Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification.

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and "pineapple-like" using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM. In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting "standard" methods when attempting to compare results between studies and to understand the significance of their differences.

An in vitro evaluation of epigallocatechin gallate (eGCG) as a biocompatible inhibitor of ricin toxin.

The catechin, epigallocatechin gallate (eGCG), found in green tea, has inhibitory activity against a number of protein toxins and was investigated in relation to its impact upon ricin toxin (RT) in vitro. The IC(50) for RT was 0.08±0.004 ng/mL whereas the IC(50) for RT+100 µM eGCG was 3.02±0.572 ng/mL, indicating that eGCG mediated a significant (p<0.0001) reduction in ricin toxicity. This experiment was repeated in the human macrophage cell line THP-1 and IC(50) values were obtained for RT (0.54±0.024 ng/mL) and RT+100 µM eGCG (0.68±0.235 ng/mL) again using 100 µM eGCG and was significant (p=0.0013). The documented reduction in ricin toxicity mediated by eGCG was found to be eGCG concentration dependent, with 80 and 100 µg/mL (i.e. 178 and 223 µM respectively) of eGCG mediating a significant (p=0.0472 and 0.0232) reduction in ricin toxicity at 20 and 4 ng/ml of RT in Vero and THP-1 cells (respectively). When viability was measured in THP-1 cells by propidium iodide exclusion (as opposed to the MTT assays used previously) 10 ng/mL and 5 ng/mL of RT was used. The addition of 1000 µM and 100 µM eGCG mediated a significant (p=0.0015 and <0.0001 respectively) reduction in ricin toxicity relative to an identical concentration of ricin with 1 µg eGCG. Further, eGCG (100 µM) was found to reduce the binding of RT B chain to lactose-conjugated Sepharose as well as significantly (p=0.0039) reduce the uptake of RT B chain in Vero cells. This data suggests that eGCG may provide a starting point to refine biocompatible substances that can reduce the lethality of ricin.

Virulence plasmid stability in environmentally occurring Bacillus anthracis from North East Turkey.

The Bacillus anthracis virulence plasmid pXO2, which encodes for a polypeptide capsule, can be lost during long term laboratory storage. To determine if pXO2 is lost in nature we screened B. anthracis isolates obtained from B. anthracis spores from contaminated animal burial sites in Turkey for their ability to express a capsule upon primary culture. A total of 672 B. anthracis colonies were examined of which ten produced a mixed mucoid (capsule +ve)/non-mucoid (capsule -ve) phenotype and a further one colony yielded non-mucoid colonies upon repeated culture. Screening by PCR using pXO2 specific primers revealed that seven of these isolates had eliminated the plasmid. Of the four colonies which were positive by PCR, one regained the ability to express a capsule upon repeated culture suggesting that the defect was reversible. This is an important observation as capsule expression is a principal marker of virulence and in the absence of PCR serves as a key diagnostic marker. The results of this preliminary study suggest that pXO2 is lost in nature and that further studies are need to determine the mechanisms by which this occurs.

Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity.

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

Human Exposure to Naturally Occurring Bacillus anthracis in the Kars Region of Eastern Türkiye.

Environmental contamination with Bacillus anthracis spores poses uncertain threats to human health. We undertook a study to determine whether inhabitants of the anthrax-endemic region of Kars in eastern Türkiye could develop immune responses to anthrax toxins without recognised clinical infection. We measured anti-PA and anti-LF IgG antibody concentrations by ELISA in serum from 279 volunteers, 105 of whom had previously diagnosed anthrax infection (100 cutaneous, 5 gastrointestinal). Of the 174 without history of infection, 72 had prior contact with anthrax-contaminated material. Individuals were classified according to demographic parameters, daily working environment, and residence type. All villages in this study had recorded previous animal or human anthrax cases. Stepwise regression analyses showed that prior clinical infection correlated strongly with concentrations at the upper end of the ranges observed for both antibodies. For anti-PA, being a butcher and duration of continuous exposure risk correlated with high concentrations, while being a veterinarian or shepherd, time since infection, and town residence correlated with low concentrations. For anti-LF, village residence correlated with high concentrations, while infection limited to fingers or thumbs correlated with low concentrations. Linear discriminant analysis identified antibody concentration profiles associated with known prior infection. Profiles least typical of prior infection were observed in urban dwellers with known previous infection and in veterinarians without history of infection. Four individuals without history of infection (two butchers, two rural dwellers) had profiles suggesting unrecognised prior infection. Healthy humans therefore appear able to tolerate low-level exposure to environmental B. anthracis spores without ill effect, but it remains to be determined whether this exposure is protective. These findings have implications for authorities tasked with reducing the risk posed to human health by spore-contaminated materials and environments.

Rapid, Point-of-Care Microwave Lysis and Electrochemical Detection of Clostridioides difficile Directly from Stool Samples.

