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.

A Stochastic Intracellular Model of Anthrax Infection With Spore Germination Heterogeneity.

We present a stochastic mathematical model of the intracellular infection dynamics of Bacillus anthracis in macrophages. Following inhalation of B. anthracis spores, these are ingested by alveolar phagocytes. Ingested spores then begin to germinate and divide intracellularly. This can lead to the eventual death of the host cell and the extracellular release of bacterial progeny. Some macrophages successfully eliminate the intracellular bacteria and will recover. Here, a stochastic birth-and-death process with catastrophe is proposed, which includes the mechanism of spore germination and maturation of B. anthracis. The resulting model is used to explore the potential for heterogeneity in the spore germination rate, with the consideration of two extreme cases for the rate distribution: continuous Gaussian and discrete Bernoulli. We make use of approximate Bayesian computation to calibrate our model using experimental measurements from in vitro infection of murine peritoneal macrophages with spores of the Sterne 34F2 strain of B. anthracis. The calibrated stochastic model allows us to compute the probability of rupture, mean time to rupture, and rupture size distribution, of a macrophage that has been infected with one spore. We also obtain the mean spore and bacterial loads over time for a population of cells, each assumed to be initially infected with a single spore. Our results support the existence of significant heterogeneity in the germination rate, with a subset of spores expected to germinate much later than the majority. Furthermore, in agreement with experimental evidence, our results suggest that most of the spores taken up by macrophages are likely to be eliminated by the host cell, but a few germinated spores may survive phagocytosis and lead to the death of the infected cell. Finally, we discuss how this stochastic modelling approach, together with dose-response data, allows us to quantify and predict individual infection risk following exposure.

Inhibition of spores to prevent the recurrence of Clostridioides difficile infection - A possibility or an improbability?

Clostridioides difficile is one of the most common nosocomial gastrointestinal pathogens, and recurrence is a problematic issue because approximately 20-30% of patients experience at least one episode of recurrence, even after treatment with a therapeutic drug of choice for C. difficile infection (CDI), such as vancomycin. CDI recurrence has a multifactorial complex mechanism, in which gut microbiota disruption coincident with viable C. difficile spores, is considered the most important factor. The effectiveness of an anti-C. difficile antimicrobial agent against CDI cannot guarantee its inhibitory effect on C. difficile spores and vice versa. However, an antimicrobial agent, such as fidaxomicin, which has a good inhibitory effect on both C. difficile vegetative cells and spores is assumed to not only treat CDI but also prevent its recurrence. Prolonged adherence to the exosporium has been proposed as a possible mechanism of inhibiting spores, and as a result, redesigning anti-C. difficile antimicrobial agents with the ability to adhere to the exosporium may provide another pathway for the development of anti-C. difficile spore agents. For example, vancomycin lacks an inhibitory effect against C. difficile spores, but a vancomycin-loaded spore-targeting iron oxide nanoparticle that selectively binds to C. difficile spores has been developed to successfully delay spore germination. Some new antimicrobial agents in phase II clinical trials, including cadazolid and ridinilazole, have shown exceptional anti-C. difficile and spore-inhibiting effects that can be expected to not only treat CDI but also prevent its recurrence in the future.

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 use of ozone gas for the inactivation of Bacillus anthracis and Bacillus subtilis spores on building materials.

A study was conducted to assess the efficacy of ozone gas in inactivating spores of both Bacillus anthracis and Bacillus subtilis inoculated onto six building materials (glass, wood, carpet, laminate, galvanized metal, and wallboard paper). Testing conditions consisted of ozone gas concentrations ranging from 7,000-12,000 parts per million (ppm), contact times from 4 to 12 h, and two relative humidity (RH) levels of 75 and 85%. Results showed that increasing the ozone concentration, contact time, and RH generally increased decontamination efficacy. The materials in which the highest decontamination efficacy was achieved for B. anthracis spores were wallboard paper, carpet, and wood with ≥ 6 log10 reduction (LR) occurring with 9,800 ppm ozone, 85% RH, for 6 h. The laminate and galvanized metal materials were generally more difficult to decontaminate, requiring 12,000 ppm ozone, 85% RH, and 9-12 h contact time to achieve ≥6 LR of B. anthracis. Lastly, overall, there were no significant differences in decontamination efficacy between the two species.

