Persistence Of Spores Research Articles

Soil Sample Analysis of Bacillus anthracis Contaminated Animal Burial Sites.

Environmental contamination with Bacillus anthracis spores poses clear threats to livestock that play key roles in the economies of pastoral communities. Regular monitoring of contaminated sites is particularly important in anthrax-endemic parts of the world, such as Kars province in eastern Türkiye, where the Veterinary Microbiology Department of Kafkas University has conducted an anthrax surveillance programme for over 30 years. We reviewed the microbiological results of 232 soil samples collected during 2009-2023, from sites known to be contaminated with B. anthracis spores following burial or butchering of infected animal carcasses. Twenty-five contaminated sites in 16 villages were studied. Samples were taken from a total of 61 different positions within these sites and viable spores were detected in 136 (58.6%) of the samples examined. Of the 96 samples from which spores were not recovered, subsequent samples from the same positions proved positive on 21 occasions. Using a standardised sampling plan, it was discovered that samples taken 1-2 m on a downward slope from the centre-point of contamination had higher (p < 0.001) spore concentrations than those taken from other positions. Although spore concentrations at some sampling positions varied over time, the overall values remained stable. This finding contrasts with observations in other parts of the world where spore concentrations tend to decline with time and may reflect regional differences in soil composition that permit more prolonged spore persistence. Concentrations of >100 spores/g soil were found in 10 (66.7%) of the 15 samples taken 10-13 years following a contamination event. These results demonstrate the longevity of viable anthrax spores in the soil of agricultural environments following decomposition of infected animal carcasses, and therefore the need for prolonged bacteriological monitoring of contaminated sites. Furthermore, they underline the importance of appropriate decontamination, as burial on its own does not eliminate all spores.

Materials Engineering to Help Pest Control: A Narrative Overview of Biopolymer-Based Entomopathogenic Fungi Formulations.

Biopolymer-based formulations show great promise in enhancing the effectiveness of entomopathogenic fungi as bioinsecticides. Chitosan and starch, among other biopolymers, have been utilized to improve spore delivery, persistence, and adherence to target insects. These formulations offer advantages such as target specificity, eco-friendliness, and sustainability. However, challenges related to production costs, stability, and shelf life need to be addressed. Recently, biomimetic lure and kill approaches based on biopolymers offer cost-effective solutions by leveraging natural attractants. Further research is needed to optimize these formulations and overcome challenges. Biopolymer-based formulations have the potential to revolutionize pest control practices, providing environmentally friendly and sustainable solutions for agriculture.

The effect of cold storage and cooking on the viability of Clostridioides difficile spores in consumer foods.

The increased detection of clinical cases of Clostridioides difficile coupled with the persistence of clostridial spores at various stages along the food chain suggest that this pathogen may be foodborne. This study examined C. difficile (ribotypes 078 and 126) spore viability in chicken breast, beef steak, spinach leaves and cottage cheese during refrigerated (4°C) and frozen (-20°C) storage with and without a subsequent sous vide mild cooking (60°C, 1h). Spore inactivation at 80°C in phosphate buffer solution, beef and chicken were also investigated to provide D80°C values and determine if PBS was a suitable model system for real food matrices. There was no decrease in spore concentration after chilled or frozen storage and/or sous vide cooking at 60°C. Non-log-linear thermal inactivation was observed for both C. difficile ribotypes at 80°C in phosphate buffer solution (PBS), beef and chicken. The predicted PBS D80°C values of 5.72±[2.90, 8.55] min and 7.50±[6.61, 8.39] min for RT078 and RT126, respectively, were in agreement with the food matrices D80°C values of 5.65min (95% CI range from 4.29 to 8.89min) for RT078 and 7.35min (95% CI range from 6.81 to 7.01min) for RT126. It was concluded that C. difficile spores survive chilled and frozen storage and mild cooking at 60°C but may be inactivated at 80°C. Moreover thermal inactivation in PBS was representative of that observed in real food matrices (beef and chicken).

