Evaluation and Modifications of Media for Enumeration of Clostridium perfringens

The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose.     

Radiation Resistance of Spores of Some Clostridium perfringens Strains

Clostridium perfringens spores (eight strains) were irradiated in a model system with 60Co gamma rays at -30 C. The quantal response data obtained were analyzed with extreme value statistics. It was found (at the 95% confidence level) that all eight strains followed the same rate of death and that this rate was probably (at the 95% level) not exponential. The statistics did not exclude, however, a normal, lognormal, Weibull, or related rate of spore kill. A more definitive study would be required to distinguish between the latter distributions.     

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.     

Comparison of Media for the Enumeration of Clostridium perfringens

For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 10(4) organisms per g, but with 10(6) organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested.     

Response of Clostridium perfringens Spores and Vegetative Cells to Temperature Variation

The vegetative cells and spores of four strains of Clostridium perfringens were examined to determine the effect of lowered and elevated temperatures. Spores were produced by following the method of Ellner, and vegetative cells were obtained from thioglycolate cultures. After exposure to freezing or refrigeration temperatures (-17.7 and 7.1 C, respectively), only small numbers of the vegetative cells were recovered. After similar treatment, 16 to 58% of the spores were recovered. Essentially no vegetative cells and few spores survived holding at 80 C for 10 min. Although all strains were isolated from food, only one strain of the four studied had its origin in a food-poisoning outbreak, and it had been carried on laboratory media for approximately 10 years.     

Recovery of Heated Clostridium perfringens Type A Spores on Selective Media

The enumeration of Clostridium perfringens spores on sulfite-polymyxin-sulfadiazine agar (SPS), tryptone-sulfite-neomycin agar (TSN), Shahidi-Ferguson-perfringens agar (SFP), tryptone-sulfite-cycloserine agar (TSC), and TSN lacking antibiotics (BASE) was studied. The spores were heated at 105 to 120 C by the capillary-tube method. The media were about equally efficient for the enumeration of heat-activated spores. Efficiency of the media for the recovery of spores surviving heat treatments at ultrahigh temperatures varied as follows: TSC >/= SFP > BASE > SPS > TSN. Greater recovery when survivors were enumerated on TSC or SFP was attributed to germination of injured spores by the lysozyme present in the egg yolk emulsion used in these media. Low recovery of survivors on TSN and SPS was due to both the absence of lysozyme and inhibition of injured spores by the selective agents of these media. Recovery of heated spores was reduced greatly by polymyxin, neomycin, and kanamycin, and slightly by sulfadiazine and D-cycloserine. The addition of lysozyme to SPS or TSN did not improve the percentage of heat-injured spores recovered because the selective agents of these media interfered with the action of lysozyme. The suitability of the selective media for the enumeration of survivors was greatly affected by the presence of certain foods.     

Improved Medium for Enumeration of Clostridium perfringens

An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective.     

Mechanism of Nitrite-Induced Germination of Clostridium perfringens Spores

A study has been undertaken to understand the mechanism(s) of the nitrite-induced germination of Clostridium perfringens S40 spores. An increase in germination rates of the spores in response to increasing NaNO2 concentrations was entirely dependent on both pH and temperature of incubation. Low pH and high temperature were effective in accelerating the germination rate, the maximal germination level being reached at pH 4.0 and 60°C in the presence of 0.5 M NaNO₂. On the basis of germination rate, the activation energy (μ) for the nitrite-induced germination calculated was approximately 9.9 kcal/mol. Germination was greatly stimulated after pretreatment of spores with DTT at pH 10.5 to remove the coats. Furthermore, cortical fragments prepared from spores of the same organism were lysed not only by lysozyme but also by NaNO_2. Hexosamine-containing material was also solubilized by these reagents. However, nitrite, unlike lysozyme, released a considerable amount of free hexosamine as well. These results suggest that nitrite-induced germination may involve an interaction of sodium nitrite as nitrous acid with some component of the cortex. A possible mechanism of nitrite-induced germination is discussed.     

Rapid Technique for the Enumeration of Clostridium perfringens

A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar.     

