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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.

The microbial safety concern associated with thermally processed extended shelf life (ESL) refrigerated foods is based on adequate elimination of spore-forming pathogens such as nonproteolytic Clostridium botulinum types B, E, and F. These pathogens are traditionally regarded as targets for validation of thermally processed ESL foods. However, their use for research is restricted due to their designation as select agents. In this study, the thermal resistances of spores of 10 nonproteolytic C. botulinum types B and F and seven psychrotrophic Bacillus cereus strains were evaluated in ACES (N-(2-acetamido)-2-aminoethanesulfonic acid) buffer (0.05 M, pH 7.00) and compared to determine whether any of the B. cereus strains could serve as a nonselect agent for establishing thermal processes for ESL refrigerated foods. Thermal decimal reduction times (DT-values) of both nonproteolytic C. botulinum types B and F and psychrotrophic B. cereus strains decreased as process temperature increased from 80 to 91°C, and the highest values were obtained at 80°C. All psychrotrophic B. cereus strains tested were more thermally resistant than nonproteolytic C. botulinum types B and F. DT-values of nonproteolytic C. botulinum types B and F decreased to

The measurement of Clostridium botulinum type E toxin in fish was accomplished using an amplified immunoassay (enzyme-linked immunosorbent assay-enzyme-linked coagulation assay [ELISA-ELCA]) based on the coagulation cascade. Fresh catfish fillets inoculated with a mixture of spores from five strains of C. botulinum type E were packaged in high barrier film with air, vacuum and modified atmosphere and stored at 4, 8 or 16°C for up to 75 days. Toxin production was monitored during storage by both mouse bioassay (trypsin and non-trypsin treated) and ELISA-ELCA on the non-trypsinized samples. All 26 inoculated products that were positive by the mouse bioassay were also positive by ELISA-ELCA. Of 35 uninoculated samples which were not toxic in mouse bioassay, none were positive by ELISA-ELCA; of 73 inoculated samples which were not toxic by mouse bioassay, 14 had toxin measurable by the ELISA-ELCA. The position of these immunoassay-positives in the sampling sequence indicated that the toxin was identified by the immunoassay before it was found in the mouse bioassay. These results suggest that the ELISA-ELCA technique is a usable alternative to the mouse bioassay for monitoring C. botulinum type E toxin production in fish challenge studies.

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies., Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies. We report here the closed genome sequence of Clostridium botulinum strain CFSAN064329 (62A).

The impact of high pressure processing on the inactivation of spores of nonproteolytic Clostridium botulinum is important in extended shelf life chilled low-acid foods. The three most resistant C. botulinum strains (Ham-B, Kap 9-B, and 610-F) were selected for comparison of their thermal and pressure-assisted thermal resistance after screening 17 nonproteolytic C. botulinum strains (8 type B, 7 type E, and 2 type F). Spores of strains Ham-B, Kap 9-B, and 610-F were prepared using a biphasic media method, diluted in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.00) to 105 to 106 CFU/mL, placed into a modified sterile transfer pipette, heat sealed, and subjected to a combination of high pressures (600 to 750 MPa) and high temperatures (80 to 91°C) using laboratory and pilot-scale pressure test systems. Diluted spores from the same crops were placed in nuclear magnetic resonance tubes, which were heat sealed, and subjected to 80 to 91°C in a Fluke 7321 high precision bath with Duratheram S oil as the heat transfer fluid. After incubation for 3 months, survivors in both studies were determined by the five-tube most-probable-number method using Trypticase-peptone-glucose-yeast extract broth. The highest (>5.0) log reductions in spore counts for Ham-B, Kap 9-B, and 610-F occurred at the highest temperature and pressure combination tested (91°C and 750 MPa). Thermal D-values of Ham-B, Kap 9-B, and 610-F decreased as the process temperature increased from 80 to 87°C, decreasing to

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinum type A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase-peptone-glucose-yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41°C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P < 0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P < 0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinum spores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinum spores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

