Low Spore Levels Research Articles

Efficient Secretion of Murine IL-2 From an Attenuated Strain of Clostridium sporogenes, a Novel Delivery Vehicle for Cancer Immunotherapy.

Despite a history dating back to the 1800s, using Clostridium bacteria to treat cancer has not advanced beyond the observation that they can colonise and partially destroy solid tumours. Progress has been hampered by their inability to eradicate the viable portion of tumours, and an instinctive anxiety around injecting patients with a bacterium whose close relatives cause tetanus and botulism. However, recent advances in techniques to genetically engineer Clostridium species gives cause to revisit this concept. This paper illustrates these developments through the attenuation of C. sporogenes to enhance its clinical safety, and through the expression and secretion of an immunotherapeutic. An 8.6 kb sequence, corresponding to a haemolysin operon, was deleted from the genome and replaced with a short non-coding sequence. The resultant phenotype of this strain, named C. sporogenes-NT, showed a reduction of haemolysis to levels similar to the probiotic strain, C. butyricum M588. Comparison to the parental strain showed no change in growth or sporulation. Following injection of tumour-bearing mice with purified spores of the attenuated strain, high levels of germination were detected in all tumours. Very low levels of spores and vegetative cells were detected in the spleen and lymph nodes. The new strain was transformed with four different murine IL-2-expressing plasmids, differentiated by promoter and signal peptide sequences. Biologically active mIL-2, recovered from the extracellular fraction of bacterial cultures, was shown to stimulate proliferation of T cells. With this investigation we propose a new, safer candidate for intratumoral delivery of cancer immunotherapeutics.

Detecting Heterobasidion irregulare in Minnesota and Assessment of Indigenous Fungi on Pines

Heterobasidion irregulare is one of the most problematic forest pathogens in the northern hemisphere, but has only been found relatively recently in the north central United States. Discovered in Wisconsin in 1993, but probably established sometime before that, it quickly spread throughout the state. In November 2014, it was found in southeastern Minnesota. Field surveys were then conducted throughout Minnesota with the focus in the southeast near the initial discovery. To find additional infection sites, surveys were conducted with accompanying aerial imagery of red pine (Pinus resinosa Aiton) stands that were previously thinned. Samples were collected from selected sites with dead and dying trees as well as samples from stumps in recently thinned pine stands. These samples were processed first with a nested polymerase chain reaction (PCR) protocol, which was replaced by a real-time PCR assay after its development. No samples tested positive for H. irregulare using these methods and no cultures from isolations were obtained outside the original infection area. Other indigenous fungi were also identified. The majority were wood decay fungi in the Basidiomycota. A spore collection study was also conducted after field surveys. Automated rotary arm spore collectors were used and assayed with an ITS TaqMan real-time PCR assay. Collectors were placed strategically in different areas of Minnesota. A positive control was used in an infected red pine plantation in Wisconsin and this location had the highest number of spores trapped, with 63,776 over a week period. Spores of H. irregulare were detected at several sites in Minnesota, with the highest spore total observed in traps at 413 over a week period. All other locations sampled also had some spores collected except Itasca State Park located in northwestern Minnesota. The weekly deposition of spores ranged from 0 to 1.26 m−2 h−1. Low spore levels occurring in Minnesota indicate that some spores are present, but they are currently being detected in amounts that may not be sufficient for colonization to be successful.

Evaluating Efficacy of Fumagilin-B® Against Nosemosis and Tracking Seasonal Trends of Nosema spp. in Nova Scotia Honey Bee Colonies

Abstract The efficacy of the antimicrobial Fumagilin-B® against nosemosis was evaluated in both spring and autumn feeding treatments following label directions in seventy-two honey bee (Apis mellifera) colonies across three apiaries in Nova Scotia, Canada. The seasonal trend of Nosema spp. spore loads was also tracked in these same colonies throughout a thirteen-month period (February 2018 – March 2019). We found the spring Fumagilin-B® treatment to be effective at significantly suppressing Nosema spp. spore levels below the recommended treatment threshold. There was no effect of Fumagilin-B® treatment in the autumn based on low spore levels at this time. We detected a drastic increase in Nosema spp. spore loads as May progressed but a decline in spores in summer (June–September). By October, there was another increase in spore levels, but this increase did not exceed the economic treatment threshold. Across seventeen collection periods in both control and Fumagilin-B® colonies, 74% (25) of samples tested positive for Nosema ceranae, while 26% (9) contained no Nosema spp. spores. No Nosema apis spores were detected during this trial. Our results indicate that Fumagilin-B® is an effective management practice in the spring, but colonies should still be monitored in the autumn. Our data also support that the Nosema species profile is shifting to be exclusively N. ceranae and the treatment threshold for Fumagilin-B® may need to be updated to reflect this, as the threshold was originally developed for N. apis.