An integrated experimental-computational approach for predicting virulence in New Zealand white rabbits and humans following inhalation exposure to Bacillus anthracis spores.

Inhalation of Bacillus anthracis spores can lead to an anthrax infection that can be fatal. Previously published mathematical models have extrapolated kinetic rates associated with bacterial growth in New Zealand White (NZW) rabbits to humans, but to date, actual measurements of the underlying processes associated with anthrax virulence between species have not been conducted. To address this knowledge gap, we have quantified species-specific rate constants associated with germination, proliferation, and immune cell inactivation of B. anthracis Sterne using an in vitro test platform that includes primary lung epithelial and immune cells. The generated data was then used to develop a physiologically based biokinetic model (PBBK) which quantitatively compares bacterial growth and mean time to death under lethal conditions in rabbits and humans. Simulations based upon our in vitro data and previously published in vivo data from rabbits indicate that disease progression is likely to be faster in humans than in NZW rabbits under comparable total deposited dose conditions. With the computational framework established, PBBK parameters can now be refined using experimental data for lethal B. anthracis strains (e.g. Ames) under identical conditions in future studies. The PBBK model can also be linked to existing aerosol dosimetry models that account for species-specific differences in aerosol deposition patterns to further improve the human health risk assessment of inhalation anthrax.

A label-free impedimetric aptasensor for the detection of Bacillus anthracis spore simulant.

Herein, we report an impedimetric DNA-based aptamer sensor for a single-step detection of B. anthracis spore simulant (B. cereus spore). Specifically, we designed a miniaturized label-free aptasensor for B. cereus spores based on a gold screen-printed electrode functionalized with B. cereus spores-binding aptamer (BAS-6R). Several parameters were optimized to fabricate the aptasensor such as the concentration of DNA aptamer solution (0.5 µM), the time (48 h), the temperature (4 °C), and the pH (7.5) for aptamer immobilization on the working electrode surface. Once the aptasensor was developed, it was tested against B. cereus spores 14579 evaluating the effect of incubation time and MgCl2 concentration. Under the optimized conditions (incubation time equal to 3 h and absence of MgCl2), B. cereus spores 14579 were detected with a linear range between 104 CFU/ml and 5 × 106 CFU/ml and a detection limit of 3 × 103 CFU/ml. Furthermore, the study of selectivity toward B. cereus 11778, B. subtilis, Legionella pneumophila, and Salmonella Typhimurium has demonstrated the capability of this sensor to detect B. cereus spores, proving the suitability of the DNA-based sensing element combined with a portable instrument for a label-free measurement on site of B. anthracis spore simulant.

Hydrogen Peroxide-Assisted Ultrasonic Synthesis of BCNO QDs for Anthrax Biomarker Detection.

A facile 3% hydrogen peroxide-assisted ultrasonic synthetic strategy is demonstrated to successfully synthesize fluorescent boron carbon oxynitride quantum dots (BCNO QDs). The obtained BCNO QDs exhibit intense blue fluorescence and favorable biocompatibility and water solubility. The quantum yield of the BCNO QDs is 19.9%. Owing to the absorbance energy-transfer emission effect, an efficient ratiometric fluorescence biosensor is developed for anthrax biomarker detection based on the BCNO QD-ethylenediaminetetraacetic acid disodium salt-Eu3+ complex. Under optimal conditions, the detection limit of the anthrax biomarker is 0.5 nM. Furthermore, the sensitivity of the system was evaluated by Bacillus subtilis spores and with the detection limit as low as 1.95 × 106 spores. On combining a smartphone with the home-made BCNO QD test paper, the lowest recorded visual detection limit of 1.0 µM anthrax biomarker was achieved using a portable UV lamp. The fast response speed, excellent sensitivity, and selectivity of the approach show potential applications in clinical analysis.

Increased sporulation underpins adaptation of Clostridium difficile strain 630 to a biologically-relevant faecal environment, with implications for pathogenicity.

Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically-relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile-derived metabolites in culture supernatants, including hexanoyl- and pentanoyl-amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300-fold increases in the expression of sporulation-associated genes were observed in FW media-grown cells, along with reductions in motility and toxin genes' expression. Moreover, the expression of classical stress-response genes did not change, showing that C. difficile is well-adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility.

