Co-culture with potentially probiotic microorganisms antagonises virulence factors of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in vitro

Toxigenic strains of Clostridium difficile were co-cultured with different strains of bifidobacteria and lactobacilli. Spent culture supernatants were tested for biological activity on cultured Vero cells. Co-culture of C. difficile with some potentially probiotic strains lead to a reduction of the biological activity of spent culture supernatants. The observed effects cannot be ascribed either to secreted factors from the probiotic strains or to toxin adsorption by bacterial cells. Immunological assays showed that there was significant diminution of both clostridial toxins (TcdA and TcdB) in spent culture supernatants of co-cultures as compared with pure clostridial cultures. Even though co-cultured clostridial cells showed a slight increase of intracellular toxins, this increase did not completely explains the reduction of toxin concentration in culture supernatants. The evidence suggests that the antagonism could be due to the diminution of the synthesis and/or secretion of both clostridial toxins. Our findings provide new insights into the possible mechanisms involved in the protective effect of probiotics in the context of C. difficile infection.     

Incidence of Mesophilic Clostrodium Spores in Raw Pork, Beef, and Chicken in Processing Plants in the United States and Canada

The anaerobic film pouch technique was used to quantitate and isolate clostridial spores in 2,358 samples of raw meat (1,078 of chicken, 624 of beef, 656 of pork). Of 19,727 putrefactive anaerobic (PA) sporeformers isolated, 1 was confirmed by mouse protection testing to be Clostridium botulinum type C. This isolate was obtained from a Western Canada chicken sample which contained 5.33 clostridia per gram. These data indicate a very low incidence of botulinal contamination in raw meats at the packing-plant level (0.042% of 2,358 samples) and an almost 20,000:1 ratio of nonbotulinal PA sporeformers to mesophilic C. botulinum spores. The mean level of PA contamination was 2.8 PA sporeformers per gram of meat; 77% of the samples contained three or less PA sporeformers per gram. Small but statistically significant differences in the incidence of clostridial spores were noted for season, geographical region, and type of meat.     

Solvent Production and Morphological Changes in Clostridium acetobutylicum

The morphological and cytological changes which occurred in Clostridium acetobutylicum P262 during the production of acetone, butanol, and ethanol in an industrial fermentation medium were identified and correlated with the growth and physiological changes. The swollen, cigar-shaped clostridial forms were involved in the conversion of acids to neutral solvents, and there was a correlation between the number of clostridial forms and the production of solvents. Sporulation mutants which were unable to form clostridial stages (cls mutants) did not produce solvents. Oligosporogenous mutants which showed reduced clostridial stage formation produced intermediate levels of solvents. Sporulation mutants blocked after the clostridial stage, which were unable to form mature spores (spo mutants), produced normal levels of solvents.     

Characterization of Ribosomes from Dormant Spores of Bacillus cereus

Characterization of ribosomes from dormant spores and vegetative cells of Bacillus cereus strain T has been carried out. Polyuridylic acid binding activity, ribonuclease activity associated with ribosomes, thermal denaturation profile, and sedimentation coefficients are essentially identical for both ribosomal preparations. However, ribosomal protein content of dormant spore ribosomes is about 70% of that of vegetative ribosomes. Polyacrylamide gel electrophoresis of ribosomal proteins shows that some ribosomal proteins are missing from dormant spore ribosomes. Sucrose density gradient centrifugation of ribosomes shows the existence of defective ribosomal subunits, in addition to 30S and 50S subunits, in dormant spore ribosomes. These results indicate that the ribosomes from dormant spores are distinctively different from those of vegetative cells.     

Spores of the mycorrhizal fungus <Emphasis Type="Italic">Glomus mosseae</Emphasis> host yeasts that solubilize phosphate and accumulate polyphosphates

The present paper reports the presence of bacteria and yeasts tightly associated with spores of an isolate of Glomus mosseae. Healthy spores were surface disinfected by combining chloramine-T 5%, Tween-40, and cephalexin 2.5 g L⁻¹ (CTCf). Macerates of these spores were incubated on agar media, microorganisms were isolated, and two yeasts were characterized (EndoGm1, EndoGm11). Both yeasts were able to solubilize low-soluble P sources (Ca and Fe phosphates) and accumulate polyphosphates (polyPs). Sequence analysis of 18S ribosomal deoxyribonucleic acid showed that the yeasts belong to the genera Rhodotorula or Rhodosporidium (EndoGm1) and Cryptococcus (EndoGm11). Results from inoculation experiments showed an effect of the spore-associated yeasts on the root growth of rice, suggesting potential tripartite interactions with mycorrhizal fungi and plants.     