Short communication: Clostridial spore counts in vat milk of Alpine dairies

One of the most severe quality defects in hard and semi-hard cheese, the late blowing defect, is caused by endospore-forming bacteria of the genus Clostridium. To minimize financial losses and waste of resources due to cheese spoilage, raw milk with elevated clostridial spore counts should not be used for the production of certain cheese types. In this context, threshold values of clostridial spore concentrations that cause quality defects in cheese are still under debate. To improve our understanding about late blowing defects, further information on the correlation between clostridial spore concentrations in milk and cheese quality is indispensable. Thus, the aim of this study was to monitor the microbiological quality of milk used for Alpine cheese production regarding clostridial endospore levels to facilitate the establishment of threshold spore concentrations that guarantee the absence of quality defects in Austrian cheese. For this purpose, we monitored clostridial endospore levels in vat milk of 4 Alpine dairies throughout the summer grazing period in 2018. Surprisingly, we observed almost complete absence of butyric acid-producing clostridia in milk and no blowing defects in cheese. Hence, critical clostridial spore concentrations could not be verified. Moreover, the observed low spore levels reveal that the prohibition of silage feeding and good farming practices effectively minimize clostridial endospore counts in milk and ensure the manufacture of high-quality cheese even if technological possibilities are limited.

Tetracapsuloides bryosalmonae abundance in river water.

Tetracapsuloides bryosalmonae is a myxozoan parasite of freshwater bryozoans and salmonids, causing proliferative kidney disease in the latter. To date, detection of the parasite has required collection of hosts and subsequent molecular or histological examination. The release of infectious spores from both hosts offers an opportunity to detect the parasite in water samples. We developed a novel SYBR® Green quantitative real-time PCR (qPCR) assay for T. bryosalmonae in water samples which provides an estimation of bryozoan malacospore numbers and tested the assay in 3 rivers in southern England (UK) over a period of 5 wk. The assay proved to be both highly sensitive and specific to the parasite, detecting low levels of spores throughout the study period. Larger-volume samples afforded greater detection likelihood, but did not increase the number of spores detected, possibly as a result of low and patchy spore distributions and lack of within-site replication of large-volume samples. Based on point-measurements, temperature was positively associated with the likelihood of detecting spores, possibly reflecting the temperature dependence of spore shedding from bryozoan hosts. The presence of T. bryosalmonae in water samples was predominantly influenced by spatial (sites within rivers, amongst rivers) and temporal (sampling dates) factors, while the latter also influenced quantification cycle (Cq) values and spore abundance. Environmental monitoring for infectious stages can complement traditional methods, providing faster and easier detection and avoiding potentially prolonged searching, collecting and destructive sampling of invertebrate and vertebrate hosts.

Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wβ::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wβ::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wβ::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Different management practices are associated with mesophilic and thermophilic spore levels in bulk tank raw milk

Bacterial endospores (also referred to as spores) present in raw milk are capable of surviving pasteurization and other adverse conditions encountered during dairy powder production. Therefore, requiring low spore levels in raw ingredients (e.g., raw milk) may be necessary for producing dairy powders with low spore counts. To identify potential associations between management practices and spore levels in raw milk, we sampled bulk tank raw milk from 33 farms throughout New York State every other month for 1yr. Following spore pasteurization (80°C for 12min), samples were incubated at 3 different temperatures to enumerate psychrotolerant (6°C for 10 d), mesophilic (32°C for 48h), and thermophilic (55°C for 48h) spores. An additional enrichment procedure was used to detect spores present at low levels (<10 spores/mL). Overall, psychrotolerant, mesophilic, and thermophilic spores were detected (at levels ≥10 spores/mL) in 1, 74, and 58% of bulk tank raw milk samples, respectively. Although thermophilic spore levels could not be quantified (due to bacterial swarming), mesophilic spore levels ranged from below detection (<10 spores/mL) to 680 spores/mL. Data collected through surveys were used to identify management practices associated with either mesophilic or thermophilic spore levels. We found that different management practices are associated with mesophilic and thermophilic spore levels. Low mesophilic spore levels in bulk tank raw milk samples were associated with (1) large herd size, (2) use of sawdust or sand bedding, and (3) not fore stripping during the premilking routine. Management practices that were associated with lower odds of having a thermophilic spore level ≥10 spores/mL are (1) large herd size, (2) spray-based application of the postmilking disinfectant, (3) dry massaging the udder during the premilking routine, and (4) the use of straw bedding. Collectively, these results suggest that different management practices may influence mesophilic and thermophilic spore levels in raw milk.