Spore-forming bacteria in the gut: an interview with Hilary Browne.

Hilary Browne speaks to Alice Greenway, Commissioning Editor of Future Microbiology, at the Microbiology Society Annual Conference 2018. Hilary Browne is a Staff Scientist at the Wellcome Sanger Institute in Cambridge (UK). His research focuses on culturing, curating and whole genome sequencing of the human gut microbiota and using these resources to study the human intestinal microbiota. At the Microbiology Society Annual Conference 2018, we asked him a few questions regarding his past and current research focuses, including the offered paper he presented at the conference entitled: 'Host adaptation of commensal bacteria through loss of sporulation and genome reduction'.

Linking sex differences to the evolution of infectious disease life-histories.

Sex differences in the prevalence, course and severity of infection are widespread, yet the evolutionary consequences of these differences remain unclear. Understanding how male-female differences affect the trajectory of infectious disease requires connecting the contrasting dynamics that pathogens might experience within each sex to the number of susceptible and infected individuals that are circulating in a population. In this study, we build on theory using genetic covariance functions to link the growth of a pathogen within a host to the evolution and spread of disease between individuals. Using the Daphnia-Pasteuria system as a test case, we show that on the basis of within-host dynamics alone, females seem to be more evolutionarily liable for the pathogen, with higher spore loads and greater divergence among pathogen genotypes as infection progresses. Between-host transmission, however, appears to offset the lower performance of a pathogen within a male host, making even subtle differences between the sexes evolutionarily relevant, as long as the selection generated by the between-host dynamics is sufficiently strong. Our model suggests that relatively simple differences in within-host processes occurring in males and females can lead to complex patterns of genetic constraint on pathogen evolution, particularly during an expanding epidemic.This article is part of the theme issue 'Linking local adaptation with the evolution of sex differences'.

Modeling the macrophage-anthrax spore interaction: Implications for early host-pathogen interactions.

Inhalational anthrax, caused by the gram positive bacteria Bacillus anthracis, is a potentially fatal form of anthrax infection. It is initiated after inhaled spores are deposited in the lung, phagocytosed by immune cells, and subsequently transported to nearby lymph nodes. Intracellular spores that successfully germinate and become vegetative bacteria can lyse their host cell and contribute to bacterial outgrowth and toxin production. To better understand the early disease dynamics of the host-pathogen interaction, we develop a mathematical model of ordinary differential Equations and estimate parameters using available data. The model which consists of two subsystems is designed in accordance with an in vitro experimental protocol in which macrophages were challenged with varying doses of spores at spore-to-macrophage ratios of 1:1, 1:2, 1:10, 1:20. Initial modeling results suggested the need to consider two distinct subpopulations of anthrax bacteria: newly germinated bacteria which cannot replicate immediately and fully vegetative bacteria that can. Additional modeling results provide insights into possible reasons why macrophage-induced killing is more effective at the 1:20 ratio.

Recurrent Anthrax Outbreaks in Humans, Livestock, and Wildlife in the Same Locality, Kenya, 2014-2017.

Epidemiologic data indicate a global distribution of anthrax outbreaks associated with certain ecosystems that promote survival and viability of Bacillus anthracis spores. Here, we characterized three anthrax outbreaks involving humans, livestock, and wildlife that occurred in the same locality in Kenya between 2014 and 2017. Clinical and epidemiologic data on the outbreaks were collected using active case finding and review of human, livestock, and wildlife health records. Information on temporal and spatial distribution of prior outbreaks in the area was collected using participatory epidemiology. The 2014-2017 outbreaks in Nakuru West subcounty affected 15 of 71 people who had contact with infected cattle (attack rate = 21.1%), including seven with gastrointestinal, six with cutaneous, and two with oropharyngeal forms of the disease. Two (13.3%) gastrointestinal human anthrax cases died. No human cases were associated with infected wildlife. Of the 54 cattle owned in 11 households affected, 20 died (attack rate = 37%). The 2015 outbreak resulted in death of 10.5% of the affected herbivorous wildlife at Lake Nakuru National Park, including 745 of 4,500 African buffaloes (species-specific mortality rate = 17%) and three of 18 endangered white rhinos (species-specific mortality rate = 16%). The species mortality rate ranged from 1% to 5% for the other affected wildlife species. Participatory epidemiology identified prior outbreaks between 1973 and 2011 in the same area. The frequency and severity of outbreaks in this area suggests that it is an anthrax hotspot ideal for investigating risk factors associated with long-term survival of anthrax spores and outbreak occurrence.