Decay and synthesis of ribosomal proteins during Dictyostelium discoideum development

Summary Ribosome turnover is a prominent process during cell differentiation in Dictyostelium discoideum. At the end of 24 h of development on filters, the cells contain only 30% of the ribosome content of vegetatively growing cells. We determined the relative rates of synthesis and decay of each of the ribosomal proteins during this period. Approximately 80% of the total vegetative cell ribosomal proteins were degraded during the course of fruiting body construction. Ribosomal RNA and protein degradation apparently occurred coordinately during development. Although all ribosomal proteins decayed during development, some were more stable and a few less stable than the average. In addition, all the ribosomal proteins were synthesized during this period. Most ribosomal proteins were synthesized at the same rate as other cellular proteins, although a number were made at lower or higher rates. It was estimated that about 35% of the ribosomes in developed cells represented those, that were made during cell differentiation. Differential decay and/or synthesis of ribosomal proteins could account for the observed difference in protein content of ribosomes from growing amoebae and late development cells and spores.Paper No. 4 in the series, Studies on Ribosomal Proteins in Dictyostelium discoideum. Paper No. 3 is Ramagopal and Ennis (1982)     

Growth of Paenibacillus larvae,the causative agent of American foulbrood in honey bees and Bacillus popilliae,the causative agent of milky disease in beetle larvae in lepidopteran cell cultures

TNM-FH Lepidopteran insect cell culture medium containing 10% fetal bovine serum (FBS), while allowing limited vegetative growth of Paenibacillus larvae (wild-type strain), the causative agent of American foulbrood, contained no viable vegetative cells upon subculture, nor were any heat resistant spores produced in this medium alone. However, TNM-FH medium cotaining embryonic or midgut cells from Trichoplusia ni, hemocytes from Estigmene acrea, ovarian and embryonic cells from Spodoptera frugiperda, embryonic cells from Plutella xylostella, Spodoptera exigua and Pseudaletia unipuncta or ovarian cells from Lymantria dispar, supported both heavy vegetative cell growth and moderate production of heat resistant spores. EX-CELL 405 serum-free insect cell culture medium alone appeared to contain the appropriate nutrients required for both vegetative growth and sporulation of P. larvae. However, in the presence of embryonic cells from T. ni, limited vegetative growth occurred and the P. larvae cells appeared to die off. This was confirmed by the fact that no colony growth occurred upon subculture, nor were any heat resistant spores detected. This was true also in the presence of fat body cells from T. ni, except that a limited number of spores (4,000/ml) were detected in the form of cology-forming units (CFU) on plates following heating to 80°C for 20 minutes. In a parallel study with a wild-type strain of Bacillus popilliae, vegetative cells grew only in TNM-FH medium in the presence of mid-gut BTI-Tn-MG and ovarian (Tn-368) cells of T. ni. No heat resistant spores, however, were detected in any of the cultures. When BTI-Tn-MG and Tn-368 cells were further challenged with four variant cultures of B. popilliae, vegetative growth and limited sporulation were achieved. The BTI-Tn-MG cell line in TNM-FH medium produced as many as 12,000 spores/ml after 21 days in culture.     

Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage

Late blowing defect (LBD) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall‐binding domains (EAD and CBD) attached to green fluorescent protein (GFP) to detect dairy‐related Clostridium species by fluorescence microscopy. GFP‐CTP1L and GFP‐CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP‐EAD did not label any Clostridium strain tested. Unexpectedly, GFP‐CTP1L and GFP‐CBD were also able to bind to clostridial spores. In addition, GFP‐CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site‐directed mutants of GFP‐CTP1L and GFP‐CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP‐CTP1L and GFP‐CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

A genomic update on clostridial phylogeny: Gram‐negative spore formers and other misplaced clostridia