The natural product chlorotonil A preserves colonization resistance and prevents relapsing Clostridioides difficile infection

Clostridioides difficile infections (CDIs) remain a healthcare problem due to high rates of relapsing/recurrent CDIs (rCDIs). Breakdown of colonization resistance promoted by broad-spectrum antibiotics and the persistence of spores contribute to rCDI. Here, we demonstrate antimicrobial activity of the natural product class of chlorotonils against C.difficile. In contrast to vancomycin, chlorotonil A (ChA) efficiently inhibits disease and prevents rCDI in mice. Notably, ChA affects the murine and porcine microbiota to a lesser extent than vancomycin, largely preserving microbiota composition and minimally impacting the intestinal metabolome. Correspondingly, ChA treatment does not break colonization resistance against C.difficile and is linked to faster recovery of the microbiota after CDI. Additionally, ChA accumulates in the spore and inhibits outgrowth of C.difficile spores, thus potentially contributing to lower rates of rCDI. We conclude that chlorotonils have unique antimicrobial properties targeting critical steps in the infection cycle of C.difficile.

S133 An Open-Label Study (ECOSPOR IV) to Evaluate the Safety, Efficacy and Durability of SER-109, an Investigational Oral Microbiome Therapeutic, in Adults With Recurrent Clostridioides difficile Infection (rCDI)