A bivalent protein r-PB, comprising PA and BclA immunodominant regions for comprehensive protection against Bacillus anthracis.

Anthrax infection is primarily initiated by B. anthracis endospores that on entry into the host germinate to vegetative cells and cause severe bacteremia and toxaemia employing an array of host colonisation factors and the lethal tripartite toxin. The protective efficacy of conventional protective antigen (PA) based anthrax vaccines is improved by co-administration with inactivated spores or its components. In the present study, using structural vaccinology rationale we synthesized a bivalent protein r-PB encompassing toxin (PAIV) and spore components (BclACTD) and characterized its protective efficacy against B. anthracis infection. Active immunization of mice with r-PB generated high titer circulating antibodies which facilitated the phagocytic uptake of spores, inhibited their germination to vegetative cells and completely neutralized anthrax toxins in vivo resulting in 100 % survival against anthrax toxin challenge. Proliferation of CD4+ T cell subsets with up-regulation of Th1 (IFN-γ, IL-2, and IL-12), Th2 (IL-5, IL-10) cytokines and balanced expression of IgG1:IgG2a antibody isotypes indicated the stimulation of both Th1 and Th2 subsets. The immunized mice exhibited 100 % survival upon challenge with B. anthracis spores or toxin indicating the ability of r-PB to provide comprehensive protection against anthrax. Our results thus demonstrate r-PB an efficient vaccine candidate against anthrax infection.

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 parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med-fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well-known as invertebrate-active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N-terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid-cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

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.

Clostridium difficile infection: new approaches to prevention, non-antimicrobial treatment, and stewardship.

INTRODUCTION: Despite the large amount of scientific publications exploring the epidemiology and the clinical management of Clostridium difficile (CD) infection, some issues remain unsolved or need further studies. The aim of this review is to give an update on the hot topics on CD prevention, including stewardship programs, and on the non-microbiological treatment of CD infection. Areas covered: This article will review the importance of minimizing the CD spore shedding in the healthcare environment for potentially reducing CD transmission. Moreover, antimicrobial stewardship programs aimed to reduce CD incidence will be reviewed. Finally, new strategies for reducing CD infection recurrence will be described. Expert commentary: Besides the basic infection control and prevention practices, including hand hygiene, contact isolation and environmental cleaning, in the prevention of CD infection other issues should be addressed including minimizing the spread of CD in the healthcare setting, and implementing the best strategy for reducing CD infection occurrence, including tailored antimicrobial stewardship programs. Regarding new advancements in treatment and management of CDI episodes, non-antimicrobial approaches seem to be promising in reducing and managing recurrent CD infection.

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.

An in silico evaluation of treatment regimens for recurrent Clostridium difficile infection.

BACKGROUND: Clostridium difficile infection (CDI) is a significant nosocomial infection worldwide, that recurs in as many as 35% of infections. Risk of CDI recurrence varies by ribotype, which also vary in sporulation and germination rates. Whether sporulation/germination mediate risk of recurrence and effectiveness of treatment of recurring CDI remains unclear. We aim to assess the role of sporulation/germination patterns on risk of recurrence, and the relative effectiveness of the recommended tapered/pulsing regimens using an in silico model. METHODS: We created a compartmental in-host mathematical model of CDI, composed of vegetative cells, toxins, and spores, to explore whether sporulation and germination have an impact on recurrence rates. We also simulated the effectiveness of three tapered/pulsed vancomycin regimens by ribotype. RESULTS: Simulations underscored the importance of sporulation/germination patterns in determining pathogenicity and transmission. All recommended regimens for recurring CDI tested were effective in reducing risk of an additional recurrence. Most modified regimens were still effective even after reducing the duration or dosage of vancomycin. However, the effectiveness of treatment varied by ribotype. CONCLUSION: Current CDI vancomycin regimen for treating recurrent cases should be studied further to better balance associated risks and benefits.