The purpose of this study was to determine the inactivation kinetics of the spores of the most resistant proteolytic Clostridium botulinum strains (Giorgio-A and 69-A, as determined from an earlier screening study) and of Clostridium sporogenes PA3679 and to compare the thermal and pressure-assisted thermal resistance of these spores. Spores of these strains were prepared using a biphasic medium method. C. sporogenes PA3679 spores were heat treated before spore preparation. Using laboratory-scale and pilot-scale pressure test systems, spores of Giorgio-A, 69-A, and PA3679 suspended in ACES [N-(2-acetamido)-2-aminoethanesulfonic acid] buffer (pH 7.0) were exposed to various combinations of temperature (93 to 121°C) and pressure (0.1 to 750 MPa) to determine their resistance. More than a 5-log reduction occurred after 3 min at 113°C for spores of Giorgio-A and 69-A and after 5 min at 117°C for spores of PA3679. A combination of high temperatures (93 to 121°C) and pressures yielded greater log reductions of spores of Giorgio-A, 69-A, and PA3679 compared with reduction obtained with high temperatures alone. No survivors from initial levels (5.0 log CFU) of Giorgio-A and 69-A were detected when processed at a combination of high temperature (117 and 121°C) and high pressure (600 and 750 MPa) for1 min in a pilot-scale pressure test system. Increasing pressure from 600 to 750 MPa at 117°C decreased the time from 2.7 to 1 min for a4.5-log reduction of PA3679 spores. Thermal D-values of Giorgio-A, 69-A, and PA3679 spores decreased (i.e., 29.1 to 0.33 min for Giorgio-A, 40.5 to 0.27 min for 69-A, and 335.2 to 2.16 min for PA3679) as the temperature increased from 97 to 117°C. Pressure-assisted thermal D-values of Giorgio-A, 69-A, and PA3679 also decreased as temperature increased from 97 to 121°C at both pressures (600 and 750 MPa) (i.e., 17.19 to 0.15 min for Giorgio-A, 9.58 to 0.15 min for 69-A, and 12.93 to 0.33 min for PA3679 at 600 MPa). At higher temperatures (117 or 121°C), increasing pressure from 600 to 750 MPa had an effect on pressure-assisted thermal D-values of PA3679 (i.e., at 117°C, pressure-assisted thermal D-value decreased from 0.55 to 0.28 min as pressure increased from 600 to 750 MPa), but pressure had no effect on pressure-assisted thermal D-values of Giorgio-A and 69-A. When compared with Giorgio-A and 69-A, PA3679 had higher thermal and pressure-assisted thermal D-values. C. sporogenes PA3679 spores were generally more resistant to combinations of high pressure and high temperature than were the spores of the C. botulinum strains tested in this study.

Clostridium sporogenes PA 3679 is a nonpathogenic, nontoxic model organism for proteolytic Clostridium botulinum used in the validation of conventional thermal food processes due to its ability to produce highly heat-resistant endospores. Because of its public safety importance, the uncertain taxonomic classification and genetic diversity of PA 3679 are concerns. Therefore, isolates of C. sporogenes PA 3679 were obtained from various sources and characterized using pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. The phylogenetic relatedness and genetic variability were assessed based on 16S rRNA gene sequencing and whole-genome single nucleotide polymorphism (SNP) analysis. All C. sporogenes PA 3679 isolates were categorized into two clades (clade I containing ATCC 7955 NCA3679 isolates 1961-2, 1990, and 2007 and clade II containing PA 3679 isolates NFL, UW, FDA, and Campbell and ATCC 7955 NCA3679 isolate 1961-4). The 16S maximum likelihood (ML) tree clustered both clades within proteolytic C. botulinum strains, with clade I forming a distinct cluster with other C. sporogenes non-PA 3679 strains. SNP analysis revealed that clade I isolates were more similar to the genomic reference PA 3679 (NCTC8594) genome (GenBank accession number AGAH00000000.1 ) than clade II isolates were. The genomic reference C. sporogenes PA 3679 (NCTC8594) genome and clade I C. sporogenes isolates were genetically distinct from those obtained from other sources (University of Wisconsin, National Food Laboratory, U.S. Food and Drug Administration, and Campbell's Soup Company). Thermal destruction studies revealed that clade I isolates were more sensitive to high temperature than clade II isolates were. Considering the widespread use of C. sporogenes PA 3679 and its genetic information in numerous studies, the accurate identification and genetic characterization of C. sporogenes PA 3679 are of critical importance.

