Cells Of Clostridium Perfringens Research Articles

Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture.

Cell-immobilization is used to maintain microbial culture to produce metabolites in repeated-batch or continuous fermentations, thereby reducing the time and resources spent on delivering mass production of microbe. The technique also enables shortening of the detoxification phase and the amount of formaldehyde required due to low incidence of viable bacteria in the extract. A solution of sodium alginate containing Clostridium perfringens cells was dropped into stirring CaCl solution via a sterile syringe needle. Optimizations resulted in reasonably uniform beads containing C. perfringens. Beads were externally stabilized by poly L-lysine, followed by immersion in a solution of Na-alginate to coat them with a new layer of alginate forming an alginate-PLL-alginate cortex. This study proved successful in immobilizing C. perfringens cells inside uniform alginate microspheres. Cell loading and cell propagation inside the beads were measured. The cell loaded beads were cultivable in liquid media producing 550 minimum lethal doses per milliliter (MLD/ml) in a 72 h. The research paved the way for further investigations to optimize and establish an efficient bacterial encapsulation method. Thus, it seems possible to produce toxins from beads engulfing C. perfringens on larger scales via repeated-batch or continuous fermentation processes.

Expression of glyceraldehyde-3-phosphate dehydrogenase on the surface of Clostridium perfringens cells

During research to identify fibronectin (Fn)-binding proteins (Fbps) on the surface of Clostridium perfringens cells, we identified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a candidate Fbp. GAPDH is a glycolytic enzyme found in a wide range of prokaryotes and eukaryotes. The Fn-binding activity of recombinant C. perfringens GAPDH (rGAPDH) was investigated using both ligand blotting analysis and enzyme-linked immunosorbent assay (ELISA). rGAPDH strongly bound plasminogen but not laminin or gelatin. Although GAPDH has no signal sequence, it is expressed on the cell surface of many microorganisms. The presence of GAPDH on the surface of C. perfringens cells was analyzed using ELISA and flow cytometry analyses; purified rGAPDH bound to the surface of C. perfringens cells. As autolysin is reportedly involved in the binding of GAPDH to the cell surface, we evaluated the interaction between rGAPDH and the C. perfringens autolysin Acp by both ELISA and ligand blotting assay. These assays revealed that rGAPDH binds to the catalytic domain of Acp but not the cell wall binding domains. These results suggest that autolysin mediates expression of GAPDH on the surface of C. perfringens cells and indicate a possible moonlighting function for GAPDH in binding both Fn and plasminogen.

Determination of the Clostridium perfringens-binding site on fibronectin

The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacterium's surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10.

Pediocin A improves growth performance of broilers challenged with Clostridium perfringens

The aim of this study was to investigate the efficacy of the anticlostridial pediocin A from Pediococcus pentosaceus FBB61 to contain negative effects associated to Clostridium proliferation in broilers, through 2 subsequent investigations. In the first study, 36 Ross 508 broilers were divided into 3 groups and fed for 21 d as follows: the control diet (CTR), the control diet supplemented with supernatant filtrate of a culture of P. pentosaceus FBB61-2 (Bac−, isogenic mutant nonproducing pediocin A), and the control diet supplemented with supernatant filtrate of a culture of P. pentosaceus FBB61 (Bac+) containing pediocin A. Birds were challenged with 106 cells of Clostridium perfringens. In the second study, 216 Ross 508 broilers were allocated in 18 pens and divided into 3 groups fed the same diet for 42 d: a control group (CTR), a group challenged with 108 cells of C. perfringens (CP), and a group challenged with 108 cells of C. perfringens and receiving the control diet supplemented with P. pentosaceus FBB61 and pediocin A (PA). Broiler BW, ADG, ADFI, and feed conversion rate were measured throughout the studies. At the end of both experiments, an appropriate number of birds was killed and analyzed for necrotic enteritis lesions and microbiological examinations. In the first study, on d 9, ADG and BW were 20% higher for Bac+ compared with CTR and Bac−; on d 14, ADG was higher for Bac+ (+23%, P < 0.05), whereas BW was higher for Bac+ and Bac− compared with CTR (+23 and +14%, respectively; P < 0.05). In the second study, on d 14, ADG and BW were higher for PA compared with CTR and CP (+15% on average; P < 0.05), whereas between 15 and 42 d, there was only a tendency toward a higher ADG for PA when compared with the CP group (+4%, P = 0.08). Diet supplementation with pediocin A improved broiler growth performance during the challenge with C. perfringens and tended to restore the ADG depletion during the 42-d period.