Introduction: Antibiotics are often insufficient to treat patients with rCDI due to persistence of spores and microbiome disruption. Recurrence occurs in 20-36% of patients with 1st recurrence and ≥40% in those with ≥2 recurrences. We reported that SER-109, an investigational, oral microbiome therapeutic comprised of purified Firmicutes spores, was superior to placebo in reducing risk of rCDI at 8 weeks (12% vs 40%, respectively) in patients with ≥2 CDI recurrences (Feuerstadt, P. N Engl J Med 2022). Here, we report the results of ECOSPOR IV, an open-label trial of SER-109. Methods: Subjects with rCDI were enrolled at 72 US/Canadian sites in 2 cohorts: a) rollover subjects with rCDI in ECOSPOR III, diagnosed by toxin EIA) and b) subjects with ≥1 CDI recurrence (diagnosed by PCR or toxin EIA), inclusive of the current episode). After standard-of-care antibiotics, subjects received SER-109 (4 capsules daily x 3 days). Efficacy, including the proportion of subjects with rCDI (toxin+ diarrhea requiring treatment), and safety were evaluated through Week 8; durability of response was assessed through Week 24. Results: Of 351 subjects screened, 263 were enrolled (Cohort 1: N=29; Cohort 2: N=234; 68% female; mean age 64 years). Comorbidities included cardiac disorders (31%), neoplasms (21%), Type 2 diabetes (11%), COPD (10%), chronic kidney disease (9%), and hepatobiliary disorders (9%). Overall, 137 subjects (52.1%) experienced treatment-emergent adverse events (TEAEs) through Week 8. The majority were mild to moderate in intensity and gastrointestinal (Table). There were 6 deaths (2.3%) and 20 subjects (7.6%) with serious TEAEs, none of which were deemed treatment-related. Overall, rCDI occurred in 23 subjects (8.7%) at Week 8 (4/29 in Cohort 1 [13.8%] and 19/234 in Cohort 2 [8.1%]) and rCDI rates remained low through 24 weeks (13.7% [36/263]). CDI recurrence rates at Week 8 in subjects with first recurrence were similarly low (6.5% [5/77]) as those with ≥2 recurrences (9.7% [18/186]; Figure). Conclusion: SER-109, a potential first-in-class investigational microbiome therapeutic, was observed to be safe and well-tolerated in this population of older patients with multiple comorbidities. The rate of rCDI was low and durable, regardless of the number of prior episodes, supporting the potential benefit of microbiome repair following antibiotics for rCDI. Earlier intervention with SER-109 in first recurrence may reduce morbidity associated with rCDI. Table 1. - Summary of Treatment-Emergent Adverse Events Within 8 Weeks After Treatment, Safety Population Cohort 1 Cohort 2(N=234) n (%) Total(N=263)n (%) Randomized Treatment Arm in ECOSPOR III Total(N=29)n (%) SER-109(N=4)n (%) Placebo(N=25)n (%) Any TEAE 4 (100.0) 15 (60.0) 19 (65.5) 118 (50.4) 137 (52.1) Most Frequently Reported TEAEs by Preferred Term (≥5% in any cohort) Diarrhoea 1 (25.0) 9 (36.0) 10 (34.5) 49 (20.9) 59 (22.4) Flatulence 0 4 (16.0) 4 (13.8) 16 (6.8) 20 (7.6) Nausea 0 3 (12.0) 3 (10.3) 17 (7.3) 20 (7.6) Abdominal pain 1 (25.0) 2 (8.0) 3 (10.3) 15 (6.4) 18 (6.8) Fatigue 0 3 (12.0) 3 (10.3) 9 (3.8) 12 (4.6) Urinary tract infection 0 0 0 12 (5.1) 12 (4.6) Abdominal distension 1 (25.0) 3 (12.0) 4 (13.8) 7 (3.0) 11 (4.2) Related/Possibly Related TEAE 1 (25.0) 4 (16.0) 5 (17.2) 27 (11.5) 32 (12.2) Severe TEAEs 0 1 (4.0) 1 (3.4) 18 (7.7) 19 (7.2) Serious TEAEs 0 0 0 20 (8.5) 20 (7.6) Serious TEAEs Related/Possibly Related to Study Drug 0 0 0 0 0 Treatment-emergent AESIs 0 0 0 10 (4.3) 10 (3.8) TEAEs Leading to Death1 0 0 0 6 (2.6) 6 (2.3) Abbreviations: AESI = adverse event of special interest; TEAE = treatment-emergent adverse event Notes: Data presented are by subject. All TEAEs were collected and summarized from time of enrollment up to Week 8; Note: N is number of subjects in the Safety Population who are in the study at the beginning of the specified time interval. 1 TEAEs leading to death by preferred term included: congestive cardiomyopathy (1 subject), coronavirus infection and intestinal peforation (1 subect), death due to natural causes (1 subject), clostridium difficile infection (1 subject), necrotising fasciitis (1 subject), and pancreatic carcinoma (1 subject). Note: the start date of the fatal SAE of pancreatic carcinoma began prior to Week 8 with fatal outcome occurred after Week 12. No deaths were deemed by the investigator to be related to study drug. Figure 1.: CDI Recurrence Rate at Week 8 in the Overall Population by Number of CDI Recurrences at Study Entry Note: Subjects who are lost to follow-up, terminated the study prematurely, or died without a recorded recurrence before the end of the time interval are assumed to have had a recurrence. Numbers presented within each bar represent the number of subjects with CDI recurrence over the total number of subjects within each grouping.

Determination, distribution, and environmental fate of Bacillus thuringiensis spores in various honeybee matrices after field application as plant protection product

The increasing use of Bacillus thuringiensis (Bt)–based plant protection products (PPPs) has recently raised some concerns regarding their environmental accumulation and possible chronic exposure of non-target species, including pollinators, to higher than expected doses. The exposure level of such microbial PPPs in bee’s matrices under field conditions has not yet been described. Therefore, the current study aims at evaluating the realistic exposure level and comparing the distributions and persistence of Bt spores under field conditions. A field trial with spray application in oilseed rape (Brassica napus) as a representative bee-attractive crop was conducted. During the experimental period, different matrices, including honeybee-collected and -stored matrices as well as bee larvae and dead bees, were collected and analyzed using newly established methods. The concentration of Bt spores in the various matrices was quantified. The results show high levels of Bt spores in honey sac and pollen pellets with reduction over time but no reduction of Bt spores in the stored matrices within the colony, i.e., nectar and bee bread, over time. Our results show for the first time the exposure level of bees to Bt spores under realistic field conditions and are fundamentally important for assessing potential exposure and risks for pollinators.

Biological control of Western flower thrips, Frankliniella occidentalis using a self-sustaining granular fungal treatment.

