Tryptose Sulfite Cycloserine Research Articles

Clostridium perfringens in central Colombia: frequency, toxin genes, and risk factors

Clostridium perfringens is an opportunistic bacterium that causes intestinal diseases in both humans and animals. This study aimed to assess the frequency of C. perfringens and the presence of toxin-encoding genes in fecal samples from individuals with or without gastrointestinal symptoms in the Department of Boyacá, Colombia. Additionally, risk factors associated with carriage and disease development were analyzed. A total of 114 stool samples were analyzed using a molecular test based on specific polymerase chain reaction (PCR) targeting 16S-rRNA and alpha toxin (cpa) genes. For individuals with a positive result for the PCR test, stool samples were cultured on Tryptose Sulfite Cycloserine (TSC) agar. Two to five colonies forming units were selected based on phenotypic characteristics, resulting in 56 bacterial isolates. These isolates were then analyzed for toxin-coding genes associated with gastrointestinal diseases. In addition, sociodemographic and clinical data from 77 individuals were also analyzed. The overall frequency of C. perfringens was 19.3% (n = 22/114). The detection frequency in 77 individuals with clinical data was 16.6% (n = 5/30) among symptomatic individuals and 21.2% (n = 10/47) among asymptomatic individuals. All 56 isolates obtained carried the cpa gene, while cpb2 was present in 10.7% (n = 6/56); cpe and cpb genes were not detected. Notably, diabetes and autoimmune diseases are significantly associated with an increased risk of C. perfringens detection (adjusted OR 8.41: 95% CI 1.32–35.89). This study highlights an elevated frequency of C. perfringens and the presence of the cpb2 gene in asymptomatic individuals compared with their symptomatic counterparts. These findings offer insights into the distribution and virulence factors of C. perfringens at a micro-geographical level. This information supports the need for developing tailored prevention strategies based on local characteristics to promote active surveillance programs based on molecular epidemiology.

Evaluation of Three Different Selective Media for Enumeration of Clostridium perfringens in Untreated and Treated Wastewater.

Current and emerging legislation in North Carolina and other regions calls for the enumeration of Clostridium perfringens as a surrogate indicator for protozoan parasites in various types of waters. Past studies that have evaluated selective media for the detection of this bacterium have provided limited, conflicting, and inconclusive results. In this study, membrane filtration was used to enumerate C. perfringens as culturable spores or total culturable cells in 19 samples of untreated and 25 samples of partially treated wastewaters on 3 candidate media, Tryptose Sulfite Cycloserine Agar (TSC), CP ChromoSelect Agar (CPCS), and membrane Clostridium perfringens Agar (m-CP) in parallel, and the results were compared. Presumptive isolates from each agar were further subjected to phenotypic confirmation tests for acid phosphatase production and stormy fermentation to further determine the performance of each agar. The CPCS agar was determined to have the highest enumerative capacity of total C. perfringens cells when compared to both TSC agar and m-CP agar (p-value < 0.05), but there was no significant difference in its ability to detect spores when compared to TSC agar (p-value > 0.05). The overall specificity of CPCS agar as determined by agreement of results from both confirmation tests was 0.81, while the specificity of TSC agar was only 0.28. Based on its performance, ease of preparation and use and consistency of colony characteristics, CPCS agar is recommended as the preferred medium for C. perfringens enumeration in wastewater.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Incidence and contamination level of Clostridium perfringens in meat and meat products sold in Sakarya province of Turkey

Totally 101 meat and meat product samples obtained from local markets and restaurants were analyzed for incidence and contamination level of Clostridium perfringens. The typical colonies grown anaerobically on Tryptose Sulfite Cycloserine Agar supplemented with 4-Methyliumbelliferyl (MUP) were confirmed by biochemical tests. Forty-eight of the samples (47.5%) were contaminated with C. perfringens. The highest incidence of the pathogen was determined in uncooked meatball samples (72.2%) followed by ground beef samples (61.3%). The incidence of C. perfringens in chicken meat, cooked meat döner, cooked chicken döner and emulsified meat product samples were 33.3, 33.3, 28.6 and 16.7%, respectively. Thirteen out of 101 samples (12.9%) yielded typical colonies on TSC-MUP Agar, but could not be confirmed as C. perfringens. Average contamination levels in sample groups ranged from 8.3 to 1.5×102 cfu/g, with the highest ground beef and the lowest chicken meat.

