Nonproteolytic Type Research Articles

Rapid detection and quantitation of dipicolinic acid from Clostridium botulinum spores using mixed-mode liquid chromatography-tandem mass spectrometry.

Analysis of the dipicolinic acid (DPA) released from Clostridium botulinum spores during thermal processing is crucial to obtaining a mechanistic understanding of the factors involved in spore heat resistance and related food safety applications. Here, we developed a novel mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for detection of the DPA released from C. botulinum type A, nonproteolytic types B and F strains, and nonpathogenic surrogate Clostridium sporogenes PA3679 spores. DPA was retained on a mixed-mode C18/anion exchange column and was detected using electrospray ionization (ESI) positive mode within a 4-min analysis time. The intraday and interday precision (%CV) was 1.94-3.46% and 4.04-8.28%, respectively. Matrix effects were minimal across proteolytic type A Giorgio-A, nonproteolytic types QC-B and 202-F, and C. sporogenes PA3679 spore suspensions (90.1-114% of spiked DPA concentrations). DPA recovery in carrot juice and beef broth ranged from 105 to 118%, indicating limited matrix effects of these food products. Experiments that assessed the DPA released from Giorgio-A spores over the course of a 5-min thermal treatment at 108°C found a significant correlation (R = 0.907; P < 0.05) between the log reduction of spores and amount of DPA released. This mixed-mode LC-MS/MS method provides a means for rapid detection of DPA released from C. botulinum spores during thermal processing and has the potential to be used for experiments in the field of food safety that assess the thermal resistance characteristics of various C. botulinum spore types.

Inactivation of non-proteolytic Clostridium botulinum type E in low-acid foods and phosphate buffer by heat and pressure.

The effect of high pressure thermal (HPT) treatments on the inactivation of spores of non-proteolytic type E Clostridium botulinum TMW 2.990 was investigated at high pressures (300 to 600 MPa) and elevated temperatures (80 to 100 °C) in four low-acid foods (steamed sole, green peas with ham, vegetable soup, braised veal) and imidazole phosphate buffer (IPB). In addition, corresponding conventional thermal treatments at ambient pressure were performed to expose possible synergisms of pressure and temperature on spore inactivation. In general, spore count reduction was more efficient by combining pressure and temperatures < 100 °C and the overall process duration could be shortened due to accelerated heating rates (adiabatic effect). Processing at 90 °C and 600 MPa resulted in inactivation below the detection limit after 5 min in all foods except steamed sole. Traditional thermal processing of spores at 90 °C for 10 min, on the other hand, did not result in an estimated 6-log reduction. Additional HPT treatments in steamed sole and IPB did not reveal pronounced food matrix dependent protective effects. Here, varying pressure levels did not appear to be the driving force for spore count reduction in steamed sole at any temperature. By applying a Weibull distribution on destruction kinetics of isobaric/isothermal holding times, 6D-values were calculated. Compression and decompression phase (1 s pressure holding time) had a considerable impact on spore count reduction (max. -2.9 log units) in both, foods and buffer. Hence, compression and decompression phases should directly be included into the total lethal effect of HPT treatments to avoid prolonged holding times and overprocessing.

Combined High Pressure and Thermal Processing on Inactivation of Type E and Nonproteolytic Type B and F Spores of Clostridium botulinum

The aim of this study was to determine the resistance of multiple strains of the three nonproteolytic types of Clostridium botulinum (seven strains of type E, eight of type B, and two of type F) spores exposed to combined high pressure and thermal processing. The resistance of spores suspended in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7) was determined at a process temperature of 80°C with high pressures of 600, 650, and 700 MPa using a laboratory-scale pressure test system. Spores of C. botulinum serotype E strains demonstrated less resistance than nonproteolytic spores of type B or F strains when processed at 80°C and 600 MPa for up to 15 min. All C. botulinum type E strains were reduced by . 6.0 log units within 5 min under these conditions. Among the nonproteolytic type B strains, KAP 9-B was the most resistant, resulting in reductions of 2.7, 5.3, and 5.5 log, coinciding with D-values of 7.7, 3.4, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Of the two nonproteolytic type F strains, 610F was the most resistant, showing 2.6-, 4.5-, and 5.3-log reductions with D-values of 8.9, 4.3, and 1.8 min at 80°C and 600, 650, and 700 MPa, respectively. Pulsed-field gel electrophoresis was performed to examine the genetic relatedness of strains tested and to determine if strains with similar banding patterns also exhibited similar D-values. No correlation between the genetic fingerprint of a particular strain and its resistance to high pressure processing was observed.

