The pore structure of Clostridium perfringens epsilon toxin.

Epsilon toxin (Etx), a potent pore forming toxin (PFT) produced by Clostridium perfringens, is responsible for the pathogenesis of enterotoxaemia of ruminants and has been suggested to play a role in multiple sclerosis in humans. Etx is a member of the aerolysin family of ß-PFTs (aß-PFTs). While the Etx soluble monomer structure was solved in 2004, Etx pore structure has remained elusive due to the difficulty of isolating the pore complex. Here we show the cryo-electron microscopy structure of Etx pore assembled on the membrane of susceptible cells. The pore structure explains important mutant phenotypes and suggests that the double ß-barrel, a common feature of the aß-PFTs, may be an important structural element in driving efficient pore formation. These insights provide the framework for the development of novel therapeutics to prevent human and animal infections, and are relevant for nano-biotechnology applications.

Morphological Observation and Comparative Transcriptomic Analysis of Clostridium perfringens Biofilm and Planktonic Cells.

Bacterial biofilms can enhance survival in adverse environments and promote infection. However, little is known about biofilm formation by Clostridium perfringens. To better characterize this process, we used SEM to observe the surfaces of C. perfringens biofilms after 12, 24, 48, and 72 h of incubation. Biofilm cells appeared to be encased in a dense matrix material, and the total biomass of the biofilm increased with incubation time. To gain insight into the differentially expressed genes (DEGs) between biofilm and planktonic cells, we carried out comparative transcriptomic analysis using RNA sequencing. In total, 91 genes were significantly differentially expressed, with 40 being up-regulated and 51 down-regulated. In particular, genes encoding sortase, ribosomal proteins, and ATP synthase were up-regulated in biofilms, while genes coding for clostripain and phospholipase C were down-regulated. To validate the RNA sequencing results, qRT-PCR analysis was performed using five randomly selected DEGs. Results showed that all five genes were up-regulated, which was in accordance with the RNA sequencing results. To examine the functional differences, the DEGs were characterized by GO and KEGG pathway enrichment analyses. Results showed that the up-regulated genes were divided into 32 significantly enriched GO terms, with "macromolecular complex" being the most common. Oxidative phosphorylation was the only significantly enriched pathway, suggesting that ATP is required for biofilm stability. This study provides valuable insights into the morphology and transcriptional regulation of C. perfringens during biofilm formation, and will be useful for understanding and developing biofilm-based processes.

Environmental factors that shape biofilm formation.

Cells respond to the environment and alter gene expression. Recent studies have revealed the social aspects of bacterial life, such as biofilm formation. Biofilm formation is largely affected by the environment, and the mechanisms by which the gene expression of individual cells affects biofilm development have attracted interest. Environmental factors determine the cell's decision to form or leave a biofilm. In addition, the biofilm structure largely depends on the environment, implying that biofilms are shaped to adapt to local conditions. Second messengers such as cAMP and c-di-GMP are key factors that link environmental factors with gene regulation. Cell-to-cell communication is also an important factor in shaping the biofilm. In this short review, we will introduce the basics of biofilm formation and further discuss environmental factors that shape biofilm formation. Finally, the state-of-the-art tools that allow us investigate biofilms under various conditions are discussed.

Inactivation and ultrastructure analysis of Bacillus spp. and Clostridium perfringens spores.

Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.

Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.

Reversible monensin adaptation in Enterococcus faecium, Enterococcus faecalis and Clostridium perfringens of cattle origin: potential impact on human food safety.