Enrichment and Detection of Clostridium perfringens Toxinotypes in Retail Food Samples.

Clostridium perfringens (C. perfringens) is a prolific toxin producer and causes a wide range of diseases in various hosts. C. perfringens is categorized into five different toxinotypes, A through E, based on the carriage of four major toxin genes. The prevalence and distribution of these various toxinotypes is understudied, especially their pervasiveness in American retail food. Of particular interest to us are the type B and D strains, which produce epsilon toxin, an extremely lethal toxin suggested to be the environmental trigger of multiple sclerosis in humans. To evaluate the presence of different C. perfringens toxinotypes in various food samples, we developed an easy method to selectively culture these bacteria without the use of an anaerobic container system only involving three culturing steps. Food is purchased from local grocery stores and transported to the laboratory under ambient conditions. Samples are minced and inoculated into modified rapid perfringens media (RPM) and incubated overnight at 37 °C in a sealed, airtight conical tube. Overnight cultures are inoculated onto a bottom layer of solid Tryptose Sulfite Cycloserine (TSC) agar, and then overlaid with a top layer of molten TSC agar, creating a "sandwiched", anaerobic environment. Agar plates are incubated overnight at 37 °C and then evaluated for appearance of black, sulfite-reducing colonies. C. perfringens-suspected colonies are removed from the TSC agar using sterile eye droppers, and inoculated into RPM and sub-cultured overnight at 37 °C in an airtight conical tube. DNA is extracted from the RPM subculture, and then analyzed for the presence of C. perfringens toxin genes via polymerase chain reaction (PCR). Depending on the type of food sampled, typically 15-20% of samples test positive for C. perfringens.

Effect of grape seed extract on heat resistance of Clostridium perfringens vegetative cells in sous vide processed ground beef

The heat resistance (57.5–65 °C) of a three-strain cocktail of Clostridium perfringens vegetative cells in sous vide processed ground beef supplemented with 0–3% grape seed extract (GSE) was quantified. The surviving cell population was enumerated on tryptose–sulfite–cycloserine agar. The decimal reduction (D)-values in beef that included no GSE were 67.11, 17.15, 4.02, and 1.62 min at 57.5, 60, 62.5, and 65 °C, respectively. Addition of 1.0% GSE resulted in concomitant decrease in heat resistance as evidenced by reduced bacterial D-values. The D-values in beef with added 1.0% GSE were 62.89, 13.70, 3.47 and 1.46 min at 57.5, 60, 62.5, and 65 °C, respectively. The heat resistance was further decreased when the GSE concentration in beef was increased to 2 or 3%. The z-values in beef with or without GSE were similar, ranging from 4.41 to 4.56 °C. The results of this study would be beneficial to the retail and institutional food service establishments in estimating re-heating time and temperature for sous vide processed ground beef to ensure safety against C. perfringens.

CLOSTRIDIUM PERFRINGENS IN DUCK FARMS, RETAIL OUTLETS AND IN CONTACT WORKERS IN EGYPT INCIDENCE AND TOXIN GENOTYPING

This study investigated incidence and toxin genotyping of Clostridium perfringens isolated from ducks and farm workers. Caecal contents of 125 ducks including apparently healthy (n=100) and diseased (n=25) ducks; and stool specimens of farm workers (n=25) were randomly collected from duck farms at Sharkia governorate, Egypt. Also, caecal contents (n=25) and meat (n=25) of ducks were collected from retail outlets at Sharkia governorate, Egypt. Samples were cultured on tryptose sulfite cycloserine agar supplied with Perfringens selective supplement then isolates were biochemically identified. Toxin genotyping of 17 C. perfringensisolates was performed by multiplex PCR. In duck farms, C. perfringens was isolated from 80% of healthy ducks, 84% of diseased one and 80% of in contact workers. In retail outlets, caecal contents and meat of ducks showed infection rates of 72% and 4%, respectively. The frequency distribution of cpa gene infarms was 80% in healthy ducks, 100% in diseased birds and 66.6% in apparently healthy workers. In retail outlets, cpa had frequency distribution of 66.6% in caecal contents and 100% in duck meat. Therefore, presence of C. perfringens type A and its associated cpa gene in caecal contents and meat of ducks confirmed its zoonotic hazard and public health implications.

