Structural Integrity of the Alveolar-Capillary Barrier in Cynomolgus Monkeys Challenged with Fully Virulent and Toxin-Deficient Strains of Bacillus anthracis.

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection.

The Membrane Proteome of Spores and Vegetative Cells of the Food-Borne Pathogen Bacillus cereus.

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

Development of Protective Immunity in New Zealand White Rabbits Challenged with Bacillus anthracis Spores and Treated with Antibiotics and Obiltoxaximab, a Monoclonal Antibody against Protective Antigen.

The recommended management of inhalational anthrax, a high-priority bioterrorist threat, includes antibiotics and antitoxins. Obiltoxaximab, a chimeric monoclonal antibody against anthrax protective antigen (PA), is licensed under the U.S. Food and Drug Administration's (FDA's) Animal Rule for the treatment of inhalational anthrax. Because of spore latency, disease reemergence after treatment cessation is a concern, and there is a need to understand the development of endogenous protective immune responses following antitoxin-containing anthrax treatment regimens. Here, acquired protective immunity was examined in New Zealand White (NZW) rabbits challenged with a targeted lethal dose of Bacillus anthracis spores and treated with antibiotics, obiltoxaximab, or a combination of both. Survivors of the primary challenge were rechallenged 9 months later and monitored for survival. Survival rates after primary and rechallenge for controls and animals treated with obiltoxaximab, levofloxacin, or a combination of both were 0, 65, 100, and 95%, and 0, 100, 95, and 89%, respectively. All surviving immune animals had circulating antibodies to PA and serum toxin-neutralizing titers prior to rechallenge. Following rechallenge, systemic bacteremia and toxemia were not detected in most animals, and the levels of circulating anti-PA IgG titers increased starting at 5 days postrechallenge. We conclude that treatment with obiltoxaximab, alone or combined with antibiotics, significantly improves the survival of rabbits that received a lethal inhalation B. anthracis spore challenge dose and does not interfere with the development of immunity. Survivors of primary challenge are protected against reexposure, have rare incidents of systemic bacteremia and toxemia, and have evidence of an anamnestic response.

Gene expression profiling of primary human type I alveolar epithelial cells exposed to Bacillus anthracis spores reveals induction of neutrophil and monocyte chemokines.

The lung is the entry site for Bacillus anthracis in inhalation anthrax, the most deadly form of the disease. Spores must escape through the alveolar epithelial cell (AEC) barrier and migrate to regional lymph nodes, germinate and enter the circulatory system to cause disease. Several mechanisms to explain alveolar escape have been postulated, and all these tacitly involve the AEC barrier. In this study, we incorporate our primary human type I AEC model, microarray and gene enrichment analysis, qRT-PCR, multiplex ELISA, and neutrophil and monocyte chemotaxis assays to study the response of AEC to B. anthracis, (Sterne) spores at 4 and 24 h post-exposure. Spore exposure altered gene expression in AEC after 4 and 24 h and differentially expressed genes (±1.3 fold, p ≤ 0.05) included CCL4/MIP-1ß (4 h), CXCL8/IL-8 (4 and 24 h) and CXCL5/ENA-78 (24 h). Gene enrichment analysis revealed that pathways involving cytokine or chemokine activity, receptor binding, and innate immune responses to infection were prominent. Microarray results were confirmed by qRT-PCR and multiplex ELISA assays. Chemotaxis assays demonstrated that spores induced the release of biologically active neutrophil and monocyte chemokines, and that CXCL8/IL-8 was the major neutrophil chemokine. The small or sub-chemotactic doses of CXCL5/ENA-78, CXCL2/GROß and CCL20/MIP-3α may contribute to chemotaxis by priming effects. These data provide the first whole transcriptomic description of the human type I AEC initial response to B. anthracis spore exposure. Taken together, our findings contribute to an increased understanding of the role of AEC in the pathogenesis of inhalational anthrax.

The Spore Coat Protein CotE Facilitates Host Colonization by Clostridium difficile.