Immunization against Clostridium perfringens cells elicits protection against Clostridium tetaniin mouse model: identification of cross-reactive proteins using proteomic methodologies

BackgroundClostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs) have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization.ResultsEight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3) was the most abundant protein (43.3%), followed by methylaspartate ammonia-lyase (36.8%) and 2-phosphoglycerate dehydratase (35.6%). All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model.ConclusionMost of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG) functional category, either of posttranslational modification, protein turnover, and chaperones (O) or energy production and conversion (C). The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

DSC Analysis of foodborne bacteria

Differential scanning calorimetry (DSC) is applicable to studying the thermal properties of bacteria when treated with heat, cold, or antibiotics. Foodborne pathogens are inactivated by heat, and denaturation transitions observed by DSC indicate potential sites of cellular injury. Ribosomes, which are the sites for messenger RNA translation, are one critical component of thermal damage as evidenced by characteristic denaturation transitions in the 66-74°C range. These transitions disappear when cells of Clostridium perfringens are subjected to heat, suggesting structural or conformational changes to ribosomal proteins, and when cells of Listeria monocytogenes are cold-shocked by refrigeration, indicating ribosomal dissociation. DSC can be used to show that refrigeration followed by heat treatment improves the killing of dangerous microorganisms.

Growth behavior of Clostridium perfringens cells aggregated by bovine lactoferrin in the presence of lipoteichoic acid

The influence of lipoteichoic acid (LTA), which is the negatively charged component of the cell surface, on the bacteriostatic effect of bovine milk lactoferrin (bLF) was investigated. Cell agglutination in suspensions of 18 strains of Clostridium by iron-free and iron-saturated bLF (apo-bLF and Fe-bLF) was observed. The cell agglutination of Clostridium species by apo-bLF and Fe-bLF showed a significant correlation with the extent of the negative charge on the bacterial cell surface. The degree of cell agglutination of C. perfringens JCM1290 by bLF decreased with increasing LTA concentration. The growth suppressive ratio of bLF in the presence of LTA was also lower than that in the absence of LTA. The growth suppressive effect of bLF on C. perfringens JCM1290 was closely related to the cell agglutination by this protein. These results suggest that the direct interaction of bLF with the cell surface is necessary for the bacteriostatic effect of bLF.

Modeling and predicting non-isothermal microbial growth using general purpose software

Published experimental isothermal growth curves of Clostridium perfringens cells in ground ham were fitted with a modified three-parameter version of the logistic equation as a primary model and the temperature dependence of the three parameters by ad hoc empirical secondary models. These were used to predict the organism's non-isothermal growth curves under three different cooling regimes. The assumption has been that the organism's instantaneous (or momentary) non-isothermal growth rate is the isothermal rate at the given temperature at a time that corresponds to its instantaneous population size. This could be translated into a differential rate model equation, whose coefficients are constructed from terms that reflect the changing growth parameters with temperature and hence with time. The continuous rate equation, however, can be solved incrementally by a numerical procedure that can be implemented in similar purpose software like Microsoft Excel®. In all three cases, there was good agreement between the growth curves predicted by the model and those found experimentally. This demonstrated that the procedure can be used to generate growth curves under complicated thermal histories that may include regular and irregular temperature oscillations.

Sandwich ELISA detection of Clostridium perfringens cells and α-toxin from field cases of necrotic enteritis of poultry

Sandwich ELISAs (sELISAs) for the detection of Clostridium perfringens cells and α-toxin were developed and used to screen intestinal samples from normal broiler chickens and from clinical cases of necrotic enteritis. The assays clearly distinguished between the two sets of samples. The sELISA absorbance values from samples obtained from the majority of healthy birds were low and those from the majority of necrotic enteritis cases were high. Together, the assays provide a suitable test for the rapid screening for the diagnosis of necrotic enteritis in poultry.

Influence of butyric and lactic acids on the beta-glucuronidase activity of Clostridium perfringens.

The beta-glucuronidase activity of intact cells of Clostridium perfringens was not influenced by the presence of either 0.09 or 0.19% lactic or butyric acids. In contrast, the enhanced enzyme activity of intact cells due to sodium deoxycholate was significantly decreased by the presence of these acids. These results suggest the possibility that the development of cancer due to the intake of a high fat diet may be inhibited by the presence of organic acids produced by intestinal bacteria.

Influence of sodium chloride on the beta-glucuronidase activity of Clostridium perfringens and Escherichia coli.