Clostridium perfringens Contamination in Retail Meat and Meat-Based Products in Bursa, Turkey.

This study examined the incidence of Clostridium perfringens in raw, ready-to-cook (RTC), and ready-to-eat (RTE) meat and meat-based products (N = 306) collected from restaurants, supermarkets, and butcher shops in Bursa, Turkey. In addition, we investigated the presence of the C. perfringens enterotoxin (CPE), as well as cpe genes and their source (chromosomal or plasmid borne). In this study, tryptose sulfite cycloserine (TSC) agar for classic culture isolation and API and real-time polymerase chain reaction (RT-PCR) techniques were used to identify C. perfringens and detect cpa and cpe genes from these products, respectively. Seventeen C. perfringens isolates (5.6%) were isolated and identified with API 20A. In addition, 42 of 81 suspicious isolates (51.9%) were identified as C. perfringens using RT-PCR. Of the 81 suspicious isolates tested by RT-PCR, 22 (27.2%) carried the cpe gene either on the plasmid or chromosome. Twenty-one isolates were positive for chromosomal cpe (C-cpe), and one was positive for plasmid-borne cpe (P-cpe). CPE was detected in 31.8% (7/22) of the cpe positive isolates by the PET-RPLA test. In conclusion, C. perfringens and their CPEs were present in raw, RTC, and RTE meat and meat-based foods in this study. It is emphasized that the presence of C. perfringens and the cpe gene in these foods may be a potential risk for human health.

Antitumor effects of matrine on cancer stem like cells isolated from the human liver cancer SMMC-7721 cell line.

The existence of cancer stem cells (CSCs) or cancer stem-like cells (CSLCs) is regarded as the cause of tumor formation and recurrence. Matrine has been reported to exhibit antitumor effects in cancer cells. In the present study, a preliminary study was performed on the mechanisms of matrine on hepatocellular carcinoma (HCC) stem-like cells. The HCC SMMC-7721 cell line was cultured in tumor stem cell-specific medium to form spheres, and different concentrations (1, 2 and 5 mg/kg) of cisplatin were then used in order to purify the most drug-resistant cells, which were used as CSLCs. An MTT assay was performed to detect the inhibitory effects of matrine against CSLC proliferation. Quantitative polymerase chain reaction (qPCR) and western blot analysis were used to detect changes in cell adhesion regulating gene (CAR), E-cadherin, laminin and fibronectin. As a result, using tryptose sulfite cycloserine medium culture and cisplatin-resistance screening, CSLCs were successfully isolated from the SMMC-7721 cell line. Matrine inhibited the proliferation of CSLCs in vitro. The results of qPCR and western blot analysis demonstrated that matrine upregulated the expression of CAR, E-cadherin, laminin and fibronectin in CSLCs compared with the control treatment. A certain concentration of matrine exhibited antitumor effects on HCC stem like cells.

Comparison of methods for the enumeration of Clostridium perfringens spores in water

Four methods for enumerating Clostridium perfringens spores in water were evaluated: (1) the IMM (Iron Milk Medium) method (MPN); (2) the LS (Lactose Sulfite Broth) method (MPN); (3) the m-CP (membrane filtration Clostridium perfringens Agar) method (membrane filtration); and (4) the TSC (Tryptose Sulfite Cycloserine Agar) method (membrane filtration). The performance of these methods was compared with that of the DRCM (Differential Reinforced Clostridium Medium) method (MPN) as adopted by CETESB (Brazil's Environmental Sanitation Technology Company) for the analysis of C. perfringens spores in water. Statistical analysis was performed according to ISO 17994:2004 (Water Quality - Criteria for Establishing Equivalence between Microbiological Methods). The LS, m-CP, and TSC methods were considered not equivalent to the DRCM method, as they gave significantly lower results. The IMM showed inconclusive results and, according to ISO 17994:2004, analysis of a greater number of samples is needed to draw definitive conclusions comparing IMM and DRCM.

Novel Use of Tryptose Sulfite Cycloserine Egg Yolk Agar for Isolation of Clostridium perfringens during an Outbreak of Necrotizing Enterocolitis in a Neonatal Unit

Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.