OBJECTIVES: To determine the stability/reversibility and mechanism of monensin adaptation in monensin-treated cattle isolates compared with reference bacterial isolates, exposed in vitro to high monensin concentrations. METHODS: We evaluated the potential for cattle-origin strains of Clostridium perfringens, Enterococcus faecium and Enterococcus faecalis exposed to monensin in vivo (in vivo monensin-exposed isolates) to maintain or achieve the ability to grow in the presence of high monensin concentrations (in vitro monensin-adapted isolates). Twenty-one consecutive subcultures of the in vitro monensin-adapted strains were performed, and monensin MICs were determined for the 3rd, 7th, 14th and 21st subcultures (subcultured isolates). SDS-PAGE and transmission electron microscopy (TEM) were used to determine protein expression and visualize extracellular morphology changes. RESULTS: Monensin-non-exposed isolates did not display monensin adaptation during in vitro monensin exposure. In contrast, in vivo monensin-exposed isolates displayed monensin adaptation enabling growth at 32× MIC. Upon consecutive subculturing, monensin MICs returned to baseline, or one dilution above, for the monensin-adapted strains. SDS-PAGE identified overexpression of a 14 kDa protein (C. perfringens) and a 20.5 kDa protein (E. faecium and E. faecalis) in the monensin-adapted isolates. TEM demonstrated that in vitro monensin-adapted strains had a significantly thicker cell wall or glycocalyx compared with in vivo monensin-exposed or subcultured isolates. CONCLUSIONS: In vivo monensin-exposed isolates of C. perfringens, E. faecium and E. faecalis have the ability to grow in the presence of high monensin concentrations in vitro. This is associated with an increased thickening of the cell wall or glycocalyx that is reversible upon serial passage, suggesting a phenotypically expressed, but not genetically stable, trait.

Type IV pili and the CcpA protein are needed for maximal biofilm formation by the gram-positive anaerobic pathogen Clostridium perfringens.

The predominant organizational state of bacteria in nature is biofilms. Biofilms have been shown to increase bacterial resistance to a variety of stresses. We demonstrate for the first time that the anaerobic gram-positive pathogen Clostridium perfringens forms biofilms. At the same concentration of glucose in the medium, optimal biofilm formation depended on a functional CcpA protein. While the ratio of biofilm to planktonic growth was higher in the wild type than in a ccpA mutant strain in middle to late stages of biofilm development, the bacteria shifted from a predominantly biofilm state to planktonic growth as the concentration of glucose in the medium increased in a CcpA-independent manner. As is the case in some gram-negative bacteria, type IV pilus (TFP)-dependent gliding motility was necessary for efficient biofilm formation, as demonstrated by laser confocal and electron microscopy. However, TFP were not associated with the bacteria in the biofilm but with the extracellular matrix. Biofilms afforded C. perfringens protection from environmental stress, including exposure to atmospheric oxygen for 6 h and 24 h and to 10 mM H(2)O(2) for 5 min. Biofilm cells also showed 5- to 15-fold-increased survival over planktonic cells after exposure to 20 microg/ml (27 times the MIC) of penicillin G for 6 h and 24 h, respectively. These results indicate C. perfringens biofilms play an important role in the persistence of the bacteria in response to environmental stress and that they may be a factor in diseases, such as antibiotic-associated diarrhea and gas gangrene, that are caused by C. perfringens.

Factors contributing to heat resistance of Clostridium perfringens endospores.

The endospores formed by strains of type A Clostridium perfringens that produce the C. perfringens enterotoxin (CPE) are known to be more resistant to heat and cold than strains that do not produce this toxin. The high heat resistance of these spores allows them to survive the cooking process, leading to a large number of food-poisoning cases each year. The relative importance of factors contributing to the establishment of heat resistance in this species is currently unknown. The present study examines the spores formed by both CPE(+) and CPE(-) strains for factors known to affect heat resistance in other species. We have found that the concentrations of DPA and metal ions, the size of the spore core, and the protoplast-to-sporoplast ratio are determining factors affecting heat resistance in these strains. While the overall thickness of the spore peptidoglycan was found to be consistent in all strains, the relative amounts of cortex and germ cell wall peptidoglycan also appear to play a role in the heat resistance of these strains.

The beta-defensin gallinacin-6 is expressed in the chicken digestive tract and has antimicrobial activity against food-borne pathogens.