The class Clostridia in the phylum Firmicutes (formerly low‐G+C Gram‐positive bacteria) includes diverse bacteria of medical, environmental and biotechnological importance. The Selenomonas‐Megasphaera‐Sporomusa branch, which unifies members of the Firmicutes with Gram‐negative‐type cell envelopes, was recently moved from Clostridia to a separate class Negativicutes. However, draft genome sequences of the spore‐forming members of the Negativicutes revealed typically clostridial sets of sporulation genes. To address this and other questions in clostridial phylogeny, we have compared a phylogenetic tree for a concatenated set of 50 widespread ribosomal proteins with the trees for beta subunits of the RNA polymerase (RpoB) and DNA gyrase (GyrB) and with the 16S rRNA‐based phylogeny. The results obtained by these methods showed remarkable consistency, suggesting that they reflect the true evolutionary history of these bacteria. These data put the Selenomonas‐Megasphaera‐Sporomusa group back within the Clostridia. They also support placement of Clostridium difficile and its close relatives within the family Peptostreptococcaceae; we suggest resolving the long‐standing naming conundrum by renaming it Peptoclostridium difficile. These data also indicate the existence of a group of cellulolytic clostridia that belong to the family Ruminococcaceae. As a tentative solution to resolve the current taxonomical problems, we propose assigning 78 validly described Clostridium species that clearly fall outside the family Clostridiaceae to six new genera: Peptoclostridium, Lachnoclostridium, Ruminiclostridium, Erysipelatoclostridium, Gottschalkia and Tyzzerella. This work reaffirms that 16S rRNA and ribosomal protein sequences are better indicators of evolutionary proximity than phenotypic traits, even such key ones as the structure of the cell envelope and Gram‐staining pattern.     

The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Rapid viability assessment of spores of several fungi by an ionic intensified fluorescein diacetate method

Fluorescein diacetate (FDA) was applied to the viability assessment of spores of Aspergillus niger, Rhizopus stolonifer, Fusarium oxysporum, and Penicillium citrinum. The fluorescence of individual cells was quantitated with a charge coupled device (CCD) detector. When staining was carried out in a phosphate buffer solution (10 mM, pH 7.0), weak or no fluorescence was emitted from viable spores of A. niger and R. stolonifer, which made it difficult to distinguish between viable (nontreated) and nonviable (heat treated at 90°C for 30 min) spores. The addition of NaCl, KCl, or MgCl₂ to the staining solution caused an increase in the fluorescence intensity of A. niger viable spores, from which nonviable spores could be distinguished. The same effect of NaCl was observed in staining the spores of other species.     

Chemotherapeutic tumour targeting using clostridial spores

Abstract: The toxicity associated with conventional cancer chemotherapy is primarily due to a lack of specificity for tumour cells. In contrast, intravenously injected clostridial spores exhibit a remarkable specificity for tumours. This is because, following their administration, clostridial spores become exclusively localised to, and germinate in, the hypoxic/necrotic tissue of tumours. This unique property could be exploited to deliver therapeutic agents to tumours. In particular, genetic engineering could be used to endow a suitable clostridial host with the capacity to produce an enzyme within the tumour which can metabolise a systematically introduced, non-toxic prodrug into a toxic metabolite. The feasibility of this strategy (clostridial-directed enzyme prodrug therapy, CDEPT) has been demonstrated by cloning the Escherichia coli B gene encoding nitroreductase (an enzyme which converts the prodrug CB1954 to a highly toxic bifunctional alkylating agent) into a clostridial expression vector and introducing the resultant plasmid into Clostridium beijerinckii (formerly C. acetobutylicum ) NCIMB 8052. The gene was efficiently expressed, with recombinant nitroreductase representing 8% of the cell soluble protein. Following the intravenous injection of the recombinant spores into mice, tumour lysates have been shown, by Western blots, to contain the E. coli -derived enzyme.     

Main airborne Ascomycota spores: characterization by culture, spore morphology, ribosomal DNA sequences and enzymatic analysis

The aim of this work was to identify the main allergy-related Ascomycetes fungal spores present in the atmosphere of Porto, using different and complementary techniques. The atmospheric sampling, performed in the atmosphere of Porto (Portugal) from August 2006 to July 2008, indicated Cladosporium, Penicillium, Aspergillus and Alternaria as the main fungal spore taxa. Alternaria and Cladosporium peaks were registered during summer. Aspergillus and Penicillium highest values were registered from late winter to early spring. Additionally, the Andersen sampler allowed the culture and isolation of the collected viable spores subsequently used for different identification approaches. The internal-transcribed spacer region of the nuclear ribosomal repeat unit sequences of airborne Ascomycetes fungi isolates revealed 11 taxonomically related fungal species. Among the identified taxa, Penicillum and Aspergillus presented the highest diversity, while only one species of Cladosporium and Alternaria, respectively, were identified. All selected fungal spore taxa possessed phosphatase, esterase, leucine arylamidase and β-glucosidase enzymatic activity, while none had lipase, cystine arylamidase, trypsin or β-glucuronidase activity. The association between the spore cell wall morphology, DNA-based techniques and enzymatic activity approaches allowed a more reliable identification procedure of the airborne Ascomycota fungal spores.     

