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.     

Identification of Clostridium tyrobutyricum as the causative agent of late blowing in cheese by species-specific PCR amplification.

Butyric acid fermentation, the late-blowing defect in cheese, caused by the outgrowth of clostridial spores present in raw milk, can create considerable loss of product, especially in the production of semihard cheeses like Gouda cheese, but also in grana and Gruyère cheeses. To demonstrate the causative relationship between Clostridium tyrobutyricum and late blowing in cheese, many cheesemaking experiments were performed to provoke this defect by using spores from several strains of the major dairy-related clostridia. A method of PCR amplification of a part of the 16S rRNA gene in combination with hybridization with species-specific DNA probes was developed to allow the specific detection of clostridial sequences in DNAs extracted from cheeses. The sensitivity was increased by using nested PCR. Late blowing was provoked in experimental cheeses with 28 of the 32 C. tyrobutyricum strains tested, whereas experimental cheeses made with spores from C. beijerinckii, C. butyricum, and C. sporogenes showed no signs of butyric acid fermentation. In all experimental and commercial cheeses with obvious signs of late blowing, DNA from C. tyrobutyricum was detected; in some cheeses, signals for C. beijerinckii were also found. It was concluded that only C. tyrobutyricum strains are able to cause butyric acid fermentation in cheese.     

Development and validation of PCR primers to assess the diversity of Clostridium spp. in cheese by temporal temperature gradient gel electrophoresis

A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing. Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates. TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species. PCR-TTGE was applied to analyze commercial cheeses with defects. All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species. The direct identification of Clostridium spp. was confirmed by sequencing of excised bands. C. tyrobutyricum and C. beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C. butyricum and C. sporogenes were detected in one cheese sample. Most-probable-number counts and volatile fatty acid were determined for comparison purposes. Results obtained were in agreement, but only two species, C. tyrobutyricum and C. sporogenes, could be isolated by the plating method. In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C. tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation. These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses. The sensitivity of the method was estimated to be 100 CFU/g.     

Enumeration of Fecal Clostridium perfringens Spores in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The Shahidi-Ferguson perfringens, tryptose-sulfite-cycloserine (TSC), and egg yolk-free TSC agars have been tested for their suitability to enumerate fecal spores of Clostridium perfringens. When these spores comprised at least 20% of the total anaerobe spores, equally accurate counts were obtained in the three media. With lower ratios of C. perfringens spores, the most accurate counts were obtained in egg yolk-free TSC agar. The median C. perfringens spore count of 60 normal fecal specimens was log 3.4/g. A nonmotile, sulfite- and nitrate-reducing Clostridium, not identifiable with any known clostridial species, was isolated from 14 out of 60 fecal specimans. It was not differentiated from C. perfringens in the nitrite motility test, but could be distinguished by its inability to liquefy gelatin.     

Selection of high affine peptide ligands for detection of Clostridium Tyrobutyricum spores

Clostridium tyrobutyricum is the main agent responsible for “late blowing” in cheese, which causes severe economic losses. Nowadays, the reference method for its detection is the Most-Probable-Number (MPN), however, is time consuming and non-specific. Thus, in order to check milk contamination with spores of C. tyrobutyricum, a more specific and rapid method would be required. The objective of this work was to obtain a ligand to establish the basis to develop a biomagnetic separation method for detection of C. tyrobutyricum spores. This study describes the selection of thirteen highly affine peptides to C. tyrobutyricum spores from a phage-display peptide library. In order to test the ability of the peptides attached to a solid support to bind the spores, the most frequent peptide was synthesised and used to coat paramagnetic beads.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37 degrees C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37 degrees C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37 degrees C for 48 h is recommended.     

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 × 10⁴, 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.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.     

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.     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.     

Progesterone analogs influence germination of Clostridium sordellii and Clostridium difficile spores in vitro

Clostridium sordellii and Clostridium difficile are closely related anaerobic Gram-positive, spore-forming human pathogens. C. sordellii and C. difficile form spores that are believed to be the infectious form of these bacteria. These spores return to toxin-producing vegetative cells upon binding to small molecule germinants. The endogenous compounds that regulate clostridial spore germination are not fully understood. While C. sordellii spores require three structurally distinct amino acids to germinate, the occurrence of postpregnancy C. sordellii infections suggests that steroidal sex hormones might regulate its capacity to germinate. On the other hand, C. difficile spores require taurocholate (a bile salt) and glycine (an amino acid) to germinate. Bile salts and steroid hormones are biosynthesized from cholesterol, suggesting that the common sterane structure can affect the germination of both C. sordellii and C. difficile spores. Therefore, we tested the effect of sterane compounds on C. sordellii and C. difficile spore germination. Our results show that both steroid hormones and bile salts are able to increase C. sordellii spore germination rates. In contrast, a subset of steroid hormones acted as competitive inhibitors of C. difficile spore germination. Thus, even though C. sordellii and C. difficile are phylogenetically related, the two species' spores respond differently to steroidal compounds.     