Operational Evaluation of the Rapid Viability PCR Method for Post-Decontamination Clearance Sampling

The Rapid Viability Polymerase Chain Reaction (RV-PCR) method was evaluated during the Bio-Response Operational Testing and Evaluation (BOTE), an interagency project to evaluate field-level facility biological remediation, using leading decontamination technologies. The tests were performed using an intentional release (aerosolization) of spores of Bacillus atrophaeus subspecies globigii (BG), as a surrogate for Bacillus anthracis, the etiologic agent for anthrax. Three decontamination methods were assessed including fumigation with vaporized hydrogen peroxide (VHP), fumigation with chlorine dioxide (CD), and a surface treatment process using pH-adjusted bleach.The RV-PCR method was developed to rapidly detect live B. anthracis spores during a bioterrorism event. The method uses a change in realtime PCR response before and after a nine hour incubation step, to determine the presence of viable bacterial spores in the sample; the method was recently verified for air filter, wipe and water samples at the 10-spore level for B. anthracis Ames spores, and was also developed for swab, sponge-stick, and vacuum sock/filter samples. In the method, high throughput sample processing is combined with PCR-based analysis before and after a rapid culture step to speed viability determination, especially for complex surface and environmental samples that present challenges to current culture-based methods. In the BOTE project, a total of 159 surface wipe samples from post-decontamination events were analyzed by splitting the suspension after spore recovery into two equal parts, with one part analyzed by RV-PCR and the other part by culture after concentrating to the same volume. In the BOTE project, the RV-PCR method provided rapid results for post-decontamination samples that were 98% (156/159 samples) consistent with results from culture analysis. The percentage agreement was noteworthy, given the large number of samples containing low spore levels. For the Post-VHP, Post-Bleach, and Post-CD event samples, the percentage agreement was 93% (41/44 samples), 100% (47/47 samples), and 100% (68/68 samples), respectively. The RV-PCR method performed well for the surrogate BG spores exposed to decontaminants at real-world application levels, and with wipe samples containing background debris and indigenous microbial populations.

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10 4, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.

Antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts.

Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88(-/-)) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures.

Toxicity of Bacillus thuringiensisδ‐ endotoxins toward the potato aphid in an artificial diet bioassay

AbstractRecent investigations indicated that Bacillus thuringiensis delta‐endotoxins (DET) possess aphidicidal activity in an artificial diet bioassay. Crystalline preparations of CryIIA, CryIIIA and CryIVD solubilized in a slightly alkaline sucrose/amino acid diet clearly imparted toxicity toward adults of potato aphid, Macrosiphum euphorbiae (Thomas) (Homoptera: Aphididae) after 4–5 days of continuous feeding. No obvious feeding deterrence was noted in these assays, as copious honeydew was produced and aphids often died in a feeding position. CryIIIA which was solubilized in aphid diet, but filtered to remove spores or crystalline toxin lacked aphidicidal activity. Spores from an acrystalliferous strain (EG2205) were not toxic by themselves at 7.75 × 105 spores/ml aphid diet, but did restore toxicity to the filtered CryIIIA solution. Therefore, low levels of spores may be very effective in concert with DET for aphicidal activity. Results also clearly demonstrated that a suspension of crystalline CryIIIA alone, without spores, exhibited toxicity. Therefore, DET may be more toxic to the aphids when imbibed as a fine suspension, perhaps indicating the need for slow solubilization into the aphid midgut.

Growth of Proteolytic Clostridium botulinum in Process Cheese Products: I. Data Acquisition for Modeling the Influence of pH, Sodium Chloride, Emulsifying Salts, Fat Dry Basis, and Temperature

Outgrowth of proteolytic Clostridium botulinum type A and B spores in pasteurized process cheese products was assessed to acquire data for improved models of botulinum stability. High-moisture (58.5%) products were made with different levels of pH (5.45 to 5.9), sodium chloride (1.1 to 2.8%, wt/wt) and citrates or phosphates as emulsifying salts (1.5 to 2%, wt/wt), and held at 15 to 30°C. Supplemental experiments were carried out to address the effect of lactic acid concentration originating from the nonfat and 50% fat dry basis (FDB) cheese raw materials, of moisture (50 to 69%), and of total fat (0.1 to 41%, wt/wt). Colony counts were recorded as substitutes for the traditional times to toxin formation. In the last experimental series a polyclonal ELISA against type A and B toxins was carried out as an alternative to the mouse challenge test. Very low spore levels could lead to detectable toxin formation. Temperature strongly influenced outgrowth. At 18°C outgrowth only occurred in 3 months at favorable aw (0.966) and pH (5.9). At 25°C, outgrowth occurred within one week under favorable conditions. No growth occurred within 3 months when aw and pH were 0.95 and 5.55 respectively. Polyphosphate appeared to be more inhibitory than citrate. Moisture is a frequently used indicator of botulinum stability, but when the FDB deviates from 50%, moisture is actually a poor indicator. Components such as NaCl, emulsifying salts, and lactic acid determine stability. Fat does not contribute to stability. Increased fat levels can reduce moisture without a concomitant increase in stability.