Western flower thrips (WFT), Frankliniella occidentalis, is one of the most destructive pests of vegetables, fruits and ornamental crops worldwide, causing extensive damage by direct feeding of the crop and transmitting economically important viruses. Despite the successes of biocontrol agents to control WFT, more efficient and cost-effective ways must be found to encourage grower adoption of integrated pest management. A sustainable fungal treatment was developed to preserve fungal inoculum in potting soil and reduce thrips populations. Combining cooked, oven-dried millet with BotaniGard® (a commercial form of Beauveria bassiana strain GHA) to potting soil increased spore production and persistence of the fungus in the soil. In treated pots with millet, spore concentrations were 3-4 times greater after 30 days compared with spore yields at 10 days. The number of WFT adults was significantly lower in the marigold pots treated with GHA mix + millet than untreated controls, 12% and 10% in treated pots and 70% and 68% in untreated pots in sterile and non-sterile soil, respectively. Incorporation of millet in the potting mix enhanced the effect of the fungal treatments by providing a nutritive substrate on which the fungus could become established. This method is relatively inexpensive and easy for growers to use in greenhouses because granular formulations of B. bassiana are not commercially available.

Entry of spores into intestinal epithelial cells contributes to recurrence of Clostridioides difficile infection

Clostridioides difficile spores produced during infection are important for the recurrence of the disease. Here, we show that C. difficile spores gain entry into the intestinal mucosa via pathways dependent on host fibronectin-α5β1 and vitronectin-αvβ1. The exosporium protein BclA3, on the spore surface, is required for both entry pathways. Deletion of the bclA3 gene in C. difficile, or pharmacological inhibition of endocytosis using nystatin, leads to reduced entry into the intestinal mucosa and reduced recurrence of the disease in a mouse model. Our findings indicate that C. difficile spore entry into the intestinal barrier can contribute to spore persistence and infection recurrence, and suggest potential avenues for new therapies.

In vitro Production and Immunogenicity of a Clostridium Difficile Spore-Specific BclA3 Glycopeptide Conjugate Vaccine.

The BclA3 glycoprotein is a major component of the exosporangial layer of Clostridium difficile spores and in this study we demonstrate that this glycoprotein is a major spore surface associated antigen. Here, we confirm the role of SgtA glycosyltransferase (SgtA GT) in BclA3 glycosylation and recapitulate this process by expressing and purifying SgtA GT fused to MalE, the maltose binding protein from Escherichia coli. In vitro assays using the recombinant enzyme and BclA3 synthetic peptides demonstrated that SgtA GT was responsible for the addition of β-O-linked GlcNAc to threonine residues of each synthetic peptide. These peptide sequences were selected from the central, collagen repeat region of the BclA3 protein. Following optimization of SgtA GT activity, we generated sufficient glycopeptide (10 mg) to allow conjugation to KLH (keyhole limpet hemocyanin) protein. Glycosylated and unglycosylated versions of these conjugates were then used as antigens to immunize rabbits and mice. Immune responses to each of the conjugates were examined by Enzyme Linked Immunosorbent Assay ELISA. Additionally, the BclA3 conjugated peptide and glycopeptide were used as antigens in an ELISA assay with serum raised against formalin-killed spores. Only the glycopeptide was recognized by anti-spore polyclonal immune serum demonstrating that the glycan moiety is a predominant spore-associated surface antigen. To determine whether antibodies to these peptides could modify persistence of spores within the gut, animals immunized intranasally with either the KLH-glycopeptide or KLH-peptide conjugate in the presence of cholera toxin, were challenged with R20291 spores. Although specific antibodies were raised to both antigens, immunization did not provide any protection against acute or recurrent disease.

Linking Geospatial and Laboratory Sciences to Define Mechanisms behind Landscape Level Drivers of Anthrax Outbreaks.

