation to the next varied between generations while the presence of a fluidizer had no statistical impact on the transfer of material. Likewise, the presence or absence of a fluidizer had no statistically significant impact on cross-contamination levels or reaerosolization from letter opening.

]]>
<![CDATA[The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme]]> https://www.researchpad.co/article/5989da07ab0ee8fa60b76560

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

]]>
<![CDATA[Redefining the Australian Anthrax Belt: Modeling the Ecological Niche and Predicting the Geographic Distribution of Bacillus anthracis]]> https://www.researchpad.co/article/5989da22ab0ee8fa60b7f6f7

The ecology and distribution of B. anthracis in Australia is not well understood, despite the continued occurrence of anthrax outbreaks in the eastern states of the country. Efforts to estimate the spatial extent of the risk of disease have been limited to a qualitative definition of an anthrax belt extending from southeast Queensland through the centre of New South Wales and into northern Victoria. This definition of the anthrax belt does not consider the role of environmental conditions in the distribution of B. anthracis. Here, we used the genetic algorithm for rule-set prediction model system (GARP), historical anthrax outbreaks and environmental data to model the ecological niche of B. anthracis and predict its potential geographic distribution in Australia. Our models reveal the niche of B. anthracis in Australia is characterized by a narrow range of ecological conditions concentrated in two disjunct corridors. The most dominant corridor, used to redefine a new anthrax belt, parallels the Eastern Highlands and runs from north Victoria to central east Queensland through the centre of New South Wales. This study has redefined the anthrax belt in eastern Australia and provides insights about the ecological factors that limit the distribution of B. anthracis at the continental scale for Australia. The geographic distributions identified can help inform anthrax surveillance strategies by public and veterinary health agencies.

]]>
<![CDATA[Engineering, and production of functionally active human Furin in N. benthamiana plant: In vivo post-translational processing of target proteins by Furin in plants]]> https://www.researchpad.co/article/5c915f83d5eed0c48420aa15

A plant expression platform with eukaryotic post-translational modification (PTM) machinery has many advantages compared to other protein expression systems. This promising technology is useful for the production of a variety of recombinant proteins including, therapeutic proteins, vaccine antigens, native additives, and industrial enzymes. However, plants lack some of the important PTMs, including furin processing, which limits this system for the production of certain mammalian complex proteins of therapeutic value. Furin is a ubiquitous proprotein convertase that is involved in the processing (activation) of a wide variety of precursor proteins, including blood coagulation factors, cell surface receptors, hormones and growth factors, viral envelope glycoproteins, etc. and plays a critical regulatory role in a wide variety of cellular events. In this study, we engineered the human furin gene for expression in plants and demonstrated the production of a functional active recombinant truncated human furin in N. benthamiana plant. We demonstrate that plant produced human furin is highly active both in vivo and in vitro and specifically cleaved the tested target proteins, Factor IX (FIX) and Protective Antigen (PA83). We also demonstrate that both, enzymatic deglycosylation and proteolytic processing of target proteins can be achieved in vivo by co-expression of deglycosylating and furin cleavage enzymes in a single cell to produce deglycosylated and furin processed target proteins. It is highly expected that this strategy will have many potential applications in pharmaceutical industry and can be used to produce safe and affordable therapeutic proteins, antibodies, and vaccines using a plant expression system.

]]>
<![CDATA[Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar]]> https://www.researchpad.co/article/5989da4eab0ee8fa60b8d6c4

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.

]]>
<![CDATA[Composite Sampling of a Bacillus anthracis Surrogate with Cellulose Sponge Surface Samplers from a Nonporous Surface]]> https://www.researchpad.co/article/5989daa0ab0ee8fa60ba57df

A series of experiments was conducted to explore the utility of composite-based collection of surface samples for the detection of a Bacillus anthracis surrogate using cellulose sponge samplers on a nonporous stainless steel surface. Two composite-based collection approaches were evaluated over a surface area of 3716 cm2 (four separate 929 cm2 areas), larger than the 645 cm2 prescribed by the standard Centers for Disease Control (CDC) and Prevention cellulose sponge sampling protocol for use on nonporous surfaces. The CDC method was also compared to a modified protocol where only one surface of the sponge sampler was used for each of the four areas composited. Differences in collection efficiency compared to positive controls and the potential for contaminant transfer for each protocol were assessed. The impact of the loss of wetting buffer from the sponge sampler onto additional surface areas sampled was evaluated. Statistical tests of the results using ANOVA indicate that the collection of composite samples using the modified sampling protocol is comparable to the collection of composite samples using the standard CDC protocol (p  =  0.261). Most of the surface-bound spores are collected on the first sampling pass, suggesting that multiple passes with the sponge sampler over the same surface may be unnecessary. The effect of moisture loss from the sponge sampler on collection efficiency was not significant (p  =  0.720) for both methods. Contaminant transfer occurs with both sampling protocols, but the magnitude of transfer is significantly greater when using the standard protocol than when the modified protocol is used (p<0.001). The results of this study suggest that composite surface sampling, by either method presented here, could successfully be used to increase the surface area sampled per sponge sampler, resulting in reduced sampling times in the field and decreased laboratory processing cost and turn-around times.