MODIFICATION OF FUNG DOUBLE TUBE AND CP ANASELECT OXYPLATE METHODS TO IMPROVE THEIR PERFORMANCE IN ENUMERATING CLOSTRIDIUM PERFRINGENS FROM SEWAGE AND ENVIRONMENTAL WATERS

ABSTRACT The Fung Double Tube (FDT) and the newly developed CP AnaSelect Oxyplates methods can enumerate concentrations of Clostridium perfringens without need for external anaerobic generating systems. Because Clostridium perfringens is a reliable indicator of fecal contamination and it is one of the fastest growing fecal bacteria, these two methods were evaluated as feasible methods to screen recreational waters for sewage contamination. To increase the sensitivity and selectivity of these methods, three modifications were evaluated. The first modification was to pretreat the water samples using a microwave oven to attain high temperature (70C) for a short time (2.5 min) to reduce the interfering growth of non‐C. perfringens colonies on the selective media. The second modification was the addition of phosphatase reaction, thus enumerated colonies could be confirmed as C. perfringens. The third modification was to increase the sample volume for the FDT test from 5 to 10 mL/tube. The data collected showed that these modifications improved the selectivity and sensitivity of these two methods to enumerate C. perfringens from sewage‐contaminated water samples as well as environmental water samples such as streams, harbors, canals and coastal swimming beaches. The recovery efficiencies of the FDT and experimental CP AnaSelect Oxyplate for C. perfringens were similar to traditionally used membrane C. perfringens, tryptose sulfite cycloserine and Shahidi Ferguson perfringens agar media by membrane filtration technology, followed by incubation in anaerobic chambers. These results show that the modified FDT and CP AnaSelect Oxyplate methods are feasible and reliable methods to monitor environmental waters for C. perfringens. The FDT method is especially promising because it is simple, inexpensive and can produce results in 5–6 h.PRACTICAL APPLICATIONS Clostridium perfringens is currently being evaluated as a reliable indicator of sewage contamination in recreational waters. However, traditional methods to assay for C. perfringens are expensive and cumbersome because of the need for external anaerobic generating systems. In addition, target colonies must be confirmed by a second test. In this study, two methods are described, with capabilities to self‐generate anaerobic conditions and to immediately confirm the target colonies as C. perfringens. Moreover, one of the methods meets the difficult criterion of obtaining results in 6 h so decision on closing the beach can be reached on the same day the beach water sample is tested. Since these two methods are feasible and reliable, it will encourage many laboratories to assay their recreational waters for C. perfringens and a national database to determine the quality of recreational waters based on concentrations of C. perfringens can be developed.

Evaluation of an alternative method for the enumeration and confirmation of Clostridium perfringens from treated and untreated sewages

Clostridium perfringens is recommended as a suitable indicator bacterium for human enteric viruses, Giardia cysts and Cryptosporidium oocysts in finished water and in the assessment and evaluation of water treatment. Several agars and confirmation procedures were evaluated in parallel with the Australian/New Zealand Standard (AS/NZ) Method for the enumeration of Cl. perfringens from treated and untreated sewage samples. The current AS/NZ method utilizes tryptose sulfite cycloserine agar (TSC), lactose gelatin medium (LG) and nitrate motility medium (NM) at an incubation temperature of 37 degrees C. Sixty treated and untreated sewage samples were used to evaluate TSC agar, membrane Cl. perfringens agar (mCP), Perfringens agar (OPSP) and Perfringens agar with 4-methylumbelliferyl phosphate (OPSP-MUP) for enumeration of Clostridium. An incubation temperature of 44 degrees C for 24 h was used for comparison. Confirmation procedures were also evaluated using 103 isolates and included LG and NM, ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) with MUP (ONPG-MUP) and phosphatase reagent (PR). OPSP compared favourably with TSC agar. One false negative result was obtained from each of the LG/NM and ONPG-MUP procedures. No false results were obtained using the PR confirmation procedure. OPSP agar and PR were determined as suitable replacements for the AS/NZ Standard procedure with no interference from spreading organisms. This is a simple and rapid method for isolating and enumerating Cl. perfringens from sewage samples and confirmed results can be reported more quickly due to shorter analytical turnaround times.