Background: A seasonal predictor of anthrax outbreaks is rainfall, which may be approximated by NDVI using remote sensing. How rainfall or vegetative green-up influences bacterial physiology or microecology to drive anthrax outbreaks is not known. Methods: Rainfall and NDVI dependency of anthrax epizootics was demonstrated with global and local phenological analysis. Growth analysis of B. anthracis in response to pH and calcium gradients was carried out. The influence of pH and calcium levels on expression of toxin and sporulation related proteins in broth culture models was characterized using engineered B. anthracis luminescent reporter strains. Results: Short-term bacterial growth and longer-term bacterial survival were altered by pH and calcium. These conditions also played a major role in pagA and sspB promoter-driven luminescent expression in B. anthracis. Conclusions: Rainfall induced cycling of pH and calcium in soils plays a plausible role in amplifying spore load and persistence in endemic anthrax zones. Observed evidence of B. anthracis favoring soil alkalinity and high soil calcium levels in the environment were linked to physiological conditions that promote bacterial growth, survival, toxin secretion and spore formation; illustrating the utility of bringing laboratory-based (controlled) microbiology experiments into the fold of zoonotic disease ecology.

Persistence and Lethality of a Fungal Biopesticide (Aprehend) Applied to Insecticide-impregnated and Encasement-type Box Spring Covers for Bed Bug Management.

The newly developed fungal biopesticide Aprehend, containing spores of Beauveria bassiana, is the first biological control agent to be incorporated into management programs to control the common bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae). Aprehend is sprayed as barriers where bed bugs are likely to walk and pick up spores as they search for a bloodmeal. A key application target for Aprehend is the box spring, which may be covered by encasement-type or insecticide-impregnated covers. Since some insecticides can reduce the persistence of fungal spores, we tested the efficacy and spore germination percentages of Aprehend when applied to the two types of box spring covers. We found that spore germination was about 11% lower on the permethrin-impregnated ActiveGuard cover than on the encasement-type AllerEase cover. However, bed bugs exposed for 15 min to Aprehend on the two box spring covers suffered similarly high levels of mortality irrespective of the cover material. Thus, there was no inhibitory or additive effect of the ActiveGuard cover on bed bug mortality. Lastly, overall mortality was higher if bed bugs were exposed to Aprehend-treated ActiveGuard than the ActiveGuard cover alone. Our findings indicate that if pest managers are using ActiveGuard covers in combination with Aprehend, best practice would be to use ActiveGuard on mattresses and apply Aprehend directly to the box spring or to a box spring covered by an encasement-type cover.

Membrane Proteomes and Ion Transporters in Bacillus anthracis and Bacillus subtilis Dormant and Germinating Spores.

Bacterial endospores produced by Bacillus and Clostridium species can remain dormant and highly resistant to environmental insults for long periods, but they can also rapidly germinate in response to a nutrient-rich environment. Multiple proteins involved in sensing and responding to nutrient germinants, initiating solute and water transport, and accomplishing spore wall degradation are associated with the membrane surrounding the spore core. In order to more fully catalog proteins that may be involved in spore germination, as well as to identify protein changes taking place during germination, unbiased proteomic analyses of membrane preparations isolated from dormant and germinated spores of Bacillus anthracis and Bacillus subtilis were undertaken. Membrane-associated proteins were fractionated by SDS-PAGE, gel slices were trypsin digested, and extracted peptides were fractionated by liquid chromatography and analyzed by matrix-assisted laser desorption ionization-tandem time of flight mass spectrometry. More than 500 proteins were identified from each preparation. Bioinformatic methods were used to characterize proteins with regard to membrane association, cellular function, and conservation across species. Numerous proteins not previously known to be spore associated, 6 in B. subtilis and 68 in B. anthracis, were identified. Relative quantitation based on spectral counting indicated that the majority of spore membrane proteins decrease in abundance during the first 20 min of germination. The spore membranes contained several proteins thought to be involved in the transport of metal ions, a process that plays a major role in spore formation and germination. Analyses of mutant strains lacking these transport proteins implicated YloB in the accumulation of calcium within the developing forespore.IMPORTANCE Bacterial endospores can remain dormant and highly resistant to environmental insults for long periods but can also rapidly germinate in response to a nutrient-rich environment. The persistence and subsequent germination of spores contribute to their colonization of new environments and to the spread of certain diseases. Proteins of Bacillus subtilis and Bacillus anthracis were identified that are associated with the spore membrane, a position that can allow them to contribute to germination. A set of identified proteins that are predicted to carry out ion transport were examined for their contributions to spore formation, stability, and germination. Greater knowledge of spore formation and germination can contribute to the development of better decontamination strategies.