]]>
<![CDATA[Genotype Analysis of Bacillus anthracis Strains Circulating in Bangladesh]]> https://www.researchpad.co/article/5989d9f1ab0ee8fa60b6e92d

In Bangladesh, anthrax, caused by the bacterium Bacillus anthracis, is considered an endemic disease affecting ruminants with sporadic zoonotic occurrences in humans. Due to the lack of knowledge about risks from an incorrect removal of infected carcasses, the disease is not properly monitored, and because of the socio-economic conditions, the situation is under-reported and under-diagnosed. For sensitive species, anthrax represents a fatal outcome with sudden death and sometimes bleeding from natural orifices. The most common source of infection for ruminants is ingestion of spores during grazing in contaminated pastures or through grass and water contaminated with anthrax spores. Domestic cattle, sheep and goats can also become infected through contaminated bone meal (used as feed) originating from anthrax-infected carcasses. The present investigation was conducted to isolate B. anthracis organisms from 169 samples (73 soil, 1 tissue, 4 bone and 91 bone meal samples) collected from 12 different districts of Bangladesh. The sampling was carried out from 2012 to 2015. Twelve samples resulted positive for B. anthracis. Biomolecular analyses were conducted starting from the Canonical Single Nucleotide Polymorphism (CanSNP) to analyze the phylogenetic origin of strains. The analysis of genotype, obtained through the Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) with the analysis of 15 Variable Number Tandem Repeats (VNTR), demonstrated four different genotypes: two of them were previously identified in the district of Sirajganj. The sub-genotyping, conducted with Single Nucleotide Repeats analysis, revealed the presence of eight subgenotypes. The data of the present study concluded that there was no observed correlation between imported cattle feed and anthrax occurrence in Bangladesh and that the remarkable genetic variations of B. anthracis were found in the soil of numerous outbreaks in this country.

]]>
<![CDATA[Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence]]> https://www.researchpad.co/article/5989da28ab0ee8fa60b81611

Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications.

]]>
<![CDATA[Production of Functionally Active and Immunogenic Non-Glycosylated Protective Antigen from Bacillus anthracis in Nicotiana benthamiana by Co-Expression with Peptide-N-Glycosidase F (PNGase F) of Flavobacterium meningosepticum]]> https://www.researchpad.co/article/5989d9f8ab0ee8fa60b711fc

Bacillus anthracis has long been considered a potential biological warfare agent, and therefore, there is a need for a safe, low-cost and highly efficient anthrax vaccine with demonstrated long-term stability for mass vaccination in case of an emergency. Many efforts have been made towards developing an anthrax vaccine based on recombinant protective antigen (rPA) of B. anthracis, a key component of the anthrax toxin, produced using different expression systems. Plants represent a promising recombinant protein production platform due to their relatively low cost, rapid scalability and favorable safety profile. Previous studies have shown that full-length rPA produced in Nicotiana benthamiana (pp-PA83) is immunogenic and can provide full protection against lethal spore challenge; however, further improvement in the potency and stability of the vaccine candidate is necessary. PA of B. anthracis is not a glycoprotein in its native host; however, this protein contains potential N-linked glycosylation sites, which can be aberrantly glycosylated during expression in eukaryotic systems including plants. This glycosylation could affect the availability of certain key epitopes either due to masking or misfolding of the protein. Therefore, a non-glycosylated form of pp-PA83 was engineered and produced in N. benthamiana using an in vivo deglycosylation approach based on co-expression of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum. For comparison, versions of pp-PA83 containing point mutations in six potential N-glycosylation sites were also engineered and expressed in N. benthamiana. The in vivo deglycosylated pp-PA83 (pp-dPA83) was shown to have in vitro activity, in contrast to glycosylated pp-PA83, and to induce significantly higher levels of toxin-neutralizing antibody responses in mice compared with glycosylated pp-PA83, in vitro deglycosylated pp-PA83 or the mutated versions of pp-PA83. These results suggest that pp-dPA83 may offer advantages in terms of dose sparing and enhanced immunogenicity as a promising candidate for a safe, effective and low-cost subunit vaccine against anthrax.