Draft Genome Sequences of Two Clostridium botulinum Group II (Nonproteolytic) Type B Strains (DB-2 and KAPB-3).

Clostridium botulinum is important for food safety and studies of neurotoxins associated with human botulism. We present the draft genome sequences of two strains belonging to group II type B: one collected from Pacific Ocean sediments (DB-2) and another obtained during a botulism outbreak (KAPB-3).

Arrangement of the Clostridium baratii F7 toxin gene cluster with identification of a σ factor that recognizes the botulinum toxin gene cluster promoters.

Botulinum neurotoxin (BoNT) is the most poisonous substances known and its eight toxin types (A to H) are distinguished by the inability of polyclonal antibodies that neutralize one toxin type to neutralize any of the other seven toxin types. Infant botulism, an intestinal toxemia orphan disease, is the most common form of human botulism in the United States. It results from swallowed spores of Clostridium botulinum (or rarely, neurotoxigenic Clostridium butyricum or Clostridium baratii) that germinate and temporarily colonize the lumen of the large intestine, where, as vegetative cells, they produce botulinum toxin. Botulinum neurotoxin is encoded by the bont gene that is part of a toxin gene cluster that includes several accessory genes. We sequenced for the first time the complete botulinum neurotoxin gene cluster of nonproteolytic C. baratii type F7. Like the type E and the nonproteolytic type F6 botulinum toxin gene clusters, the C. baratii type F7 had an orfX toxin gene cluster that lacked the regulatory botR gene which is found in proteolytic C. botulinum strains and codes for an alternative σ factor. In the absence of botR, we identified a putative alternative regulatory gene located upstream of the C. baratii type F7 toxin gene cluster. This putative regulatory gene codes for a predicted σ factor that contains DNA-binding-domain homologues to the DNA-binding domains both of BotR and of other members of the TcdR-related group 5 of the σ70 family that are involved in the regulation of toxin gene expression in clostridia. We showed that this TcdR-related protein in association with RNA polymerase core enzyme specifically binds to the C. baratii type F7 botulinum toxin gene cluster promoters. This TcdR-related protein may therefore be involved in regulating the expression of the genes of the botulinum toxin gene cluster in neurotoxigenic C. baratii.

Effect of media, additives, and incubation conditions on the recovery of high pressure and heat-injured Clostridium botulinum spores

The effect of additives and post-treatment incubation conditions on the recovery of high pressure and heat-injured (i.e., processed at 620 MPa and 95 and 100 °C for 5 min) spores of Clostridium botulinum strains, 62-A (proteolytic type A) and 17-B (nonproteolytic type B) was studied. High pressure and heat-injured spores were inoculated into TPGY (Trypticase–Peptone–Glucose–Yeast extract) anaerobic broth media containing additives (lysozyme, l-alanine, l-aspartic acid, dipicolonic acid, sodium bicarbonate, and sodium lactate) at various concentrations (0–10 μg/ml) individually or in combination. The spore counts of high pressure and heat-injured 62-A and 17-B recovered from TPGY broth containing lysozyme (10 μg/ml) incubated for 4 months versus that recovered from peptone–yeast extract–glucose–starch (PYGS) plating agar containing lysozyme (10 μg/ml) incubated under anaerobic conditions for 5 days were also compared. None of the additives either individually or in combination in TPGY broth improved recovery of injured spore enumeration compared to processed controls without additives. Addition of lysozyme at concentrations of 5 and 10 μg/ml in TPGY broth improved initial recovery of injured spores of 17-B during the first 4 days of incubation but did not result in additional recovery at the end of the 4 month incubation compared to the processed control without lysozyme. Adding lysozyme at a concentration of 10 μg/ml to PYGS plating agar resulted in no effect on the recovery of high pressure and heat-injured 62-A and 17-B spores. The recovery counts of high pressure and heat-injured spores of 62-A and 17-B were lower (i.e., <1.0 log units) with PYGS plating agar compared to the MPN method using TPGY broth as the growth medium.