Comparison of Two Methods for Enumeration of Anaerobe Numbers on Forages and Evaluation of Ethylene Oxide Treatment for Forage Sterilization

Experiments were conducted to (i) compare most-probable-number (MPN) procedures with roll tube procedures for enumeration of forage anaerobic bacteria and (ii) evaluate the efficacy of using ethylene oxide to sterilize wet herbage. Alfalfa, corn, and alfalfa-orchardgrass silages and alfalfa and orchardgrass herbages were analyzed for total anaerobic bacteria (medium pH, 6.8) and acid-tolerant anaerobic bacteria (medium pH, 4.5) by both roll tube and MPN procedures. No difference was found between the roll tube and MPN procedures for total bacteria; however, higher counts were obtained for acid-tolerant bacteria when the MPN procedure was used. Although MPN procedures require less time to obtain an estimate of bacterial numbers, isolation and identification of the microbial population is not possible. Alfalfa herbage was treated with ethylene oxide for 12, 24, or 36 h, incubated for 7 days at 37°C with or without addition of a bacterial inoculant, and analyzed for total bacteria by MPN procedures. Microbial growth after inoculation of ethylene oxide-treated herbage indicated that there was insufficient residual ethylene oxide to inhibit subsequent microbial growth. The results also indicated that 24 h was required to adequately sterilize fresh herbage. Thus, ethylene oxide can be used to sterilize wet herbage for use as a substrate for pure cultures of silage bacteria.     

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry

Aims: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent‐labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. Methods and Results: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10³ spores per 100 ml of milk, and results could be achieved in 2 h. Conclusions: FCM combined with fluorochrome‐conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. Significance and Impact of the Study: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.     

Genomics of clostridial pathogens: implication of extrachromosomal elements in pathogenicity

The recently decoded genomes of the major clostridial toxin-producing pathogens Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium difficile have provided a huge amount of new sequence data. Recent studies have focused on the identification and investigation of pathogenic determinants and the regulatory events governing their expression. The sequence data revealed also the genomic background of virulence genes, as well as the contribution of extrachromosomal elements to a pathogenic phenotype. This has generated new insights in clostridial pathogenesis - and will continue to do so in the future - and has deepened our understanding of the anaerobic lifestyle of clostridial species.     

Butyric acid bacteria of the genus Clostridium in the bottom sediments of inland basins of different types

The cell numbers and ecological characteristics of the distribution of certain species of butyric acid bacteria (BAB) of the genus Clostridium in the bottom sediments of inland basins of different types were studied using the optimal nutrient media. The seasonal dynamics of clostridial vegetative cells and spores in sediments with different ecological conditions were revealed. The cell numbers of the dominant BAB species were shown to depend on the redox potential of the sediments, the amount and composition of C(org), and the trophic state of the basin in general. C. pasteurianum was found to predominate in eutrophic lakes and reservoirs (5-11 x 10(6) cells/cm3), C. butyricum and C. felsineum predominated in mesotrophic ones (2-11 x 10(6) cells/cm3), and C. acetobutylicum was predominant in acidic chthionoeutrophic lakes and reservoirs (0.1-0.5 x 10(6) cells/cm3). The lowest cell numbers of BAB were found in river sediments, whereas the highest numbers were recorded in the sediments of polysaprobic zones (0.1-1.0 x 10(3) and 0.5-2.0 x 10(7) cells/cm3 respectively).     

Production of solvents (ABE fermentation) from whey permeate by continuous fermentation in a membrane bioreactor

A continuous bioreactor where cells were recycled using a cross-flow microfiltration (CFM) membrane plant was investigated for the production of solvents (ABE fermentation) from whey permeate using Clostridium acetobutylicum P262. A tubular CFM membrane plant capable of being backflushed was used.The continuous fermentations were characterized by cyclic solventogenic and acidogenic behaviour, and ultimately degenerated to an acidogenic state. Steady-state solvent production was obtained for only short periods. This degeneration is attributed to the complex morphological behaviour of this strain of organism on this substrate.It is postulated that to achieve steady-state solvent production over extended periods of time, it is necessary to maintain a balance among the various morphological cell forms, i.e. acid-producing vegetative cells, solvent-producing clostridial cells, and inert forms, e.g. spores.     

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.     

Note: Occurrence and persistence of culturable clostridial spores on the leaves of horticultural plants

Clostridial spores were found in numbers from less than 1 to over 50 colony-forming units cm^-2 on mature leaves of 19 species of horticultural plants under commercial cultivation in five localities in Apulia (SE Italy). Of 1828 clostridial isolates, 87% were identified phenotypically and ascribed to Clostridium pasteurianum, Cl. sporogenes, Cl. butyricum, Cl. roseum, Cl. perfringens, Cl. felsineum and Cl. acetobutylicum , in decreasing order of frequency. When spore suspensions of Cl. pasteurianum, Cl. perfringens, Cl. roseum and Cl. sporogenes were inoculated onto the leaves of basil, leaf-beet, lettuce, rocket-salad, spinach and tomato in the greenhouse, spore counts at first invariably declined shortly after inoculation, then rose again significantly for Cl. pasteurianum and Cl. perfringens on basil and for Cl. sporogenes on tomato in summer.     

Impedance as an alternative to MPN enumeration of coliforms in pasteurized milks

R.H. MADDEN AND A. GILMOUR. 1995. Samples (900) of pasteurized whole, semi-skimmed and skimmed milk were subjected to conventional enumeration of coliforms by a nine-tube most probable number (MPN) technique, and impedance enumeration, in parallel. Regression analysis of the positive samples (98) showed that impedance enumeration was at least as accurate as the MPN method but results were obtained faster, with all testing being completed in 20 h, rather than 48 h. Consumable requirements, and staffing levels, were also much less with the impedance system. The impedance method could therefore beneficially replace the conventional method.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.