The rapid detection of the spore form of Clostridioides difficile has remained a challenge for clinicians. To address this, we have developed a novel, precise, microwave-enhanced approach for near-spontaneous release of DNA from C. difficile spores via a bespoke microwave lysis platform. C. difficile spores were microwave-irradiated for 5 s in a pulsed microwave electric field at 2.45 GHz to lyse the spore and bacteria in each sample, which was then added to a screen-printed electrode and electrochemical DNA biosensor assay system to identify presence of the pathogen's two toxin genes. The microwave lysis method released both single-stranded and double-stranded genome DNA from the bacterium at quantifiable concentrations between 0.02 µg/mL to 250 µg/mL allowing for subsequent downstream detection in the biosensor. The electrochemical bench-top system comprises of oligonucleotide probes specific to conserved regions within tcdA and tcdB toxin genes of C. difficile and was able to detect 800 spores of C. difficile within 300 µL of unprocessed human stool samples in under 10 min. These results demonstrate the feasibility of using a solid-state power generated, pulsed microwave electric field to lyse and release DNA from human stool infected with C. difficile spores. This rapid microwave lysis method enhanced the rapidity of subsequent electrochemical detection in the development of a rapid point-of-care biosensor platform for C. difficile.

Assessing the Feasibility of Employing a Combination of a Bacteriophage-Derived Endolysin and Spore Germinants to Treat Relapsing Clostridioides difficile Infection.

Clostridioides difficile is a Gram-positive, anaerobic, spore-forming bacillus and is a major cause of healthcare-associated infections. Whereas the vegetative form of the pathogen is susceptible to treatment with antibiotics, its ability to persist in the gut as antibiotic-resistant spores means that reinfection can occur in cases were the individual fails to re-establish a protective microflora. Bacteriophages and their lysins are currently being explored as treatment options due to their specificity, which minimizes the disruption to the other members of the gut microflora that are protective. The feasibility of employing recombinant endolysins to target the vegetative form of C. difficile has been demonstrated in animal models. In this study, we cloned and expressed the enzyme active domain of LysCD6356 and confirmed its ability to lyse the vegetative forms of a diverse range of clinical isolates of C. difficile, which included members of the hypervirulent 027 ribotype. Lytic activity was adversely affected by calcium, which is naturally found in the gut and is released from the spore upon germination. Our results suggests that a strategy in which the triggering of spore germination is separated in time from the application of the lysin could be developed as a strategy to reduce the risk of relapsing C. difficile infections.

Human Anthrax: Update of the Diagnosis and Treatment.

Anthrax is one of the most important zoonotic diseases which primarily infects herbivores and occasionally humans. The etiological agent is Bacillus anthracis which is a Gram-positive, aerobic, spore-forming, nonmotile, rod-shaped bacillus. The spores are resistant to environmental conditions and remain viable for a long time in contaminated soil, which is the main reservoir for wild and domestic mammals. Infections still occur in low-income countries where they cause suffering and economic hardship. Humans are infected by contact with ill or dead animals, contaminated animal products, directly exposed to the spores in the environment or spores released as a consequence of a bioterrorist event. Three classical clinical forms of the disease, cutaneous, gastrointestinal and inhalation, are seen, all of which can potentially lead to sepsis or meningitis. A new clinical form in drug users has been described recently and named "injectional anthrax" with high mortality (>33%). The symptoms of anthrax in the early stage mimics many diseases and as a consequence it is important to confirm the diagnosis using a bacterial culture or a molecular test. With regards to treatment, human isolates are generally susceptible to most antibiotics with penicillin G and amoxicillin as the first choice, and ciprofloxacin and doxycycline serving as alternatives. A combination of one or more antibiotics is suggested in systemic anthrax. Controlling anthrax in humans depends primarily on effective control of the disease in animals. Spore vaccines are used in veterinary service, and an acellular vaccine is available for humans but its use is limited.

The influence of extrachromosomal elements in the anthrax "cross-over" strain Bacillus cereus G9241.

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes.

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.

Contribution of spores to the ability of Clostridium difficile to adhere to surfaces.

Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species.

Natural exposure to cutaneous anthrax gives long-lasting T cell immunity encompassing infection-specific epitopes.

There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.

Impact of HLA Polymorphism on the Immune Response to Bacillus Anthracis Protective Antigen in Vaccination versus Natural Infection.

The causative agent of anthrax, Bacillus anthracis, evades the host immune response and establishes infection through the production of binary exotoxins composed of Protective Antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). The majority of vaccination strategies have focused upon the antibody response to the PA subunit. We have used a panel of humanised HLA class II transgenic mouse strains to define HLA-DR-restricted and HLA-DQ-restricted CD4+ T cell responses to the immunodominant epitopes of PA. This was correlated with the binding affinities of epitopes to HLA class II molecules, as well as the responses of two human cohorts: individuals vaccinated with the Anthrax Vaccine Precipitated (AVP) vaccine (which contains PA and trace amounts of LF), and patients recovering from cutaneous anthrax infections. The infected and vaccinated cohorts expressing different HLA types were found to make CD4+ T cell responses to multiple and diverse epitopes of PA. The effects of HLA polymorphism were explored using transgenic mouse lines, which demonstrated differential susceptibility, indicating that HLA-DR1 and HLA-DQ8 alleles conferred protective immunity relative to HLA-DR15, HLA-DR4 and HLA-DQ6. The HLA transgenics enabled a reductionist approach, allowing us to better define CD4+ T cell epitopes. Appreciating the effects of HLA polymorphism on the variability of responses to natural infection and vaccination is vital in planning protective strategies against anthrax.