Role of GerKB in Germination and Outgrowth of Clostridium perfringens Spores

Previous work indicated that Clostridium perfringens gerKA gerKC spores germinate significantly, suggesting that gerKB also has a role in C. perfringens spore germination. We now find that (i) gerKB was expressed only during sporulation, likely in the forespore; (ii) gerKB spores germinated like wild-type spores with nonnutrient germinants and with high concentrations of nutrients but more slowly with low nutrient concentrations; and (iii) gerKB spores had lower colony-forming efficiency and slower outgrowth than wild-type spores. These results suggest that GerKB plays an auxiliary role in spore germination under some conditions and is required for normal spore viability and outgrowth.Spores of Bacillus and Clostridium species can break dormancy upon sensing a variety of compounds (termed germinants), including amino acids, nutrient mixtures, a 1:1 chelate of Ca²⁺ and pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]), and cationic surfactants such as dodecylamine (20). Nutrient germinants are sensed by their cognate receptors, located in the spore''s inner membrane (6), which are composed of proteins belonging to the GerA family (10, 11). In Bacillus subtilis, three tricistronic operons (gerA, gerB, and gerK) expressed uniquely during sporulation in the developing forespore each encode the three major germinant receptors, with different receptors responding to a different spectrum of nutrient germinants (5, 9, 20). Null mutations in any cistron in a gerA family operon inactivate the function of the respective receptor (9, 11). In contrast, Clostridium perfringens, a gram-positive, spore-forming, anaerobic pathogenic bacterium, has no tricistronic gerA-like operon but only a monocistronic gerAA that is far from a gerK locus. This locus contains a bicistronic gerKA-gerKC operon and a monocistronic gerKB upstream of and in the opposite orientation to gerKA-gerKC (Fig. ​(Fig.1A)1A) (16). GerAA has an auxiliary role in the germination of C. perfringens spores at low germinant concentrations, while GerKA and/or GerKC are required for l-asparagine germination and have partial roles in germination with KCl and a mixture of KCl and l-asparagine (AK) (16). In contrast to the situation with B. subtilis, where germinant receptors play no role in Ca-DPA germination (12, 13), GerKA and/or GerKC is required for Ca-DPA germination (16). The partial requirement for GerKA and/or GerKC in C. perfringens spore germination by KCl and AK suggests that the upstream gene product, GerKB, might also have some role in KCl and AK germination of C. perfringens spores. Therefore, in this study we have investigated the role of GerKB in the germination and outgrowth of C. perfringens spores.Open in a separate windowFIG. 1.Arrangement and expression of gerKB in C. perfringens SM101. (A) The arrangement of the gerK locus in C. perfringens SM101 is shown, and the locations of the primers used to amplify the upstream regions of the gerKB gene and the putative promoters of gerKB and gerKA are indicated. The gerKB promoter was predicted to be within the intergenic regions between gerKB and the gerK operon. (B) GUS specific activities from the gerKB-gusA fusion in strain SM101(pDP84) grown in TGY vegetative (filled squares) and DS sporulation (open squares) media were determined as described in the text. Data represent averages from three independent experiments with the error bars representing standard deviations, and time zero denotes the time of inoculation of cells into either TGY or DS medium.To determine if gerKB is expressed during sporulation, 485 bp upstream of the gerKB coding sequence, including DNA between gerKB and gerKA, was PCR amplified with primer pair CPP389/CPP391, which had SalI and PstI cleavage sites, respectively (see Table S2 in the supplemental material). The PCR fragment was cloned between SalI and PstI cleavage sites in plasmid pMRS127 (17) to create a gerKB-gusA fusion in plasmid pDP84 (see Table S1 in the supplemental material). This plasmid was introduced into C. perfringens SM101 by electroporation (3), and Em^r transformants were selected. The SM101 