]]>
<![CDATA[Comparative analysis of the sensitivity of metagenomic sequencing and PCR to detect a biowarfare simulant (Bacillus atrophaeus) in soil samples]]> https://www.researchpad.co/article/5989db5aab0ee8fa60bdf5f8

To evaluate the sensitivity of high-throughput DNA sequencing for monitoring biowarfare agents in the environment, we analysed soil samples inoculated with different amounts of Bacillus atrophaeus, a surrogate organism for Bacillus anthracis. The soil samples considered were a poorly carbonated soil of the silty sand class, and a highly carbonated soil of the silt class. Control soil samples and soil samples inoculated with 10, 103, or 105 cfu were processed for DNA extraction. About 1% of the DNA extracts was analysed through the sequencing of more than 108 reads. Similar amounts of extracts were also studied for Bacillus atrophaeus DNA content by real-time PCR. We demonstrate that, for both soils, high-throughput sequencing is at least equally sensitive than real-time PCR to detect Bacillus atrophaeus DNA. We conclude that metagenomics allows the detection of less than 10 ppm of DNA from a biowarfare simulant in complex environmental samples.

]]>
<![CDATA[Improving Phylogeny Reconstruction at the Strain Level Using Peptidome Datasets]]> https://www.researchpad.co/article/5989dacaab0ee8fa60bb3c3d

Typical bacterial strain differentiation methods are often challenged by high genetic similarity between strains. To address this problem, we introduce a novel in silico peptide fingerprinting method based on conventional wet-lab protocols that enables the identification of potential strain-specific peptides. These can be further investigated using in vitro approaches, laying a foundation for the development of biomarker detection and application-specific methods. This novel method aims at reducing large amounts of comparative peptide data to binary matrices while maintaining a high phylogenetic resolution. The underlying case study concerns the Bacillus cereus group, namely the differentiation of Bacillus thuringiensis, Bacillus anthracis and Bacillus cereus strains. Results show that trees based on cytoplasmic and extracellular peptidomes are only marginally in conflict with those based on whole proteomes, as inferred by the established Genome-BLAST Distance Phylogeny (GBDP) method. Hence, these results indicate that the two approaches can most likely be used complementarily even in other organismal groups. The obtained results confirm previous reports about the misclassification of many strains within the B. cereus group. Moreover, our method was able to separate the B. anthracis strains with high resolution, similarly to the GBDP results as benchmarked via Bayesian inference and both Maximum Likelihood and Maximum Parsimony. In addition to the presented phylogenomic applications, whole-peptide fingerprinting might also become a valuable complementary technique to digital DNA-DNA hybridization, notably for bacterial classification at the species and subspecies level in the future.

]]>
<![CDATA[Identification and Pathogenic Potential of Clinical Bacillus and Paenibacillus Isolates]]> https://www.researchpad.co/article/5989daf0ab0ee8fa60bc0d63

The soil-related Bacillus and Paenibacillus species have increasingly been implicated in various human diseases. Nevertheless, their identification still poses problems in the clinical microbiology laboratory and, with the exception of Bacillus anthracis and Bacillus cereus, little is known on their pathogenicity for humans. In this study, we evaluated the use of matrix-assisted laser desorption—ionization time of flight mass spectrometry (MALDI-TOF MS) in the identification of clinical isolates of these genera and conducted genotypic and phenotypic analyses to highlight specific virulence properties. Seventy-five clinical isolates were subjected to biochemical and MALDI-TOF MS identification. 16S rDNA sequencing and supplemental tests were used to solve any discrepancies or failures in the identification results. MALDI-TOF MS significantly outperformed classical biochemical testing for correct species identification and no misidentification was obtained. One third of the collected strains belonged to the B. cereus species, but also Bacillus pumilus and Bacillus subtilis were isolated at high rate. Antimicrobial susceptibility testing showed that all the B. cereus, B. licheniformis, B. simplex, B. mycoides, Paenibacillus glucanolyticus and Paenibacillus lautus isolates are resistant to penicillin. The evaluation of toxin/enzyme secretion, toxin-encoding genes, motility, and biofilm formation revealed that B. cereus displays the highest virulence potential. However, although generally considered nonpathogenic, most of the other species were shown to swim, swarm, produce biofilms, and secrete proteases that can have a role in bacterial virulence. In conclusion, MALDI-TOF MS appears useful for fast and accurate identification of Bacillus and Paenibacillus strains whose virulence properties make them of increasing clinical relevance.

]]>