Efficiency of natural substances to protect Beauveria bassiana conidia from UV radiation.

BACKGROUNDSolar radiation is assumed to be a major factor limiting the efficacy of entomopathogenic fungi used as biocontrol agents in open field applications. We evaluated 12 natural UV‐protective co‐formulants for their effect on the survival of UV‐exposed Beauveria bassiana spores on agar plates, colza leaf discs and in the field.RESULTSColony‐forming unit (CFU) counts of unformulated conidia on agar plates and leaf discs dropped to ≤ 50% after exposure to UV radiation. The highest UV protection was achieved with humic acid, which provided > 90% protection of UV‐B‐exposed conidia in laboratory experiments. In the field, 10% humic acid increased spore persistence up to 87% at 7 days after application. Sesame and colza oil also provided high UV protection in both assays (> 73% and > 70%, respectively).CONCLUSIONSThis study shows that it is possible to increase the persistence of B. bassiana spores under exposure to UV radiation by formulation with natural UV‐protective additives. UV protectants might, therefore, increase the efficacy of entomopathogenic fungi as biocontrol agents in open field applications. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Oxygen-deficiency and allelochemicals affect Sphagnum spore persistence in peatlands

To test the effects of characteristic ecological gradients in peatlands including oxygen-deficiency and allelopathy on Sphagnum spore persistence. We determined the initial viability of Sphagnum spores and then stored the spores for 60 days, either dry, in ultrapure water, peatland surface water or Sphagnum water leachate (the latter two solutions rich in allelochemicals from Sphagnum). We varied oxygen concentration by injecting air at three different concentrations during the storage experiment. After retrieval from experimental storage, spore germinability index was assessed. Germinability index after air injection was lower than under oxygen-deficiency. Germinability index in the peatland surface water and Sphagnum leachate water was higher than in ultrapure water, under oxygen-deficiency. Overall, dissolved oxygen and high pH negatively affected spore germinability and, consequently, viability. These results indicate that once dispersed onto bryophyte substrates or waterlogged hollows, Sphagnum spores can maintain viability better than when exposed to dry conditions or in water without allelochemicals. Extreme longevity of Sphagnum spores may be attributed to the oxygen-deficient, waterlogged and acidic Sphagnum-dominated peatland habitat with allelopathic substrates.

Spores and soil from six sides: interdisciplinarity and the environmental biology of anthrax (Bacillus anthracis).

Environmentally transmitted diseases are comparatively poorly understood and managed, and their ecology is particularly understudied. Here we identify challenges of studying environmental transmission and persistence with a six-sided interdisciplinary review of the biology of anthrax (Bacillus anthracis). Anthrax is a zoonotic disease capable of maintaining infectious spore banks in soil for decades (or even potentially centuries), and the mechanisms of its environmental persistence have been the topic of significant research and controversy. Where anthrax is endemic, it plays an important ecological role, shaping the dynamics of entire herbivore communities. The complex eco-epidemiology of anthrax, and the mysterious biology of Bacillus anthracis during its environmental stage, have necessitated an interdisciplinary approach to pathogen research. Here, we illustrate different disciplinary perspectives through key advances made by researchers working in Etosha National Park, a long-term ecological research site in Namibia that has exemplified the complexities of the enzootic process of anthrax over decades of surveillance. In Etosha, the role of scavengers and alternative routes (waterborne transmission and flies) has proved unimportant relative to the long-term persistence of anthrax spores in soil and their infection of herbivore hosts. Carcass deposition facilitates green-ups of vegetation to attract herbivores, potentially facilitated by the role of anthrax spores in the rhizosphere. The underlying seasonal pattern of vegetation, and herbivores' immune and behavioural responses to anthrax risk, interact to produce regular 'anthrax seasons' that appear to be a stable feature of the Etosha ecosystem. Through the lens of microbiologists, geneticists, immunologists, ecologists, epidemiologists, and clinicians, we discuss how anthrax dynamics are shaped at the smallest scale by population genetics and interactions within the bacterial communities up to the broadest scales of ecosystem structure. We illustrate the benefits and challenges of this interdisciplinary approach to disease ecology, and suggest ways anthrax might offer insights into the biology of other important pathogens. Bacillus anthracis, and the more recently emerged Bacillus cereus biovar anthracis, share key features with other environmentally transmitted pathogens, including several zoonoses and panzootics of special interest for global health and conservation efforts. Understanding the dynamics of anthrax, and developing interdisciplinary research programs that explore environmental persistence, is a critical step forward for understanding these emerging threats.