transformant carrying plasmid pDP84 was grown in TGY vegetative growth medium (3% Trypticase soy, 2% glucose, 1% yeast extract, 0.1% l-cysteine) (7) and in Duncan-Strong (DS) (4) sporulation medium and assayed for β-glucuronidase (GUS) activity as described previously (23). Vegetative cultures of strain SM101 carrying plasmid pMRS127 (empty vector) or pDP84 (gerKB-gusA) exhibited no significant GUS activity, and strain SM101 grown in DS medium also exhibited no significant GUS activity (Fig. ​(Fig.1B1B and data not shown). However, GUS activity was observed in sporulating cultures of SM101(pDP84) (Fig. ​(Fig.1B),1B), indicating that a sporulation-specific promoter is located upstream of gerKB. The expression of the gerKB-gusA fusion began ∼3 h after induction of sporulation and reached a maximum after ∼6 h of sporulation (Fig. ​(Fig.1B).1B). The decrease in GUS activity observed after ∼6 h is consistent with the GerKB-GusA protein being packaged into the dormant spore where it cannot be easily assayed and thus with gerKB being expressed in the forespore compartment of the sporulating cell (8). These results confirm that, as with the gerKA-gerKC operon (16), gerKB is also expressed only during sporulation.To investigate the role of GerKB in C. perfringens spore germination, we constructed a gerKB mutant strain (DPS108) as described previously (14-16). A 2,203-bp DNA fragment carrying 2,080 bp upstream of and 123 bp from the N-terminal coding region of gerKB was PCR amplified using primers CPP369 and CPP367, which had XhoI and BamHI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). A 1,329-bp fragment carrying 134 bp from the C-terminal and 1,195 bp downstream of the coding region of gerKB was PCR amplified using primers CPP371 and CPP370, which had BamHI and KpnI cleavage sites at the 5′ ends of the forward and reverse primers, respectively (see Table S2 in the supplemental material). These PCR fragments were cloned into plasmid pCR-XL-TOPO, giving plasmids pDP67 and pDP69, respectively (see Table S1 in the supplemental material). An ∼2.2-kb BamHI-XhoI fragment from pDP67 was cloned into pDP1 (pCR-XL-TOPO carrying an internal fragment of gerAA), giving plasmid pDP68, and an ∼1.4-kb KpnI-BamHI fragment from pDP69 was cloned in pDP68, giving pDP73 (see Table S1 in the supplemental material). The latter plasmid was digested with BamHI, the ends were filled, and an ∼1.3-kb NaeI-SmaI fragment carrying catP from pJIR418 (1) was inserted, giving plasmid pDP74. Finally, an ∼4.8-kb KpnI-XhoI fragment from pDP74 (see Table S1 in the supplemental material) was cloned between the KpnI and SalI sites of pMRS104, giving pDP75, which cannot replicate in C. perfringens. Plasmid pDP75 was introduced into C. perfringens SM101 by electroporation (3), and the gerKB deletion strain DPS108 was isolated as described previously (18). The presence of the gerKB deletion in strain DPS108 was confirmed by PCR and Southern blot analyses (data not shown). Strain DPS108 gave ∼70% sporulating cells in DS sporulation medium, similar to results with the wild-type strain, SM101 (data not shown).Having obtained evidence for successful construction of the gerKB mutant, we compared the germinations of heat-activated (80°C; 10 min) gerKB and wild-type spores as previously described (16). Both the gerKB and wild-type spores germinated identically and nearly completely in 60 min at 40°C in brain heart infusion (BHI) broth as determined by the fall in optical density at 600 nm (OD₆₀₀) of germinating cultures and phase-contrast microscopy (data not shown). This result suggests that GerKB plays no essential role in spore germination in rich medium. The role of GerKB in C. perfringens spore germination was also assessed with individual germinants identified previously (16). Heat-activated wild-type and gerKB spores germinated similarly with high (100 mM) concentrations of KCl, l-asparagine, and AK, all in 25 mM sodium phosphate (pH 7.0), and in 50 mM Ca-DPA adjusted to pH 8.0 with Tris base (Fig. 2A to D). These results were also confirmed by phase-contrast microscopy (data not shown). However, with lower (10 to 20 mM) concentrations of KCl, l-asparagine, and AK, gerKB spore