Hazard analysis and possibilities for preventing botulism originating from meat products

The paper presents the more important data on the bacteria Clostridium botulinum, the appearance of botulism, hazard analysis and the possibilities for preventing botulism. Proteolytic strains of C.botulinum Group I, whose spores are resistant to heat, create toxins predominantly in cans containing slightly sour food items, in the event that the spores are not inactivated in the course of sterilization. Non-proteolytic strains of Group II are more sensitive to high temperatures, but they have the ability to grow and create toxins at low temperatures. Type E most often creates a toxin in vacuum-packed smoked fish, and the non-proteolytic strain type B in dried hams and certain pasteurized meat products. The following plays an important role in the prevention of botulism: reducing to a minimum meat contamination with spores of clostridia, implementing good hygiene measures and production practice during the slaughter of animals, the inactivation of spores of C. botulinum during sterilization (F&gt;3), and, in dried hams and pasteurized products, the prevention of bacterial growth and toxin forming by maintaining low temperatures in the course of production and storage, as well as the correct use of substances that inhibit the multiplication of bacteria and the production of toxins (nitrites, table salt, etc.).

Inactivation of Clostridium botulinum nonproteolytic type B spores by high pressure processing at moderate to elevated high temperatures

The effect of high pressure and high temperature treatments at various process times on the inactivation of spores of Clostridium botulinum nonproteolytic type B strains, 2-B, 17-B, KAP8-B, and KAP9-B, suspended in phosphate buffer (0.067 M, pH 7.0) and a crabmeat blend was investigated. Spores of KAP8-B were less resistant to high pressure treatment than the spores of 2-B, 17-B, and KAP9-B in both phosphate buffer and crabmeat blend. No survivors of initial counts (> 4.3 log units) of KAP8-B spores were detected in these menstura after processing at 827 MPa and 60 °C for 10 min. The amount of inactivation of spores of 2-B, 17-B, and KAP9-B in phosphate buffer or crabmeat blend increased with the increase in processing time from 10 to 30 min at 827 MPa and 75 °C. Similar inactivation patterns were observed for these spores in both phosphate buffer and crabmeat blend. A reduction of > 6-log units of 2-B, 17-B, and KAP9-B spores in phosphate buffer and crabmeat blend was observed at 827 MPa and 75 °C for a processing time of between 20 and 30 min. Crabmeat blend as a suspension menstrum provided no protection against inactivation of spores of 2-B, 17-B, and KAP9-B by high pressure processing. High temperature (> 95 °C) and lower pressure (620 MPa) treatments for up to 10 min were also found to inactivate 17-B spores in phosphate buffer. Spores of nonproteolytic type B strains, 2-B, 17-B, KAP8-B, and KAP9-B in phosphate buffer and crabmeat blend can be inactivated by a combination of high pressure and temperature treatments. Spores of nonproteolytic type B strains of Clostridium botulinum are of primary concern because they have been involved in the foodborne botulism outbreaks associated with marine products. Foodborne botulism results from consumption of these foods in which C. botulinum has grown and produced neurotoxin. Recently, high pressure processing (HPP) received a great deal of interest because of its ability to destroy vegetative pathogens, viruses, and certain bacterial spores and results in a product with natural sensory, quality, and nutritional attributes. Currently, HPP is being evaluated at the National Center for Food Safety and Technology as an alternative to other traditional thermal processes for its ability to inactivate C. botulinum spores. In this study, the effects of high pressure in conjunction with moderate to elevated high temperatures on inactivation of C. botulinum nonproteolytic type B spores were investigated. Based on limited number of strains tested, HPP showed a potential of destroying spores of nonproteolytic type B strains of C. botulinum when process temperature is above 75 °C.

Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event.

Competitive Inhibition between Different Clostridium botulinum Types and Strains

Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C. botulinum were tested against each other for the purpose of selecting a mixture of compatible C. botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents. All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C. sporogenes. When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited. Nonproteolytic strains of C. botulinum also produced bacteriocin-like agents against each other. Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C. botulinum and C. sporogenes. EF4, however, was not inhibitory to the nontoxigenic E-like strains. When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production. Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30°C. On the basis of the production of these bacteriocin-like agents by different strains of C. botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.