Evaluating the long-term persistence of Bacillus spores on common surfaces.

Summary Bacillus spores resist inactivation, but the extent of their persistence on common surfaces is unclear. This work addresses knowledge gaps regarding biothreat agents in the environment to reduce uncertainty in risk assessment models. Studies were conducted to investigate the long‐term inactivation of Bacillus anthracis and three commonly used surrogate organisms – B. cereus, B. atrophaeus and B. thuringiensis on three materials: laminate countertop, stainless steel and polystyrene Petri dishes. Viable spores were measured at 1, 30, 90, 196, 304 and 1038 days. Twelve different persistence models were fit to the data using maximum likelihood estimation and compared. The study found that (1) spore inactivation was not log‐linear, as commonly modelled; (2) B. thuringiensis counts increased at 24 h on all materials, followed by a subsequent decline; (3) several experiments showed evidence of a ‘U’ shape, with spore counts apparently decreasing and then increasing between 1 and 304 days; (4) spores on polystyrene showed little inactivation; and (5) the maximum inactivation of 56% was observed for B. atrophaeus spores on steel at 196 days. Over the range of surfaces, time durations and conditions (humidity controlled vs. uncontrolled) examined, B. thuringiensis most closely matched the behaviour of B. anthracis.

Fungi in Bronchiectasis: A Concise Review.

Although the spectrum of fungal pathology has been studied extensively in immunosuppressed patients, little is known about the epidemiology, risk factors, and management of fungal infections in chronic pulmonary diseases like bronchiectasis. In bronchiectasis patients, deteriorated mucociliary clearance—generally due to prior colonization by bacterial pathogens—and thick mucosity propitiate, the persistence of fungal spores in the respiratory tract. The most prevalent fungi in these patients are Candida albicans and Aspergillus fumigatus; these are almost always isolated with bacterial pathogens like Haemophillus influenzae and Pseudomonas aeruginosa, making very difficult to define their clinical significance. Analysis of the mycobiome enables us to detect a greater diversity of microorganisms than with conventional cultures. The results have shown a reduced fungal diversity in most chronic respiratory diseases, and that this finding correlates with poorer lung function. Increased knowledge of both the mycobiome and the complex interactions between the fungal, viral, and bacterial microbiota, including mycobacteria, will further our understanding of the mycobiome’s relationship with the pathogeny of bronchiectasis and the development of innovative therapies to combat it.

Clinical, epidemiological and microbiological characteristics of relapse and re-infection in Clostridium difficile infection