germination was very slightly (Fig. ​(Fig.2A)2A) to significantly (Fig. 2B and C) slower than that of wild-type spores. These results suggest that while GerKB is not essential for germination with high concentrations of KCl, l-asparagine, or AK, it plays a significant role in germination with low l-asparagine and AK concentrations and, further, that GerKB is not required for Ca-DPA germination. This latter finding is similar to the situation with B. subtilis spores where germinant receptors play no role in Ca-DPA germination (19, 20). However, in C. perfringens spores, GerKA and/or GerKC do play a significant role in Ca-DPA germination (16).Open in a separate windowFIG. 2.Germination of spores of C. perfringens strains with various germinants. Heat-activated spores of strains SM101 (wild type) (filled symbols) and DPS108 (gerKB) (open symbols) were incubated at an OD₆₀₀ of 1 at 40°C with high (squares) and low (triangles) germinant concentrations of 100 and 10 mM KCl (A), 100 and 20 mM l-asparagine (B), 100 and 10 mM AK (C), and 50 mM Ca-DPA (D) as described in the text, and at various times the OD₆₀₀ was measured. No significant germination was observed when heat-activated spores of SM101 and DPS108 were incubated for 60 min at 40°C in 25 mM sodium phosphate buffer (pH 7.0) (data not shown). The data shown are averages from duplicate determinations with two different spore preparations, and error bars represent standard deviations.Bacterial spores can also germinate with dodecylamine, a cationic surfactant (19). In B. subtilis spores, dodecylamine induces germination most likely by opening channels composed, at least in part, of SpoVA proteins (22), allowing release of the spores'' Ca-DPA (19). Spores of B. subtilis lacking all three functional germinant receptors release DPA, as do wild-type spores, upon incubation with dodecylamine (19), while C. perfringens spores lacking GerKA-GerKC incubated with dodecylamine release DPA slower than wild-type spores (16). However, when C. perfringens gerKB spores at an OD₆₀₀ of 1.5 were incubated with 1 mM dodecylamine in Tris-HCl (pH 7.4) at 60°C (2, 16), gerKB spores released their DPA slightly faster than wild-type spores (Fig. ​(Fig.3)3) when DPA release was measured as described previously (16). These results suggest that GerKB has no role in dodecylamine germination.Open in a separate windowFIG. 3.Germination of spores of C. perfringens strains with dodecylamine. Spores of strains SM101 (wild type) (filled squares) and DPS108 (gerKB) (open squares) were germinated with dodecylamine, and germination was monitored by measuring DPA release as described in the text. There was no significant DPA release in 60 min by spores incubated similarly but without dodecylamine (data not shown). Error bars represent standard deviations.Previous work (16) found that C. perfringens spores lacking GerKA-GerKC had lower viability than wild-type spores on rich medium plates, and it was thus of interest to determine gerKB spore viability, which was measured as previously described (14, 16). Strikingly, the colony-forming ability of gerKB spores was ∼7-fold lower (P < 0.01) than that of wild-type spores after 24 h on BHI plates (Table ​(Table1),1), and no additional colonies appeared when plates were incubated for up to 3 days (data not shown). The colony-forming ability of spores lacking GerKA and GerKC determined in parallel was ∼12-fold lower than that of wild-type spores (Table ​(Table1).1). Phase-contrast microscopy of C. perfringens spores incubated in BHI broth for 24 h under aerobic conditions to prevent vegetative cell growth indicated that >90% of wild-type spores not only had germinated but had also released the nascent vegetative cell, while >85% of gerKA gerKC and gerKB spores remained as only phase-dark germinated spores with no evidence of nascent cell release (data not shown), as found previously with gerKA gerKC spores (16). The fact that >85% of gerKB spores germinated in BHI medium in 24 h but most of these germinated spores did not progress further in development strongly suggests that GerKB is needed for normal spore outgrowth (and see below) as well as for normal spore germination.