Food-borne pathogens are responsible for most cases of food poisoning in developed countries and are often associated with poultry products, including chicken. Little is known about the role of beta-defensins in the chicken digestive tract and their efficacy. In this study, the expression of chicken beta-defensin gallinacin-6 (Gal-6) and its antimicrobial activity against food-borne pathogens were investigated. Reverse transcription-PCR analysis showed high expression of Gal-6 mRNA in the esophagus and crop, moderate expression in the glandular stomach, and low expression throughout the intestinal tract. Putative transcription factor binding sites for nuclear factor kappa beta, activator protein 1, and nuclear factor interleukin-6 were found in the Gal-6 gene upstream region, which suggests a possible inducible nature of the Gal-6 gene. In colony-counting assays, strong bactericidal and fungicidal activity was observed, including bactericidal activity against food-borne pathogens Campylobacter jejuni, Salmonella enterica serovar Typhimurium, Clostridium perfringens, and Escherichia coli. Treatment with 16 mug/ml synthetic Gal-6 resulted in a 3 log unit reduction in Clostridium perfringens survival within 60 min, indicating fast killing kinetics. Transmission electron microscopy examination of synthetic-Gal-6-treated Clostridium perfringens cells showed dose-dependent changes in morphology after 30 min, including intracellular granulation, cytoplasm retraction, irregular septum formation in dividing cells, and cell lysis. The high expression in the proximal digestive tract and broad antimicrobial activity suggest that chicken beta-defensin gallinacin-6 plays an important role in chicken innate host defense.

In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production.

AIMS: Clostridium perfringens type A causes both clinical and subclinical forms of necrotic enteritis in domestic avian species. In this study the inhibitory effect of hen egg white lysozyme on the vegetative form of Cl. perfringens type A and the production of alpha-toxin in vitro was investigated. METHODS AND RESULTS: A micro-broth dilution assay was used to evaluate the minimal inhibitory concentrations (MIC) of lysozyme against three clinical isolates of Cl. perfringens type A in 96-well microtitre plates. The MIC of lysozyme against Cl. perfringens isolates was found to be 156 microg ml(-1). Scanning electron micrographs of the cells treated with 100 microg ml(-1) of lysozyme revealed extensive cell wall damage. A quantitative sandwich ELISA for alpha-toxin produced by Cl. perfringens was developed based on a commercial ELISA kit allowing only qualitative detection. Addition of 50 microg ml(-1) of lysozyme did not inhibit the growth of Cl. perfringens but significantly inhibited the toxin production. CONCLUSIONS: Lysozyme inhibited the growth of Cl. perfringens type A at 156 microg ml(-1). At sublethal levels, lysozyme was able to inhibit the alpha-toxin production. SIGNIFICANCE AND IMPACT OF STUDY: Inhibition of Cl. perfringens type A and its alpha-toxin production by hen egg white lysozyme had never previously been reported. By inhibiting this avian pathogen and its toxin production, lysozyme showed potential for use in the treatment and prevention of necrotic enteritis and other Cl. perfringens type A related animal diseases.

Susceptibility of Clostridium perfringens to C-C fatty acids.

AIMS: To determine susceptibility of Clostridium perfringens strains CCM 4435(T) and CNCTC 5459 to C(2)-C(18) fatty acids, and evaluate influence of pH in cultures grown on glucose. Straw particles were added to cultures to simulate the presence of solid phase of the digestive tract milieu. METHODS AND RESULTS: Antimicrobial activity of fatty acids was expressed as a concentration at which only 50% of the initial glucose was utilized. Lauric acid showed the highest antimicrobial activity, followed by myristic, capric, oleic and caprylic acid. Only strain CNCTC 5459 was susceptible to linoleic acid. Neither caproic acid and acids with a shorter carbon chain nor palmitic and stearic acid influenced substrate utilization. The antimicrobial activity of myristic, oleic and linoleic acid decreased when clostridia were grown in the presence of straw particles. In cultures of both strains treated with capric and lauric acid at pH 5.0-5.3, the number of viable cells was <10(2) ml(-1). Only lauric acid reduced number of viable cells of both strains below 10(2) ml(-1) at pH > 6. Transmission electron microscopy revealed separation of inner and outer membranes and cytoplasma disorganization in cells treated with lauric acid. CONCLUSIONS: Lauric acid had the highest activity towards C. perfringens among fatty acid tested. Its activity was not influenced by the presence of solid particles and did not cease at pH > 6. SIGNIFICANCE AND IMPACT OF THE STUDY: Lauric acid might be a means for control of clostridial infections in farm animals.