While the beta-glucuronidase activity of intact cells of Clostridium perfringens was higher in 0.95% sodium chloride (NaCl) than that in 0, 0.1 or 0.5%, that of Escherichia coli was higher in 0.1% NaCl than that in 0, 0.5 or 0.95% NaCl in 0.1 mol l-1 KH2PO4. However, the enzyme activity of both species of intact cells was higher in buffer containing 16 mEq sodium, 134 mEq potassium and 16 mEq chloride per litre than in that containing 146 mEq sodium, 13 mEq potassium and 146 mEq chloride. These findings suggest that bacterial cells are affected by the presence of NaCl and that the effect of NaCl on the activity of bacterial beta-glucuronidase may differ by location in the large intestine.

Influence of various bile salts on beta-glucuronidase activity of intestinal bacteria.

While the decrease of the beta-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.

Isolation and characterization of extracellular proteases ofClostridium perfringens type A

Two extracellular proteases, E-A and E-B, produced by sporulating cells ofClostridium perfringens NCTC 8798, were isolated by ammonium sulfate fractionation followed by DEAE-Sephacel and Sephacryl S-300 chromatography. E-A was further purified to homogeneity following separation on casein-agarose. E-A and E-B possessed native molecular weights of 330 kDa and 96 kDa respectively. SDS-PAGE of E-A indicated that it was composed of one major 120-kDa subunit. Both E-A and E-B hydrolyzed N-succinyl-l-phenylalanine-p-nitroanilide and were inhibited by chymotrypsin but not antipain or leupeptin, indicating that they were chymotrypsin-like enzymes. Calcium but not dithiothreitol was effective in minimizing inactivation at 50°C. Comparative analysis of E-A and I-A-1, the principal intracellular protease, indicated that they were very similar but not identical.

Application of sialidase antibodies for the diagnosis of clostridial infections

Two immunological methods, a sialidase-inhibition test and an ELISA, had been developed for a species-specific detection of sialidases (EC 3.2.1.18) secreted by growing cells of Clostridium perfringens, C. septicum, and C. sordellii. These assays were applied to samples from the infected tissues and cotton wool plugs soaked with wound exudate from patients suspected to be suffering from gas gangrene. The results from 72 patients were compared with bacteriological investigations of 67 homologous samples. From these, 35 patients were found to be infected with one or two of the clostridial species: thirty patients contained C. perfringens, six samples showed an immunologically positive reaction for C. septicum and eight for C. sordellii. In the case of C. perfringens, a high degree of coincidence between the immunological and the bacteriological analyses was obtained. However, in infections with C. septicum or C. sordellii the bacteriological and immunological tests did not agree well. The simple and rapid sialidase inhibition test or the slightly more time-consuming ELISA, which even detects low quantities of sialidase, can be applied for the specific diagnosis of infections caused by C. perfringens, thus allowing a species-specific detection of this bacterial species in two or six hours, respectively, even at an early stage of infection. This early diagnosis is a prerequisite for a specific and life-saving therapy of this severe disease.

Electroporation-induced transformation of intact cells of Clostridium perfringens.

Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMB1 and pHR106 resulted in 3.8 X 10(-5) and 4.2 X 10(-4) transformants per viable cell, respectively. With respect to shuttle plasmid pHR106, these values represent a greater than 100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.

Beef Jerky: Viability of Food-Poisoning Microorganisms on Jerky during its Manufacture and Storage

Beef jerky was made from slices of flank steak inoculated with Staphylococcus aureus, vegetative cells of Clostridium perfringens as well as Bacillus subtilis and a two species-composite of Salmonella. Slices were placed in a domestic food dehydrator for 4 h at 52.9±0.8°C (127.2°F) followed by 4 h at 48.2±0.4°C (118.8°F). Meat slices dried rapidly, reaching an aw of 0.86 and a shelf-stable moisture-protein ratio of ≤1.6 within the first 2.5–3 h of drying. Samples originally contained about 68% moisture, but this dropped to about 30% by 4 h and 20% by 8 h. Some growth of inoculated S. aureus occurred initially but total numbers of all other added microorganisms decreased rapidly from the start of drying and although significantly reduced in numbers at the end of 8 h treatment, they survived processing. C. perfringens cells were not detected at the end of the heated-drying regimen but were recovered later in an inoculated sample stored at 2.5°C for a month. Contaminated jerky stored at 20°C and high relative humidity (RH) for 26–28 d did not contain detectable added bacteria, whereas identical samples stored at 2.5°C and low RH contained viable S. aureus and B. subtilis as well as C. perfringens. Domestic preparation of jerky from beef of normal retail quality would involve little risk provided initial drying is done rapidly at temperatures equal to or greater than those used in the present study.