TABLE 1.

Colony formation by spores of C. perfringens strains^a

Detection and Enumeration of Clostridium difficile Spores in Retail Beef and Pork

Recent studies have identified Clostridium difficile in food animals and retail meat, and concern has been raised about the potential for food to act as a source of C. difficile infection in humans. Previous studies of retail meat have relied on enrichment culture alone, thereby preventing any assessment of the level of contamination in meat. This study evaluated the prevalence of C. difficile contamination of retail ground beef and ground pork in Canada. Ground beef and ground pork were purchased from retail outlets in four Canadian provinces. Quantitative and enrichment culture was performed. Clostridium difficile was isolated from 28/230 (12%) samples overall: 14/115 (12%) ground beef samples and 14/115 (12%) ground pork samples (P = 1.0). For ground beef, 10/14 samples (71%) were positive by enrichment culture only. Of the 4 ground beef samples that were positive by direct culture, 20 spores/g were present in 2 while 120 and 240 spores/g were present in 1 each. For ground pork, 10/14 (71%) samples were positive by enrichment culture only. Of the 4 ground pork samples that were positive by direct culture, 20 spores/g were present in 3 while 60 spores/g were present in 1. Ribotype 078 predominated, consistent with some previous studies of C. difficile in food animals. Ribotype 027/North American pulsotype 1 was also identified in both retail beef and pork. This study has identified relatively common contamination of retail ground beef and pork with C. difficile spores; however, the levels of contamination were very low.Clostridium difficile is an important cause of enteric disease in humans. It is the most commonly diagnosed cause of hospital- and antimicrobial agent-associated diarrhea in people, and recent evidence suggests that it may be emerging as an important community-associated pathogen (2, 5). In addition to humans, C. difficile can be found in the intestinal tracts of a variety of animal species, including food animals, such as cattle and pigs (7, 10, 13). Clostridium difficile has also been found in retail meat (11, 12, 17), and concerns about the role of food in the epidemiology of community-associated C. difficile infection (CA-CDI) have been expressed (5, 8, 15).Initial studies have reported isolation of C. difficile from 4.6 to 45% of retail meat samples (11, 12, 17). However, all studies have used broth enrichment protocols, which could detect very low spore numbers and provide no information about the number of organisms present in a sample. No studies have evaluated numbers of C. difficile spores in food. While the infectious dose is not known, an understanding of the level of contamination may be an important factor in determining the relevance of contamination of food. Additionally, the use of different methods between studies hampers comparison of results. Recently a study was performed to evaluate different methods for qualitative and quantitative detection of C. difficile (21). This study determined that the detection threshold of enrichment culture could be at least as low as 10 spores/g of meat. It also determined that quantitative culture can accurately determine the level of contamination in experimentally inoculated meat samples, albeit with a higher detection threshold. The objective of this study was to determine the prevalence of C. difficile contamination of retail ground beef and ground pork using both qualitative and quantitative methods.     

Inactivation of Clostridium perfringens Type A Spores at Ultrahigh Temperatures

The inactivation of Clostridium perfringens type A spores (three strains of different heat resistances) at ultrahigh temperatures was studied. Aqueous spore suspensions were heated at 85 to 135 C by the capillary tube method. When survivors were enumerated on the standard plating medium, the spores appeared to have been rapidly inactivated at temperatures above 100 C. The addition of lysozyme to the plating medium did not affect the recovery of spores surviving the early stages of heating, but lysozyme was required for maximal recovery of spores surviving extended heat treatments. The percentage of survivors requiring lysozyme for colony formation increased greatly with longer exposure times or increasing treatment temperature. Time-survivor curves indicated that each spore suspension was heterogeneous with respect to the heat resistance of spore outgrowth system or in the sensitivity of the spores to lysozyme. Recovery of survivors on the lysozyme containing medium revealed greater heat resistance for one strain than has been reported for spores of many mesophilic aerobes and anaerobes. The spores of all three strains were more resistant to heat inactivation when suspended in phosphate buffer, but a greater percentage of the survivors required lysozyme for colony formation.     