Behavior of Clostridium perfringens at low temperatures.

Refrigerated storage is an important step in the preparation of foods and inadequate storage is one of the main causes of food poisoning outbreaks of Clostridium perfringens. Therefore, growth and germination characteristics of C. perfringens in a temperature range of 3-42 degrees C were determined in fluid thioglycollate broth (FTG) and Dutch pea soup. To study the effect of adaptation, cells were either inoculated from a 37 degrees C pre-culture or from a temperature-adapted pre-culture. Membrane fatty acid patterns were determined at all temperatures to examine the effect of temperature on membrane composition. Spores were either inoculated with and without heat treatment. Adaptation of cells did not influence growth rate nor lag phase. Growth in pea soup, however, was slower and lag phases tended to be more extended compared to FTG. No growth was observed at temperatures < or =10 degrees C and death rates in pea soup were higher than those in FTG at these low temperatures. Cells preserved the membrane fluidity by reducing the arachidic acid content and increasing the lauric acid content when the temperature dropped. This resulted in a net reduction in chain length. Microscopic analysis of cells grown at 15 degrees C revealed a morphological change: cells were elongated compared to those grown at 37 degrees C. These data demonstrate the ability of C. perfringens to adapt to lower temperatures. However, this did not influence growth characteristics compared to non-adapted cells. Spores of C. perfringens did germinate at all temperatures with and without heat-activation. Combining this fact with the extended survival at low temperatures emphasizes the need for adequate heating of refrigerated foods before consumption to eliminate health risks due to C. perfringens.

Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia.

OBJECTIVES: The cathelicidin-derived peptide SMAP-29 exerts rapid and broad-spectrum antimicrobial activity against aerobic bacteria and fungi. In this study, the effects of the peptide against the Bacteroides fragilis group, including antibiotic-resistant isolates, Clostridium perfringens and Clostridium difficile reference and clinical isolates, were investigated. METHODS: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains. The killing mechanism was investigated further by means of a two-colour fluorescent permeabilization assay, and by scanning electron microscopy (SEM). RESULTS: The Bacteroides fragilis group, Clostridium reference strains and most clinical isolates were inhibited in vitro by 1-2 microM (3.2-6.4 mg/L) SMAP-29, and killed by 1.5- to 2-fold higher peptide concentrations. The anaerobic bacterial cells were 90%-100% permeabilized within 2 h of exposure to bactericidal concentrations of the peptide. The SEM images of bacteria exposed to SMAP-29 provide morphological evidence that the envelope is an important target of the bactericidal activity of this peptide. These results are consistent with earlier studies indicating that SMAP-29 kills aerobic bacteria with a membranolytic mechanism, and suggest that both aerobic and anaerobic bacteria share surface features that are targeted by this peptide. CONCLUSIONS: These studies demonstrate that the spectrum of antibacterial activity of SMAP-29 includes the B. fragilis group and Clostridium species, and encourage further investigations of the therapeutic potential of this peptide.

Multiple effects on Clostridium perfringens binding, uptake and trafficking to lysosomes by inhibitors of macrophage phagocytosis receptors.

Clostridium perfringens is a Gram-positive, anaerobic bacterium that is the most common cause of gas gangrene (clostridial myonecrosis) in humans. C. perfringens produces a variety of extracellular toxins that are thought to be the major virulence factors of the organism. However, C. perfringens has recently been shown to have the ability to survive in a murine macrophage-like cell line, J774-33, even under aerobic conditions. In J774-33 cells, C. perfringens can escape the phagosome and gain access to the cytoplasm. Since the receptor that is used for phagocytosis can determine the fate of an intracellular bacterium, we used a variety of inhibitors of specific receptors to identify those used by J774-33 cells to phagocytose C. perfringens. It was found that the scavenger receptor and mannose receptor(s) were involved in the phagocytosis of C. perfringens. In the presence of complement, the complement receptor (CR3) was also involved in the binding and/or uptake of C. perfringens. Since the receptor inhibition studies indicated that the scavenger receptor played a major role in phagocytosis, C. perfringens binding studies were performed with a Chinese hamster ovary (CHO) cell line expressing the mouse SR-A receptor. The cell line expressing the SR-A receptor showed a significant increase in C. perfringens binding in comparison to the non-transfected CHO cells. In the absence of opsonizing antibodies, the Fc receptor was not used to phagocytose C. perfringens. Forcing the macrophages to use a specific receptor by using combinations of different receptor inhibitors led to only a slight increase in co-localization of intracellular C. perfringens with the late endosome-lysosome marker LAMP-1. Carbohydrate analysis of C. perfringens strain 13 extracellular polysaccharide confirmed the presence of mannose and negatively charged residues of glucuronic acid, which may provide the moieties that promote binding to the mannose and scavenger receptors, respectively.