Contamination profiles and characterisation of Bacillus species in wheat bread and raw materials for bread production

The Bacillus counts in white and wholemeal wheat loaves produced without preservatives or sour dough were consistently 10 6 cfu g after two days of storage at ambient summer temperatures (25–30 °C). Identified species were B. subtilis (70%), B. licheniformis (24%), B. pumilus (2%) and B. cereus (2%). The dominance of B. subtilis in bread could be explained by the higher resistance to heat of this species as determined by inoculation studies. Among 14 species isolated from retail bread and wheat grains, B. subtilis was the only species associated with ropiness. Samples of raw materials, particularly bran, seeds and oat products, contained low levels (10 0–10 2 cfu g ) of Bacillus spores, surviving a heat treatment (100 °C, 10 min) corresponding to a baking process. Even low spore levels in raw materials, with the frequently isolated species, B. licheniformis (49%) and B. subtilis (10%), resulted in 10 7 Bacillus per g bread crumb in two days as determined by test bakings. The results indicate a need for controlling growth of Bacillus in bread.

A comparison of media and cultural conditions for identification ofBacillus larvae inhoney

SUMMARYThe sensitivity of five solid media for detection of Bacillus larvae spores in honey samples; J agar, MYPGP agar, brain heart infusion (BHIT) agar, Colombia agar and horse blood agar was compared in air or under 5% C02. MYPGP agar is superior to all other media tested for detection of low spore levels of B. larvae in honey. Incubation under 5% C02 enhances growth considerably, especially for MYPGP agar and J agar. The plating efficacy described here, where an average of 23% of inoculated spores germinated on MYPGP agar, is higher than reported elsewhere for B. larvae. Thus, contamination levels as low as 200 B. larvae spores/g of honey can be detected with more than 95% probability on MYPGP agar incubated under 5% C02 if 12 or more plates are inoculated.

Importance of spores, crystals, and δ-endotoxins in the pathogenicity of different varieties of Bacillus thuringiensis in Galleria mellonella and Pieris brassicae

Physical methods were used to produce spores containing impurities of 0.02–0.05% crystals and crystals containing impurities of 0.001–0.01% spores from cultures of Bacillus thuringiensis. In Galleria mellonella larvae, these preparations from varieties galleriae, aizawai, and wuhanensis were only moderately active compared to 1:1 mixtures of spores and crystals. Spores of an acrystalliferous aizawai mutant were inactive and did not contain a polypeptide of the same size as the potent M r 138000 δ-endotoxin present in spores and crystals of all three wild-type strains. Thus, this polypeptide probably contributed to the moderate activity of wild-type spores. Spore impurities in the crystal preparation were killed by γ irradiation without harming the crystals. The crystals without live spores were virtually inactive (LC 50s, ca. 10 10 crystals/g insect food). Addition of 10 3 spores to 10 8 crystals/g food (0.001% spores) increased the mortality of larvae from 0 to 36%, and addition of 10 4 spores (0.01% spores) killed 64% larvae. Thus, the addition of low levels of spores increased the potency of crystals in G. mellonella from virtually zero to moderate levels, suggesting that the live spore impurities in the crystal preparations were responsible for the observed moderate potency of crystals before γ irradiation, a view supported by a reduction of potency of crystal preparations following admixture of streptomycin to the insect food. In contrast to the results with G. mellonella, crystals were ca. 30 times as active as spores in Pieris brassicae larvae. Many authors have found crystals purified by physical methods to be highly active in a range of lepidopterous hosts. The present work indicates that the role of the spore impurities in these species may need further investigation. Absence of live spores of B. thuringiensis may impair the control of some insect species feeding on spore-free products and on microorganisms or plants into which endotoxins have been introduced by genetic manipulation.

Fungi in libraries: an aerometric survey.

The possible role of fungi as allergic contaminants in book collections has been investigated in eleven University of Michigan Libraries. Air in the stacks of each of the eleven libraries was sampled on three occasions (2 or 4--10 minute samples on each occasion) with Andersen Volumetric viable particle samplers. Books were handled during sampling in half the samples each day. In addition on each sampling day a location in the same building away from book storage and an outdoor location were sampled. Library spore levels were generally low. Outdoor levels consistently exceeded indoor levels. Air conditioned (AC) libraries had lower spore levels and indoor/outdoor ratios than conventionally ventilated (CV) libraries. Handling books during sampling increased spore counts in all libraries, but strikingly in CV libraries. Fungus taxa recovered were similar to those encountered in domestic interiors and outside locations in our area. The overall low spore levels and lack of a distinctive library mycoflora suggest that other sources should be sought for library-based respiratory symptoms.