Enumeration of Food-Borne Clostridium perfringens in Egg Yolk-Free Tryptose-Sulfite-Cycloserine Agar

The SFP (Shahidi-Ferguson perfringens), TSC (tryptose-sulfite-cycloserine), EY (egg yolk)-free TSC, and OPSP (oleandomycin-polymyxin-sulfadiazine perfringens) agars have been tested for their suitability to enumerate Clostridium perfringens in naturally contaminated foods. Complete recoveries of C. perfringens were obtained in each of the four media, but only the TSC and EY-free TSC agars were sufficiently selective to ensure subsequent confirmatory tests without interference from facultative anaerobes. Because of some disadvantages associated with the use of egg yolk, EY-free TSC agar is recommended for enumeration of C. perfringens in foods. Several conditions for convenient shipment of foods and C. perfringens isolates with minimum loss of viability have been tested. The highest viable counts were preserved when foods were mixed 1:1 (wt/vol) with 20% glycerol and kept in a container with dry ice. Isolated C. perfringens strains remained viable for at least 2 weeks at ambient temperatures on blood agar slopes with a 2% agar overlay in screw-cap culture tubes.     

Inhibition of Clostridium botulinum by Strains of Clostridium perfringens Isolated from Soil

Thirty-one soil samples were examined for the presence of organisms capable of inhibiting growth and toxin production of strains of Clostridium botulinum type A. Such organisms were found in eight samples of soil. Inhibiting strains of C. perfringens were found in five samples, of C. sporogenes in three and of Bacillus cereus in three. Three of the C. perfringens strains produced an inhibitor effective on all 11 strains of C. botulinum type A against which they were tested, seven of eight proteolytic type B strains, one nonproteolytic type B strain, five of nine type E strains and all seven type F strains, whether proteolytic or nonproteolytic. They did not inhibit any of 26 type C strains, 6 type D strains, 4 type E strains, or 24 C. sporogenes strains. In mixed culture, an inhibitor strain of C. perfringens repressed growth and toxin production by a C. botulinum type A strain even though it was outnumbered by the latter about 40 times. It also repressed growth and toxin production of C. botulinum in mixed culture of soils in which this latter organism naturally occurred when cooked meat medium but not when trypticase medium was used.     

Distribution of Neuraminidase among Food-poisoning Strains of Clostridium perfringens

A survey was made to determine the distribution of the enzyme neuraminidase among 76 strains of Clostridium perfringens. Representative strains from each toxigenic type (A to F) and atypical C. perfringens type A food-poisoning strains of both American and English (Hobbs types) origin were tested. Both the American food-poisoning and nonfood-poisoning associated cultures consisted of both neuraminidase-positive and -negative strains. Furthermore, American strains which could not be differentiated from the original Hobbs cultures consisted of both neuraminidase-positive and -negative representatives. In contrast, the English (Hobbs) strains uniformly failed to produce an active intracellular or extracellular neuraminidase. No enzyme activity was detected in these strains when cultures were grown in different growth media, when grown in the presence of substrate (neuraminlactose), or upon extended incubation of enzyme preparations with substrate. With the exception of a type F strain, representative strains of the other toxigenic types (A to F) produced neuraminidase; 85% of the typical type A strains contained the enzyme.     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌a, b, e, t 毒素基因cpa、cpb、etx 及iA序列合成了针对4种毒素基因的4对特异引物, 建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法。结果本所保存的A, B, C, D, E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带, 而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性; 将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段。并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定, 并与毒素中和试验鉴定结果进行了比较, 结果表明两种方法具有较高的符合率。本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义。     

多重PCR鉴定不同毒素型的产气荚膜梭菌菌落

参照文献报道的产气荚膜梭菌α,β,ε,τ毒素基因cpa、cpb,etx及iA序列合成了针对4种毒素基因的4对特异引物,建立了一种简单的产气荚膜梭菌定型的菌落多重PCR方法.结果本所保存的A,B,c,D,E各型产气荚膜梭菌参考菌株均扩增出了相应的预期条带,而诺维氏梭菌、腐败梭菌和破伤风梭菌的扩增均为阴性;将单个菌落稀释100倍利用此菌落多重PCR仍能扩增到相应的目的片段.并利用此多重PCR对13株不同动物来源的产气荚膜梭菌进行了定型鉴定,并与毒素中和试验鉴定结果进行了比较,结果表明两种方法具有较高的符合率.本方法的建立对于产气荚膜梭菌的快速检测、定型具有十分重要的意义.     