Clostridium difficile infection (CDI) is an important hospital-acquired infection resulting from the germination of spores in the intestine as a consequence of antibiotic-mediated dysbiosis of the gut microbiota. Key to this is CotE, a protein displayed on the spore surface and carrying 2 functional elements, an N-terminal peroxiredoxin and a C-terminal chitinase domain. Using isogenic mutants, we show in vitro and ex vivo that CotE enables binding of spores to mucus by direct interaction with mucin and contributes to its degradation. In animal models of CDI, we show that when CotE is absent, both colonization and virulence were markedly reduced. We demonstrate here that the attachment of spores to the intestine is essential in the development of CDI. Spores are usually regarded as biochemically dormant, but our findings demonstrate that rather than being simply agents of transmission and dissemination, spores directly contribute to the establishment and promotion of disease.

Bacterial Cooperation Causes Systematic Errors in Pathogen Risk Assessment due to the Failure of the Independent Action Hypothesis.

The Independent Action Hypothesis (IAH) states that pathogenic individuals (cells, spores, virus particles etc.) behave independently of each other, so that each has an independent probability of causing systemic infection or death. The IAH is not just of basic scientific interest; it forms the basis of our current estimates of infectious disease risk in humans. Despite the important role of the IAH in managing disease interventions for food and water-borne pathogens, experimental support for the IAH in bacterial pathogens is indirect at best. Moreover since the IAH was first proposed, cooperative behaviors have been discovered in a wide range of microorganisms, including many pathogens. A fundamental principle of cooperation is that the fitness of individuals is affected by the presence and behaviors of others, which is contrary to the assumption of independent action. In this paper, we test the IAH in Bacillus thuringiensis (B.t), a widely occurring insect pathogen that releases toxins that benefit others in the inoculum, infecting the diamondback moth, Plutella xylostella. By experimentally separating B.t. spores from their toxins, we demonstrate that the IAH fails because there is an interaction between toxin and spore effects on mortality, where the toxin effect is synergistic and cannot be accommodated by independence assumptions. Finally, we show that applying recommended IAH dose-response models to high dose data leads to systematic overestimation of mortality risks at low doses, due to the presence of synergistic pathogen interactions. Our results show that cooperative secretions can easily invalidate the IAH, and that such mechanistic details should be incorporated into pathogen risk analysis.

A Mathematical Model of Anthrax Transmission in Animal Populations.

A general mathematical model of anthrax (caused by Bacillus anthracis) transmission is formulated that includes live animals, infected carcasses and spores in the environment. The basic reproduction number [Formula: see text] is calculated, and existence of a unique endemic equilibrium is established for [Formula: see text] above the threshold value 1. Using data from the literature, elasticity indices for [Formula: see text] and type reproduction numbers are computed to quantify anthrax control measures. Including only herbivorous animals, anthrax is eradicated if [Formula: see text]. For these animals, oscillatory solutions arising from Hopf bifurcations are numerically shown to exist for certain parameter values with [Formula: see text] and to have periodicity as observed from anthrax data. Including carnivores and assuming no disease-related death, anthrax again goes extinct below the threshold. Local stability of the endemic equilibrium is established above the threshold; thus, periodic solutions are not possible for these populations. It is shown numerically that oscillations in spore growth may drive oscillations in animal populations; however, the total number of infected animals remains about the same as with constant spore growth.

Protection of farm goats by combinations of recombinant peptides and formalin inactivated spores from a lethal Bacillus anthracis challenge under field conditions.

BACKGROUND: Bacillus (B.) anthracis, the causal agent of anthrax, is effectively controlled by the Sterne live spore vaccine (34F2) in animals. However, live spore vaccines are not suitable for simultaneous vaccination and antibiotic treatment of animals being at risk of infection in an outbreak situation. Non-living vaccines could close this gap. RESULTS: In this study a combination of recombinant protective antigen and recombinant Bacillus collagen-like antigen (rBclA) with or without formalin inactivated spores (FIS), targeted at raising an immune response against both the toxins and the spore of B. anthracis, was tested for immunogenicity and protectiveness in goats. Two groups of goats received from local farmers of the Kars region of Turkey were immunized thrice in three weeks intervals and challenged together with non-vaccinated controls with virulent B. anthracis, four weeks after last immunization. In spite of low or none measurable toxin neutralizing antibodies and a surprisingly low immune response to the rBclA, 80% of the goats receiving the complete vaccine were protected against a lethal challenge. Moreover, the course of antibody responses indicates that a two-step vaccination schedule could be sufficient for protection. CONCLUSION: The combination of recombinant protein antigens and FIS induces a protective immune response in goats. The non-living nature of this vaccine would allow for a concomitant antibiotic treatment and vaccination procedure. Further studies should clarify how this vaccine candidate performs in a post infection scenario controlled by antibiotics.