PEER REVIEWED PAPERS

Alaska pollock, fillets, washed, strained and dewatered minced fish (mince), and surimi with cryoprotectants, were analyzed for the presence of Clostridium botulinum. Two of the 912 surimi sub-samples tested were contaminated with type E, and type A was shown to be present in two other sub-samples (0.4% incidence). C. botulinum was not present in 72 sub-samples of mince, 7 fish fillets, or 130 whole unevis-cerated pollock. Surimi is distributed in frozen blocks and used in the manufacturing of imitation crab legs, scallops, shrimp and lobster. The products are usually pasteurized after packaging. Since both the non-proteolytic and proteolytic types were found in the surimi, emphasis should be placed on the combination of time and temperature of pasteurization, water-phase salt of the finished product and on storage of the finished product under continuous refrigeration (below 3°C), or as a frozen product.

Prevalence of Clostridium botulinum types B, E and F in faecal samples from Swedish cattle

Faeces were collected from 60 cows at three slaughterhouses situated in southern and central Sweden. The faecal samples were collected during two sampling periods over the year, summer and winter. All samples were analysed for the presence of Clostridium botulinum spores, according to a combined selection and enrichment PCR procedure. One PCR assay was specific for part of the type B neurotoxin gene, while the other assay was specific for both type E and F neurotoxin genes. The prevalence of C. botulinum in Swedish cattle was established to be 73% for non-proteolytic type B and less than 5% for types E and F. Twenty-eight (64%) of the positive faecal samples had a spore load of less than 4 spores/g. Statistical analysis (ANOVA) showed that seasonal variation (summer and winter) had a significant effect on the prevalence of C. botulinum type B in cattle, whereas the effect of geographical location of rearing of the cattle (southern and central Sweden) was less significant.

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (104/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.

Screening for Clostridium botulinum Type A, B, and E in Cooked Chilled Foods Containing Vegetables and Raw Material Using Polymerase Chain Reaction and Molecular Probes

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

Comparison of Amplified ELISA and Mouse Bioassay Procedures for Determination of Botulinal Toxins A, B, E, and F

The amplified enzyme-linked immunosorbent assay (amp-ELISA) was compared to the mouse bioassay for determination of botulinal neurotoxin types A, B, E, and F. Twelve different toxin-producing type A, 13 proteolytic type B, 9 nonproteolytic type B, 16 type E, 8 proteolytic type F, 5 nonproteolytic type F, and 6 nontoxigenic clostridial strains were tested. The cultures were inoculated into cooked meat medium (CMM) and tryptone-peptone-glucose-yeast extract (TPGY) medium, incubated for 5 days, and then examined for biological toxicity in mice and amp-ELISA endpoints. The amp-ELISA was less sensitive in detecting toxins produced by nonproteolytic than proteolytic strains of type B and F organisms. All of the toxin-producing strains tested were positive by the AOAC method and the amp-ELISA in either undiluted TPGY or CMM culture fluids regardless of mouse toxicity level, source, or strain. Cross-reactivity was observed between some but not all of the botulinal strains tested. None of the nontoxigenic strains were positive by the amp-ELISA. Purified botulinal toxins were also assayed using these 2 methods. The sensitivity of the amp-ELISA using purified neurotoxins was about 0.1 ng/mL for types A, B, and E and about 1.0 ng/mL for type F.

Differentiation between Types and Strains of Clostridium botulinum by Riboprinting

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35°C. Plates were rinsed with buffer (2mM Tris + 20mM EDTA) to remove vegetative cells that were heated for 10 min at 80°C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinum most commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages. This degree of differentiation between strains of C. botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.