A microfuge tube technique for disrupting microquantities of fungal spores

A practical apparatus was assembled and through its use a technique was developed to disrupt microquantities of Ceratocystis ulmi spores. spores were placed in a microfuge tube with buffer and glass beads. The microfuge tube was sealed and placed inside a holding tube and then subjected to rapid vibrations over a Vortex mixer. By this technique 80–90% of the spores were disrupted and it was possible to extract a high-quality ribosomal RNA fraction from the supernatant fluid of the disrupted spore sample. The technique has also proved applicable to spores of fungi other than C. ulmi.     

Immunomodulatory effects of Bacillus subtilis (natto) B4 spores on murine macrophages

To investigate the immunomodulatory effects of Bacillus subtilis (B. subtilis) (natto) B4 spores on murine macrophage, RAW 264.7 cells were cultured alone or with B subtilis (natto) B4 spores at 37°C for 12 hrs, then both cells and culture supernatants were collected for analyses. Exposure of RAW 264.7 cells to B. subtilis (natto) B4 spores had no significant effects on macrophage viability and amounts of extracellular lactate dehydrogenase (LDH). However, it remarkably increased the activities of acid phosphatase (ACP), lactate dehydrogenase (LDH) and inducible nitric oxide synthase (iNOS) in cells and the amounts of nitric oxide (NO) and cytokines (tumor necrosis factor‐alpha, interferon‐gamma, interleukin [IL]‐1 beta, IL‐6, IL‐12, IL‐10 and macrophage inflammatory protein‐2) in culture supernatants. These results demonstrate that B. subtilis (natto) B4 spores are harmless to murine macrophages and can stimulate their activation through up‐regulation of ACP and LDH activities and enhance their immune function by increasing iNOS activity and stimulating NO and cytokine production. The above findings suggest that B. subtilis (natto) B4 spores have immunomodulatory effects on macrophages.     

Paranucleospora theridion n. gen., n. sp. (Microsporidia,Enterocytozoonidae) with a Life Cycle in the Salmon Louse (Lepeophtheirus salmonis,Copepoda) and Atlantic Salmon (Salmo salar)

ABSTRACT. Paranucleospora theridion n. gen, n. sp., infecting both Atlantic salmon (Salmo salar) and its copepod parasite Lepeophtheirus salmonis is described. The microsporidian exhibits nuclei in diplokaryotic arrangement during all known life‐cycle stages in salmon, but only in the merogonal stages and early sporogonal stage in salmon lice. All developmental stages of P. theridion are in direct contact with the host cell cytoplasm or nucleoplasm. In salmon, two developmental cycles were observed, producing spores in the cytoplasm of phagocytes or epidermal cells (Cycle‐I) and in the nuclei of epidermal cells (Cycle‐II), respectively. Cycle‐I spores are small and thin walled with a short polar tube, and are believed to be autoinfective. The larger oval intranuclear Cycle‐II spores have a thick endospore and a longer polar tube, and are probably responsible for transmission from salmon to L. salmonis. Parasite development in the salmon louse occurs in several different cell types that may be extremely hypertrophied due to P. theridion proliferation. Diplokaryotic merogony precedes monokaryotic sporogony. The rounded spores produced are comparable to the intranuclear spores in the salmon in most aspects, and likely transmit the infection to salmon. Phylogenetic analysis of P. theridion partial rDNA sequences place the parasite in a position between Nucleospora salmonis and Enterocytozoon bieneusi. Based on characteristics of the morphology, unique development involving a vertebrate fish as well as a crustacean ectoparasite host, and the results of the phylogenetic analyses it is suggested that P. theridion should be given status as a new species in a new genus.     

Germination and outgrowth of Bacillus subtilis and Bacillus licheniformis spores in the gastrointestinal tract of pigs

Aims: To determine if orally ingested Bacillus spores used as probiotics or direct‐fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. Methods and Results: Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore‐feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4–6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid‐colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2·14‐fold compared to time zero. Conclusions: The experiments showed that 70–90% of dietary‐supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. Significance and Impact of the Study: A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.