Bile acid sensitivity and in vivo virulence of clinical Clostridium difficile isolates.

Clostridium difficile is an anaerobic bacterium that causes diarrheal illnesses. Disease onset is linked with exposure to oral antibiotics and consequent depletion of secondary bile acids. Here we investigate the relationship between in vitro secondary bile acid tolerance and in vivo disease scores of diverse C. difficile strains in mice.

Clostridium perfringens: Comparative effects of heat and osmotic stress on non-enterotoxigenic and enterotoxigenic strains.

Clostridium perfringens isolates associated with food poisoning carries a chromosomal cpe gene, while non-foodborne human gastrointestinal disease isolates carry a plasmid cpe gene. The enterotoxigenic strains tested produced vegetative cells and spores with significantly higher resistance than non-enterotoxigenic strains. These results suggest that the vegetative cells and spores have a competitive advantage over non-enterotoxigenic strains. However, no explanation has been provided for the significant associations between chromosomal cpe genotypes with the high resistance, which could explain the strong relationship between chromosomal cpe isolates and C. perfringens type A food poisoning. Here, we analyse the action of physical and chemical agent on non-enterotoxigenic and enterotoxigenic regional strains. And this study tested the relationship between the sensitivities of spores and their levels SASPs (small acid soluble proteins) production in the same strains examined.

Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains.

Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.

Clostridium difficile infection (CDI) following antibiotic therapy is a major public health threat. While antibiotic disruption of the indigenous microbiota underlies the majority of cases of CDI, the early dynamics of infection in the disturbed intestinal ecosystem are poorly characterized. This study defines the dynamics of infection with C. difficile strain VPI 10463 throughout the gastrointestinal (GI) tract using a murine model of infection. After inducing susceptibility to C. difficile colonization via antibiotic administration, we followed the dynamics of spore germination, colonization, sporulation, toxin activity, and disease progression throughout the GI tract. C. difficile spores were able to germinate within 6 h postchallenge, resulting in the establishment of vegetative bacteria in the distal GI tract. Spores and cytotoxin activity were detected by 24 h postchallenge, and histopathologic colitis developed by 30 h. Within 36 h, all infected mice succumbed to infection. We correlated the establishment of infection with changes in the microbiota and bile acid profile of the small and large intestines. Antibiotic administration resulted in significant changes to the microbiota in the small and large intestines, as well as a significant shift in the abundance of primary and secondary bile acids. Ex vivo analysis suggested the small intestine as the site of spore germination. This study provides an integrated understanding of the timing and location of the events surrounding C. difficile colonization and identifies potential targets for the development of new therapeutic strategies.

The spore-associated protein BclA1 affects the susceptibility of animals to colonization and infection by Clostridium difficile.

The BclA protein is a major component of the outermost layer of spores of a number of bacterial species and Clostridium difficile carries three bclA genes. Using insertional mutagenesis each gene was characterized and spores devoid of these proteins had surface aberrations, reduced hydrophobicity and germinated faster than wild-type spores. Therefore the BclA proteins were likely major components of the spore surface and when absent impaired the protective shield effect of this outermost layer. Analysis of infection and colonization in mice and hamsters revealed that the 50% infectious dose (ID50 ) of spores was significantly higher (2-logs) in the bclA1(-) mutant compared to the isogenic wild-type control, but that levels of toxins (A and B) were indistinguishable from animals dosed with wild-type spores. bclA1(-) spores germinated faster than wild-type spores yet mice were less susceptible to infection suggesting that BclA1 must play a key role in the initial (i.e. pre-spore germination) stages of infection. We also show that the ID50 was higher in mice infected with R20291, a 'hypervirulent' 027 strain, that carries a truncated BclA1 protein.

Efficacy of Single and Combined Antibiotic Treatments of Anthrax in Rabbits.