Bactericidal properties of the chloroform fraction from rhizomes of Aristolochia paucinervis Pomel.

The deffated chloroform fraction (APRC) obtained from the rhizomes of Aristolochia paucinervis Pomel (Aristolochiaceae) has a high bacteriostatic activity against bacterial strains like Clostridium perfringens ATCC 13124 and Enterococcus faecalis ATCC 29212. Here, we report the bactericidal activity of APRC against both strains which was evaluated by using time-to kill assays. The results showed that APRC produced an intense time-dependent bactericidal effect against C. perfringens, achieving over a 24 h-period a 5log10-unit decrease in CFU/ml at a concentration > or =1.25 x MIC. In contrast, when tested against E. faecalis, APRC exhibited a concentration-dependent killing activity at concentrations of 1.25 x MIC and 2.5 x MIC, yielding to a decrease of 1.5 and 2.5log10-unit in CFU/ml at 4 h, respectively. However, substantial regrowth of E. faecalis occurred within 24 h. Ultrastructural alterations were observed for both exposed microorganisms by scanning and transmission electron microscopy.

[Physico-chemical and immunobiological properties of the ribosomal preparations of Clostridium perfringens, type A]. / Fiziko-khimicheskie i immunobiologicheskie svoistva ribosomal'nykh preparatov Clostridium perfringens tipa A.

Ribosomes, sufficiently purified by physicochemical criteria (with the RNA/protein ratio equal to 1.6 - 1.7), were isolated from disintegrated C.perfringens cells, type A, strain BP6K, by ultracentrifugation or by fractionation with polyethylene glycol. The purified conjugates of ribosomes and C.perfringens toxoid, absorbed on aluminum hydroxide, were obtained. The ribosomal preparations had low toxicity and high protective activity: when used in a dose of 6 binding units, they protected guinea pigs and mice inoculated with 8 - 9 LD50 of C.perfringens culture.

Stimulation of the onset of sporulation of Clostridium perfringens type A by netropsin and distamycin.

The basic peptide antibiotics, netropsin and distamycin, previously shown to inhibit sporulation of Bacillus subtilis, stimulated low levels of sporulation of Clostridium perfringens strain NCTC 8798 at concentrations of 1.0 and 0.1 microgram/ml respectively. Most sporulating cells produced in the presence of the antibiotics were defective. These were blocked at Stage III of sporulation, and many possessed forespores exterior to the sporangium. The same antibiotics could also inhibit the caffeine-induced stimulation of sporulation of this strain.

Isolation of inclusion bodies from vegetative Clostridium perfringens: partial purification of a 47 kDa inclusion protein.

A refractile inclusion body produced by vegetative cells of Clostridium perfringens at temperatures above 40 degrees C was isolated and partially characterized. The inclusion was composed of protein and could be solubilized by sodium dodecyl sulphate plus either dithiothreitol or beta-mercaptoethanol. The solubilized inclusion showed no antigenic relationship with Cl. perfringens enterotoxin. One major band with an apparent MW of 47 kDa was demonstrated after polyacrylamide gel electrophoresis of the solubilized inclusion. Both enterotoxin-positive and enterotoxin-negative strains produced the inclusion body. No effect on the morphology of several eucaryotic cell lines was observed when solubilized or intact inclusion was added to the cell cultures.

Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C.

Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C. Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C. There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C).