A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge.

The health and economic burden of infectious diseases in general and bioterrorism in particular necessitate the development of medical countermeasures. One proven approach to reduce the disease burden and spread of pathogen is treatment with monoclonal antibodies (mAb). mAbs can prevent or reduce severity of the disease by variety of mechanisms, including neutralizing pathogen growth, limiting its spread from infected to adjacent cells, or by inhibiting biological activity of toxins, such as anthrax lethal toxin. Here, we report the production of glycosylated (pp-mAb (PA) ) and non-glycosylated (pp-mAb (PANG) ) versions of a plant-derived mAb directed against protective antigen (PA) of Bacillus anthracis in Nicotiana benthamiana plants using agroinfiltration. Both forms of the antibody were able to neutralize anthrax lethal toxin activity in vitro and protect mice against an intraperitoneal challenge with spores of B. anthracis Sterne strain. A single 180 µg intraperitoneal dose of pp-mAb (PA) or pp-mAb (PANG) provided 90% and 100% survival, respectively. When tested in non-human primates, pp-mAb (PANG) was demonstrated to be superior to pp-mAb (PA) in that it had a significantly longer terminal half-life and conferred 100% protection against a lethal dose of aerosolized anthrax spore challenge after a single 5 mg/kg intravenous dose compared to a 40% survival rate conferred by pp-mAb (PA) . This study demonstrates the potential of a plant-produced non-glycosylated antibody as a useful tool for the treatment of inhalation anthrax.

Bacillus anthracis endospores regulate ornithine decarboxylase and inducible nitric oxide synthase through ERK1/2 and p38 mitogen-activated protein kinases.

Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide ((•)NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing L-ornithine at the expense of (•)NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.

Development of a multiple-antigen protein fusion vaccine candidate that confers protection against Bacillus anthracis and Yersinia pestis.

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.

Extraction and detection of DNA from Bacillus anthracis spores and the vegetative cells within 1 min.

The use of a combination of low-cost technologies to both extract and detect anthrax DNA from spores and vegetative cells in two steps within 1 min is described. In a cavity, microwave energy is highly focused using thin-film aluminum "bow-tie" structures, to extract DNA from whole spores within 20 s. The detection of the released DNA, from less than 1000 vegetative cells, without additional preprocessing steps is accomplished in an additional 30 s by employing the microwave-accelerated metal-enhanced fluorescence technique. The new platform technology presented here is a highly attractive alternative method for DNA extraction and the fast detection of gram-positive bacteria and potentially other pathogenic species and cells as well.

Target discovery focused approaches to overcome bottlenecks in the exploitation of antimycobacterial natural products.

Tuberculosis is a major global health hazard. The search for new antimycobacterials has focused on such as screening combinational chemistry libraries or designing chemicals to target predefined pockets of essential bacterial proteins. The relative ineffectiveness of these has led to a reappraisal of natural products for new antimycobacterial drug leads. However, progress has been limited, we suggest through a failure in many cases to define the drug target and optimize the hits using this information. We highlight methods of target discovery needed to develop a drug into a candidate for clinical trials. We incorporate these into suggested analysis pipelines which could inform the research strategies to accelerate the development of new drug leads from natural products.

Untargeted metabolomics reveals a new mode of action of pretomanid (PA-824).

Pretomanid is a promising anti-tubercular drug currently at clinical phase III, but its mechanisms of action are currently unclear. This study aimed to: (i) reveal the metabolome of Mycobacterium smegmatis under pretomanid treatment; (ii) compare major sources of metabolite variation in bacteria treated with pretomanid treatment and other antibiotics; and (iii) to target metabolites responsible for the killing activity of pretomanid in mycobacteria. Untargeted high-resolution metabolite profiling was carried out using flow infusion electrospray ion high resolution mass spectrometry (FIE-HRMS) to identify and quantify metabolites. The identification of key metabolites was independently confirmed by gas-chromatography time-of flight mass spectrometry (GC-tofMS) in comparison to standards. Pretomanid treatments generated a unique distinctive metabolite profile when compared to ampicillin, ethambutol, ethionamide, isoniazid, kanamycin, linezolid, rifampicin and streptomycin. Metabolites which differed significantly only with pretomanid treatment were identified and mapped on to bacterial metabolic pathways. This targeted the pentose phosphate pathway with significant accumulation seen with fructose-6-phosphate, ribose-5-phosphate and glyceraldehyde-3-phosphate. These effects were linked to the accumulation of a toxic metabolite methylglyoxal. This compound showed significant antimicrobial activity (MIC 0.65 mM) against M. smegmatis.

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

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