Genetic Characterization of Type A Enterotoxigenic Clostridium perfringens Strains

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Contrasting Effects of Heat Treatment and Incubation Temperature on Germination and Outgrowth of Individual Spores of Nonproteolytic Clostridium botulinum Bacteria

In this study, we determined the effects of incubation temperature and prior heat treatment on the lag-phase kinetics of individual spores of nonproteolytic Clostridium botulinum Eklund 17B. The times to germination (t_germ), one mature cell (t_C1), and two mature cells (t_C2) were measured for individual unheated spores incubated at 8, 10, 15, or 22°C and used to calculate the t_germ, the outgrowth time (t_C1 − t_germ), and the first doubling time (t_C2 − t_C1). Measurements were also made at 22°C of spores that had previously been heated at 80°C for 20 s. For unheated spores, outgrowth made a greater contribution to the duration and variability of the lag phase than germination. Decreasing incubation temperature affected germination less than outgrowth; thus, the proportion of lag associated with germination was less at lower incubation temperatures. Heat treatment at 80°C for 20 s increased the median germination time of surviving spores 16-fold and greatly increased the variability of spore germination times. The shape of the lag-time (t_C1) and outgrowth (t_C1 − t_germ) distributions were the same for unheated spores, but heat treatment altered the shape of the lag-time distribution, so it was no longer homogeneous with the outgrowth distribution. Although heat treatment mainly extended germination, there is also evidence of damage to systems required for outgrowth. However, this damage was quickly repaired and was not evident by the time the cells started to double. The results presented here combined with previous findings show that the stage of lag most affected, and the extent of any effect in terms of duration or variability, differs with both historical treatment and the growth conditions.Clostridium botulinum is a group of four physiologically and phylogenetically distinct anaerobic spore-forming bacteria (known as groups I, II, III, and IV) that produce the highly toxic botulinum neurotoxin (12). The severity of the intoxication, botulism, ensures considerable effort is directed at preventing the growth of this pathogen in food. Nonproteolytic (group II) C. botulinum is one of the two groups most frequently associated with food-borne botulism. It forms heat-resistant spores and can germinate, grow, and produce toxin at 3°C (8); thus, nonproteolytic C. botulinum is a particular concern in mild heat-treated chilled foods (16, 17).Spores formed by pathogens such as C. botulinum are a significant food safety issue since they are able to resist many of the processes, such as cooking, used to kill vegetative cells. Understanding the transformation from a dormant spore to active vegetative cells is an important part of quantifying the risk associated with such organisms. Considerable effort has been targeted at measuring and relating the kinetic responses of populations of C. botulinum to environmental conditions and such data have been used to create predictive models, for example, ComBase (www.combase.cc). Such approaches have made a considerable contribution to ensuring food safety but problems with using population based predictions may arise when an initial inoculum is very small or additional information beyond point values is required. Spores typically contaminate foods at low concentrations so that growth of C. botulinum, when it occurs, is likely to initiate from just a few spores. In these circumstances the distribution of times to growth in packs will reflect the heterogeneity of times to growth from the contaminating individual spores. There is an intrinsic variability between individual spores within a population, and the relationship between population lag and individual lag is complex. Consequently, individual lag times cannot be predicted from population measurements (3). Knowledge of the underlying distribution would allow greater refinement of risk assessments.The lag period between a spore being exposed to conditions suitable for growth and the start of exponential growth will reflect the combined times of germination, emergence, elongation, and first cell division. Currently, very little is known about the variability and duration of these stages and any relationships between them. Measuring the kinetics of spore germination is usually achieved by measuring a population to identify time to percent completion. Such germination curves represent the summation of responses by individual spores. Some authors have measured the biovariability associated with individual spores, but most studies have examined only germination (4-7, 11, 22) and not subsequent outgrowth. More recently, we have used phase-contrast microscopy and image analysis to follow individual spores of nonproteolytic C. botulinum from dormancy, through germination and emergence, to cell division (21, 23). These experiments showed there is very little, or no, relationship between the time spent in each stage by individual spores. We have now extended this work to determine distributions of times for different stages in lag phase as affected by heat treatment and incubation temperature.