Heat-Pasteurization Process for Inactivation of Nonproteolytic Types of Clostridium botulinum in Picked Dungeness Crabmeat

Development of a heat-pasteurization process is described for picked meat of Dungeness crabs ( Cancer magister ) contained in oxygen-impermeable flexible pouches, For each time-temperature treatment, 30 samples, each inoculated with an equal mixture of three strains of C. botulinum nonproteolytic type B, for a total of 107 spores, provided the basis for calculation of the thermal resistance (a 7D process). Following heat processing, the crabmeat was removed from the pouches and transferred to enrichment medium where it was incubated anaerobically for 150 days. Endpoints at which spores survived were determined by the presence of toxin in the enrichment medium. Process times ranged from 90 min at 88.9°C to 20.3 min at 94.4°C. D values (the time at each temperature required to reduce the inoculum by 1 log) ranged from 12.9 for the 88.9°C process to 2.9 for the 94.4°C process. The relative sterilization value, F0 was .054 and the pasteurization value, , was 240. This pasteurization process safely extends refrigerated shelf life by inactivating spores of Clostridium botulinum nonproteolytic types B, E, and F and also non-spore-forming pathogens such as Listeria monocytogenes . The process does not, however, inactivate the heat-resistant proteolytic strains of C. botulinum or other more heat-resistant spore-formers. The packages and master cartons of the pasteurized product, therefore, should be labeled "Keep refrigerated-Continuous refrigeration below 38°F (3.3°C) required."

Time-to-turbidity model for non-proteolytic type B Clostridium botulinum

A model to predict the time for growth to turbidity from spores of non-proteolytic type B strains of Clostridium botulinum was developed in broth media with varying temperatures (4–28°C), pH values, (5–7), NaCl additions (0–4%) and total spores (10 1−10 5). The model estimates the probability that a sample will have growth on a given day for up to 90 days of storage. The parameters of the model include the probability of growth after 90 days ( P max) and the mean time of growth (τ) for those tubes that showed growth. The 95% confidence interval (CI 95%) for τ was also determined. The τ decreased with increasing temperature and pH, but NaCl levels below 3% had little effect. Decreasing the number of spores in a sample increased both τ and the confidence intervals about τ, reflecting the increasing uncertainty about the estimation of growth times for low spore numbers.

Potential for Growth from Spores of Bacillus cereus and Clostridium botulinum and Vegetative Cells of Staphylococcus aureus, Listeria monocytogenes, and Salmonella Serotypes in Cooked Ground Beef during Cooling

The ability of 16 foodborne pathogens, representative of 5 different species, to grow during cooling of previously sterilized cooked beef was studied to determine a safe cooling rate. Auto-claved ground beef samples (3 g) were inoculated with heat-shocked spores of Bacillus cereus (strain BH 86) or Clostridium botulinum (nonproteolytic type B strains CBW 25, 17B, and KAP B5 and type E strains Whitefish, Saratoga, and Alaska) or vegetative cells of Listeria monocytogenes (strains HO-VJ-S, V-7, and Scott A), Staphylococcus aureus (strains 196E, B121, and B 124), or Salmonella serotypes (S. dublin, S. enteritidis, and S. typhimurium), vacuum-packaged, and cooked in a stirred water bath to an internal temperature of 60°C in I h. In some experiments combinations of C. botulinum and B. cereus spores or S. aureus and salmonellae vegetative cells were used. Heated samples were cooled through the temperature range of 54.4 to 7.2°C at rates varying from 6 to 21 h. Samples were removed at various times during cooling to determine if growth of the pathogens had occurred. No growth was observed with cooling periods of up to 21h. This study with the model meat system (3 g autoclaved ground beef inoculated with selected pathogens and then pasteurized) indicated that cooling from 52.4 to 7.2°C in up to 21 h would not pose a food safety hazard from growth of these pathogens.

Type A and B Neurotoxin Genes in a Clostridium botulinum Type AB Strain

The genetic structure of neurotoxin genes in a Clostridium botulinum strain producing both type A and B neurotoxins (type AB) was investigated. Analyses by polymerase chain reaction (PCR) using type-specific primers corresponding to the coding regions for N-terminals of light-chains and C-terminals of heavy-chains of type A and type B neurotoxins, and Southern hybridization of total DNA showed that the type AB strain I.P.7212 carries at least one copy each of type A and B neurotoxin genes. Partial nucleotide sequences obtained by direct sequencing of the PCR products indicate that the type A and B genes carried by this strain are not classical type A and non-proteolytic type B genes, but are similar to the type A gene present in a strain which had caused infant botulism in Kyoto and the type B gene present in a proteolytic type B strain.