Respiratory anthrax is a fatal disease in the absence of early treatment with antibiotics. Rabbits are highly susceptible to infection with Bacillus anthracis spores by intranasal instillation, succumbing within 2 to 4 days postinfection. This study aims to test the efficiency of antibiotic therapy to treat systemic anthrax in this relevant animal model. Delaying the initiation of antibiotic administration to more than 24 h postinfection resulted in animals with systemic anthrax in various degrees of bacteremia and toxemia. As the onset of symptoms in humans was reported to start on days 1 to 7 postexposure, delaying the initiation of treatment by 24 to 48 h (time frame for mass distribution of antibiotics) may result in sick populations. We evaluated the efficacy of antibiotic administration as a function of bacteremia levels at the time of treatment initiation. Here we compare the efficacy of treatment with clarithromycin, amoxicillin-clavulanic acid (Augmentin), imipenem, vancomycin, rifampin, and linezolid to the previously reported efficacy of doxycycline and ciprofloxacin. We demonstrate that treatment with amoxicillin-clavulanic acid, imipenem, vancomycin, and linezolid were as effective as doxycycline and ciprofloxacin, curing rabbits exhibiting bacteremia levels of up to 10(5) CFU/ml. Clarithromycin and rifampin were shown to be effective only as a postexposure prophylactic treatment but failed to treat the systemic (bacteremic) phase of anthrax. Furthermore, we evaluate the contribution of combined treatment of clindamycin and ciprofloxacin, which demonstrated improvement in efficacy compared to ciprofloxacin alone.

Lethal factor, but not edema factor, is required to cause fatal anthrax in cynomolgus macaques after pulmonary spore challenge.

Inhalational anthrax is caused by inhalation of Bacillus anthracis spores. The ability of B. anthracis to cause anthrax is attributed to the plasmid-encoded A/B-type toxins, edema toxin (edema factor and protective antigen) and lethal toxin (lethal factor and protective antigen), and a poly-d-glutamic acid capsule. To better understand the contribution of these toxins to the disease pathophysiology in vivo, we used B. anthracis Ames strain and isogenic toxin deletion mutants derived from the Ames strain to examine the role of lethal toxin and edema toxin after pulmonary spore challenge of cynomolgus macaques. Lethal toxin, but not edema toxin, was required to induce sustained bacteremia and death after pulmonary challenge with spores delivered via bronchoscopy. After intravenous challenge with bacilli to model the systemic phase of infection, lethal toxin contributed to bacterial proliferation and subsequent host death to a greater extent than edema toxin. Deletion of protective antigen resulted in greater loss of virulence after intravenous challenge with bacilli than deletion of lethal toxin or edema toxin alone. These findings are consistent with the ability of anti-protective antigen antibodies to prevent anthrax and suggest that lethal factor is the dominant toxin that contributes to the escape of significant numbers of bacilli from the thoracic cavity to cause anthrax after inhalation challenge with spores.

Characterization of the spore-forming Bacillus cereus sensu lato group and Clostridium perfringens bacteria isolated from the Australian dairy farm environment.

BACKGROUND: The Bacillus cereus sensu lato group and Clostridium perfringens are spore-forming bacteria often associated with food spoilage and which can cause emetic and diarrheal syndromes in humans and ruminants. This study characterised the phenotypes and genotypes of 50 Bacillus cereus s. l. isolates and 26 Clostridium perfringens isolates from dairy farms environments in Victoria, Australia. RESULTS: Five of the seven B. cereus s. l. species were isolated, and analysis of the population diversity using Pulsed-Field Gel Electrophoresis (PFGE) suggested that the populations are largely distinct to each farm. Enterotoxin production by representative isolates of each B. cereus s. l. species identified was typically found to be reduced in milk, compared with broth. Among the C. perfringens isolates, only two different toxin types were identified, type A and D. Bovine and ovine farms harbored only type A whereas both type A and D were found on two of the three caprine farms. CONCLUSIONS: This study showed that the B. cereus s. l. populations on the sampled farms exhibit a broad diversity in both species and genotypes. The risk of toxin-induced diarrheal illness through consumption of contaminated milk may be limited, in comparison with other food matrices. Type A strains of C. perfringens were the most abundant on dairy farms in Victoria, however type D may be of concern on caprine farms as it can cause enterotoxemia in goats.

In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.

A possible route for foodborne transmission of Clostridium difficile?

Spores of toxigenic Clostridium difficile and spores of food-poisoning strains of Clostridium perfringens show a similar prevalence in meats. Spores of both species are heat resistant and can survive cooking of foods. C. perfringens is a major cause of foodborne illness; studies are needed to determine whether C. difficile transmission by a similar route is a cause of infection.

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes.

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

Progress and novel strategies in vaccine development and treatment of anthrax.

The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.