Recurrent diarrhea is a common complication of Clostridium difficile infection (CDI). Recurrent CDI (r-CDI) may be produced by the persistence of spores (relapse) or by the acquisition of a new strain (reinfection). In this study, we analyze epidemiological, clinical, microbiological and laboratory data from patients with r-CDI, relapse, and reinfection-CDI over 5 years and compared with a control group (non r-CDI). Among 60 patients with r-CDI, 36 patients had stool samples collected from two or more episodes, which were molecularly analyzed. Based on ribotyping, 63.9% of the samples were relapse, and 36.1% reinfection. In a multivariable logistic regression analysis, previous antibiotic exposure was found to be a risk factor for r-CDI (OR: 2.23; 95% CI: 1.0–4.9; p = 0.04). Patients with relapse had previous antibiotic exposure more frequently than did patients with reinfection (p = 0.03), and patients with reinfection suffered more frequently from chronic liver disease (p = 0.02) than did relapse patients. Relapse patients compared with the control group had a higher percentage of previous antibiotic exposure, although the difference was statistically no significant (73.9% vs. 91.3 p = 0.06). No significant differences for the selected variables were observed between the reinfection and control groups, although we observed a higher percentage of patients with chronic liver disease (30.8% vs 13.3%; p = 0.08). All isolates were sensitive to metronidazole and vancomycin. No significant differences in antibiotic susceptibility were found between the different groups. Sporulation and germination frequency of r-CDI were higher than non r-CDI (p = 0.02 and p < 0.01, respectively). Nevertheless, there were statistically not significant differences between the relapse and reinfection groups. Both frequencies were compared between the first and second episode of CDI for the relapse and reinfection groups, but differences were not observed to be statistically significant. In conclusion, our study showed that the recurrence of CDI was associated with antibiotic use and sporulation/germination frequency, regardless of relapse or reinfection. The use of antibiotics would produce a dysbiosis and favor the persistence of the C. difficile spores and relapse. A possible alteration of the intestinal microbiota and the bile salts produced by chronic liver disease could favor reinfection.

The effect of hospital biocide sodium dichloroisocyanurate on the viability and properties of Clostridium difficile spores.

This study is the first to report on changes in Clostridium difficile spore surface property after exposure to sublethal levels of the commonly used biocide sodium dichloroisocyanurate. The implications of these changes to the spore surface include increased adherence of the spores to inorganic surfaces which can directly contribute to persistence and spread of spores within the hospital environment.

Construction of Bacillus thuringiensis Simulant Strains Suitable for Environmental Release

For a surrogate bacterium to be used in outdoor studies, it is important to consider environmental and human safety and ease of detection. Recently, Bacillus thuringiensis, a popular bioinsecticide bacterium, has been gaining attention as a surrogate bacterium for use in biodefense. In this study, we constructed simulant strains of B. thuringiensis with enhanced characteristics for environmental studies. Through transposon mutagenesis, pigment genes were inserted into the chromosome, producing yellow-colored colonies for easy detection. To prevent persistence of spores in the environment, a genetic circuit was designed to produce a spore without sporulation capability. Two loxP sites were inserted, one on each side of the spo0A gene, which encodes a sporulation master regulator, and a sporulation-dependent Cre expression cassette was inserted into the chromosome. This genetic circuit successfully deleted spo0A during sporulation, producing spores that lacked the spo0A gene. In addition, two major α/β-type small acid-soluble spore protein (SASP) genes, predicted by synteny analysis, were deleted. The spores of the mutant strain showed increased UV-C sensitivity and quickly lost viability when tested in a solar simulator. When the spores of the mutant strain were administered to the lungs of BALB/c mice, cells were quickly removed from the body, suggesting enhanced in vivo safety. All strains constructed in this study contain no antibiotic resistance markers and all heterologous genes were inserted into the chromosome, which are useful features for simulants to be released into the environment.IMPORTANCEB. thuringiensis has recently been receiving increasing attention as a good spore simulant in biodefense research. However, few studies were done to properly address many important features of B. thuringiensis as a simulant in environmental studies. Since spores can persist in the environment for years after release, environmental contamination is a big problem, especially when genetically engineered strains are used. To solve these problems, we report here the development of B. thuringiensis simulant strains that are capable of forming yellow colonies for easy detection, incapable of forming spores more than once due to a genetic circuit, and lacking in two major SASP genes. The genetic circuit to produce a spore without sporulation capability, together with the deletion of SASP genes, ensures the environmental and human safety of the simulant strains developed in this study. All of these features will allow wider use of B. thuringiensis as a simulant for Bacillus anthracis in environmental release studies.