Evaluation of acid phosphatase as a confirmation test for Clostridium perfringens isolated from water

To evaluate testing for acid phosphatase as an alternative method for the confirmation of Clostridium perfringens isolated from water. Sixty-two reference strains of Clostridium were tested for their ability to produce acid phosphatase, as well as reduction of sulfite on tryptose sulfite cycloserine agar (TSC) and production of fluorescence in TSC supplemented with 4-methylumbelliferylphosphate (MUP). Additionally 155 environmental presumptive C. perfringens isolates from TSC incubated at 44 degrees C were identified and tested for acid phosphatase production and by the conventional MNLG (testing for motility, nitrate reduction, lactose fermentation and gelatin liquefaction) confirmation procedure. Twenty-seven strains from 15 species of Clostridium-reduced sulfite to some extent on TSC incubated at 44 degrees C, with a significant number of species being able to grow well at this temperature, indicating that a confirmation step is needed for the enumeration of C. perfringens on this medium. All 10 strains of C. perfringens tested, together with one strain each of Clostridium baratii and Clostridium rectum produced acid phosphatase. These also produced fluorescence on MUP supplemented TSC, as did 13 strains of acid phosphatase negative, sulfite-reducing clostridia, representing nine species. Of the environmental isolates, 114 were identified as C. perfringens of which 108 (94.7%) were confirmed by the acid phosphatase test compared with 104 (91.2%) by the MNLG tests. Testing for acid phosphatase production is at least as reliable, and much simpler to perform, than the current standard confirmation MNLG procedure. Incorporation of MUP into TSC does not reliably improve the identification of presumptive C. perfringens. Application of testing for acid phosphatase as a confirmation test for C. perfringens would substantially simplify the analysis for this bacterium from water samples, and reduce the analysis time to confirmed counts.

The effect of grapefruit extract and temperature abuse on growth of Clostridium perfringens from spore inocula in marinated, sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag, marinated chicken breast that included additional 1.0% NaCl. The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 19 °C, C. perfringens levels were consistently about 2.5 log 10 CFU/g until 9.5 h regardless of the presence or absence of Citricidal®. However, storage of the unsupplemented marinated chicken samples and those with 50 or 100 ppm Citricidal® samples at 25 °C for more than 6 h resulted in rapid growth of C. perfringens, exhibiting 2–3 log 10 CFU/g increase at 7 h. Citricidal® at 200 ppm significantly ( p < 0.05) reduced the growth of C. perfringens at both 19 and 25 °C. The D-values obtained at 90 °C were significantly decreased ( p < 0.05) from 14.07 (no Citricidal®) to 9.20 min (200 ppm Citricidal®). Supplementing marinated chicken products with Citricidal® and the temperature abuses had no consistent effect on color, shear force or lipid oxidation. However, the organism may grow to unsafe levels if sous-vide products are poorly handled or temperature abused for a relatively long period. An extra degree of safety may be assured in such products by supplementation with 100 or 200 ppm Citricidal®. Industrial relevance: Since temperature abuse is a common occurrence during trasportation, distribution, storage or handling in grocery stores or by consumers, an extra degree of safety may be assured in marinated chicken products by supplementation 100–200 ppm Citricidal®. In such products, the temperature abuse should not have any consistent effect on color, shear force or lipid oxidation of products.

Sporulation and Heat Resistance of Spores from a Clostridium sp. RKD

ABSTRACT:A Clostridium sp. RKD isolated from the intestine of decaying fish, showing 99% sequence identity with Clostridium tetani at a 16S rRNA level, produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E). It also showed an amplification of near‐expected size when polymerase chain reaction (PCR) was performed using group‐ and type‐specific primers for botulinum neurotoxin type B. The isolate exhibited differences with both C. tetani and Clostridium botulinum with respect to phenotypic characteristics and chemotaxo‐nomic markers. Spore production was optimized with respect to media composition and stage of growth. Time‐dependant examination of sporulation revealed 2.6% to 49.0% spores in the late stationary phase culture when grown in different broth media. A simpler method for spore production and isolation from culture grown in tryptose sulfite cycloserine (TSC)/anaerobic agar sandwich resulted in &gt;95% sporulation, which could be purified to near homogeneity by a simple 2‐step procedure. Thermal resistance of spores revealed a biphasic inactivation at lower temperatures with D values for linear inactivation varying from 26.6, 8.0, and 4.3 min at 70 °C, 80 °C, and 90 °C, respectively. The z values of 7.86 °C and 10.47 °C were obtained in the linear and tail regions, respectively. The Weibull parameter b values at 70 °C, 80 °C, and 90 °C were 27.38, 3.55, and 0.99, respectively, with a z’ value of 13.869 °C. The shape parameter n at 70 °C, 80 °C, and 90 °C were 0.63, 0.55, 0.45, respectively. Spores produced on 2 different media (cooked meat medium [CMM] and trypticase peptone yeast‐extract glucose [TPYG] agar) exhibited differences in heat resistance. The addition of lysozyme (50 jj.g/mL) before heat treatment resulted in increased thermal resistance of spores.

Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products

Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag marinated chicken breast that included 0%, 1.5%, 3%, or 4.8% sodium lactate (NaL; w/w). The packages were processed to an internal temperature of 71.1 °C, ice chilled and stored at 4, 19, and 25 °C. The total C. perfringens population was determined by plating diluted samples on Tryptose–sulfite–cycloserine agar followed by anaerobic incubation for 48 h at 37 °C. At 25 °C, addition of 1.5% NaL was effective in delaying growth for 29 h. Increasing the NaL level to 4.8%, C. perfringens growth from a spore inoculum during storage at 25 °C for 480 h was not observed. At 19 °C, the growth was >6 log 10 cfu/g by 288 h in control samples. In samples with 3.0% or 4.8% NaL, the growth of C. perfringens from spores was dramatically restricted with little or no growth in 648 h at 19 °C. C. perfringens growth was not observed at 4 °C regardless of NaL concentration. The D-values at 55 °C ranged from 47.40 (no NaL) to 57.58 min (1.5% NaL). Cyclic and static temperature abuse of refrigerated products for ⩽20 h did not permit C. perfringens growth. However, temperature abuse of products for periods 24 h or longer in the absence of NaL led to growth of C. perfringens from a spore inoculum. An extra degree of safety may be assured in such products by supplementation with NaL at 1.5–4.8% NaL level.

Comparison of media for enumeration of Clostridium perfringens from foods

Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar] were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial. Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial. Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (±30%) of IS, which was set as a reference medium. Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological–analytical relevance. Food matrices did not affect the recovery of C. perfringens in general. DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common. Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C. perfringens from foods.

Feasibility of using food-grade additives to control the growth of Clostridium perfringens

Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria. This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C. perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM). Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C. perfringens on tryptose sulfite cycloserine (TSC) agar was >150 ppm at 37°C. However, in FTM, a SLEB (1:1:1, v/v/v) concentration of >100 ppm inhibited C. perfringens during an incubation (anaerobic) period of 196 h at 37°C. The sensitivity of C. perfringens to different combination ratios was also investigated in FTM. The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C. perfringens growth increased initially during the first 24 h of incubation (37°C) but remained constant during the next 48 h. Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C. perfringens viability decreased in all of the treated samples during 72-h incubation at 37°C. The results indicated that SLEB was an effective inhibitor of C. perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs.

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed

A multiplex polymerase chain reaction (PCR) assay, developed to detect the alpha-toxin and enterotoxin genes ( cpa and cpe, respectively) of Clostridium perfringens, was used to identify enterotoxigenic isolates of this organism from feces and intestinal contents of pigs and from feed samples from pig farms in Iowa. The organism was grown on tryptose-sulfite-cycloserine (TSC) agar, TSC agar without egg-yolk, sheep blood agar, or in brain heart infusion broth or cooked meat medium. DNA was extracted by boiling and the PCR assay was carried out using reagents from a commercial kit. The 319 bp amplification product of cpa and the 364 bp product of cpe were visualized under UV light after electrophoresis in a 2% agarose gel containing ethidium bromide. The average sensitivity of the assay, determined on artificially contaminated feces, was 9.2×10 4 colony forming units per gram. Assay of 97 isolates from feces and intestinal contents revealed cpa in 89, but all were negative for cpe. While 28% of the 442 total samples cultured yielded C. perfringens, only 5% of 298 fecal or intestinal contents samples were positive upon direct examination by the PCR assay. Ninety-one and eight-tenths % of isolates with the phenotype of C. perfringens were cpa positive by PCR. Forty-three percent of feed samples were culture positive, while 48.3% were PCR positive for cpa. None of these were cpe positive. We conclude that PCR is a useful assay for rapid detection of C. perfringens in feed, and for confirmation of the identity of isolates presumed to be C. perfringens.