SPORULATION OF Clostridium perfringens IN MEATS SUPPLEMENTED WITH SOY MATERIALS

ABSTRACTCooked meat samples and meat supplemented with 30% hydrated isolated soy protein (ISP), soy protein concentrate (SPC), or textured soy protein (TSP) were inoculated with vegetative cells of Clostridium perfringens and incubated anaerobically at 3 7°C for 24 hr. Of six C. perfringens strains, six sporulated in chicken thigh meat, two in beef, and none in pork sausage. Sporulation was significantly less (P &lt; 0.01) in chicken thigh meat or beef supplemented with SPC or TSP than in chicken or beef controls. Sporulation was significantly higher (P &lt; 0.01) in beef supplemented with ISP than in beef. Of the three pork sausage‐soy protein combinations, sporulation was infrequently detected in pork sausage‐ISP samples. The changes in sporulation in chicken or beef by addition of ISP, SPC and TSP were apparently due to a pH effect. With the pH adjusted to 7.5, sporulation of C. perfringens in Duncan‐Strong (D‐S) sporulation medium with added ISP, SPC, or TSP was similar to or greater than that in D‐S medium.

Method for Maintaining Viability of Clostridium perfringens in Foods During Shipment and Storage: Collaborative Study

A collaborative study was conducted in 12 laboratories to determine the effectiveness of a new method for maintaining vegetative cells of Clostridium perfringens in viable condition during storage and transport of food specimens to the laboratory. The collaborative results showed that treatment of brown gravy and roast beef samples with an equal amount by weight of sterile buffered glycerol-sodium chloride solution to give a final 10% glycerol concentration and storage with Dry Ice for 10 days at -56 degrees C resulted in plate counts of C. perfringens which were 2-4 log cycles higher with 2 different strains than counts with untreated specimens stored by the usual method at -20 degrees C. Plate counts obtained with the treated specimens stored with Dry Ice were less than 1 log cycle lower than counts made with identical specimens before freezing for storage and shipment to the collaborators. The results with treated specimens were also more uniform among the different laboratories. Because the new method for storage and shipment of food samples was so effective for maintaining viability of the organism, the official first action method for C. perfringens (46.B01) was changed to incorporate these procedures as part of the method.

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The extracellular matrix protein fibronectin (Fn) is known to bind to the surface of Clostridium perfringens cells. Fn is a disulfide-linked homodimer protein, with each Fn polypeptide consisting of three types of repeating modules: 12 type I, 2 type II, and 15-17 type III modules. To determine the epitope on Fn recognized by C. perfringens cells, anti-Fn monoclonal antibodies (mAbs) and various Fn fragments (III2-10, rIII2-4, rIII5-7, rIII8, rIII9, rIII10) were employed. Although two C. perfringens-derived Fn-binding proteins, FbpA and FbpB, have been reported, they appear not to be the bacterium's surface Fn receptor. Moreover, both FbpA and FbpB were found to bind to C. perfringens cells. To avoid confusion, a mutant C. perfringens lacking both the fbpA and fbpB genes (MW5) was prepared using an in-frame deletion system. MW5 cells bound Fn on their surface, suggesting the presence of a putative Fn receptor(s) on C. perfringens cells. Of several anti-Fn mAbs, both HB39 and MO inhibited the binding of Fn to MW5 cells. HB39 reacted strongly with III2-10 and rIII9, and weakly with rIII2-4, rIII10 and rIII5-7 in Western blotting analysis. Binding of HB39 to Fn was inhibited in the presence of either rIII9 or rIII10, but not in the presence of rIII2-4, rIII5-7, or rIII8. Binding of Fn to MW5 cells was strongly inhibited by both III2-10 and rIII9, marginally inhibited by rIII2-4, but not affected by rIII5-7, rIII8, or rIII10. Significant binding of MW5 cells to immobilized rIII9 and rIII10 as well as immobilized III2-10 was observed. The region of Fn recognized by C. perfringens was thus mapped to the region encompassed by III9 and III10.

The Interaction of Clostridium perfringens and Its Toxins in the Production of Necrotic Enteritis of Chickens

The intraduodenal administration of large numbers of Clostridium perfringens cells harvested from broth cultures and resuspended in PBS or fresh sterile thioglycollate broth produced a very mild form of necrotic enteritis. Administering an appropriate number of cells in culture supernatant, however, produced typical field-type disease. Alpha toxin was shown to be the significant toxin recoverable from broth-culture supernatant fluids. Requirements to produce the disease are minor intestinal damage and sufficient numbers of toxigenic C. perfringens in the intestine.