The potential threat of terrorist attacks against the United States food supply using neurotoxin produced by Clostridium botulinum (BoNT) has resulted in the need for studying the effect of various food process operations on the bioavailability of this toxin. The objective of this study was to evaluate C. botulinum type A neurotoxin bioavailability after a simulated hot fill juice bottling operation. C. botulinum type A acid mud toxin (∼10(6) mouse lethal dose [MLD50]/ml) was deposited into juice bottles at an experimentally determined fastest cooling spot. Bottles (12 or 20 oz [355 and 592 ml]) were filled with either apple juice or an orange drink, at 80 or 85°C, in either upright or inverted orientations. Toxicity of the juice was evaluated as a function of holding time (1 to 2 min) by the mouse bioassay. The fastest cooling point in the upright orientation was determined to be at a bottle's bottom rim. In the inverted orientation, the fastest cooling point was in the bottle cap region. With respect to these two points, the upright bottle cooled faster than the inverted bottle, which was reflected in a higher inactivation of BoNT in the latter. For the orange drink (pH 2.9) toxicity was reduced by 0.5 × 10(6) MLD50/ml to a nondetectable level after 1 min in all bottle sizes, orientations, and temperatures as measured by the mouse bioassay. This indicates that there was at least a 0.5 × 10(6) MLD50/ml reduction in activity. Inactivation in apple juice (pH 4.0), to the same degree as in the orange drink, was found only for the inverted orientation at 85°C. Complete inactivation in apple juice for all conditions was found at a lower added toxin level of 0.25 × 10(5) MLD50/ml. In general, bottle inversion and filling at 85°C provided complete inactivation of BoNT to the 0.5 × 10(6) MLD50/ml level. All experiments resulted in the inactivation of 2.5 × 10(4) MLD50/ml of BoNT regardless of juice type, fill temperature, or bottle orientation and size.

The purpose of this study was to determine the effect of sporulation temperature on the resistance of Clostridium botulinum type A spores of strains 62A and GiorgioA to thermal and high pressure processing (HPP). Spore crops produced in Trypticase-peptone-glucose-yeast extract broth at four incubation temperatures (20, 27, 37, and 41°C) were harvested, and heat resistance studies were conducted at 105°C (strain 62A) and 100°C (strain GiorgioA). Resistance to HPP was evaluated by subjecting the spores to a high pressure (700 MPa) and temperature combination (105°C, strain 62A; 100°C strain GiorgioA) in a laboratory-scale pressure test system. The decimal reduction time (D-value) was calculated using the log-linear model. Although the time to sporulation for GiorgioA was shorter and resulted in higher spore concentrations than for 62A at 20, 27, and 37°C, GiorgioA did not produce a sufficient spore crop at 41°C to be evaluated. The heat resistance of 62A spores was greatest when produced at 27°C and decreased for spore crops produced above or below 27°C (D105°C-values: 20°C, 1.9 min; 27°C, 4.03 min; 37°C, 3.66 min; and 41°C, 3.5 min; P0.05). Unlike 62A, the heat resistance behavior of GiorgioA spores increased with rising sporulation temperature, and spores formed at the organism's optimum growth temperature of 37°C were the most resistant (D100°C-values: 20°C, 3.4 min; 27°C, 5.08 min; and 37°C, 5.65 min; P0.05). Overall, all spore crops were less resistant to pressure-assisted thermal processing than thermal treatment alone. Sporulation temperature has an effect on the resistance of C. botulinum spores to heat and HPP, and is characteristic to a particular strain. Knowledge of the effect of sporulation temperature on the resistance of C. botulinum spores is vital for the production of spores utilized in thermal and high pressure inactivation studies.

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.

The aim of this study was to determine the resistance of multiple strains of Clostridium botulinum type A and proteolytic type B spores exposed to combined high pressure and thermal processing and compare their resistance with Clostridium sporogenes PA3679 and Bacillus amyloliquefaciens TMW-2.479-Fad-82 spores. The resistance of spores suspended in N-(2acetamido)-2-aminoethanesulfonic acid (ACES) buffer (0.05 M, pH 7.0) was determined at a process temperature of 105°C, with high pressures of 600, 700, and 750 MPa by using a laboratory-scale pressure test system. No surviving spores of the proteolytic B strains were detected after processing at 105°C and 700 MPa for 6 min. A . 7-log reduction of B. amyloliquefaciens spores was observed when processed for 4 min at 105°C and 700 MPa. D-values at 105°C and 700 MPa for type A strains ranged from 0.57 to 2.28 min. C. sporogenes PA3679 had a D-value of 1.48 min at 105°C and 700 MPa. Spores of the six type A strains with high D-values along with C. sporogenes PA3679 and B. amyloliquefaciens were further evaluated for their pressure resistance at pressures 600 and 750 MPa at 105°C. As the process pressure increased from 600 to 750 MPa at 105°C, D-values of some C. botulinum strains and C. sporogenes PA3679 spores decreased (i.e., 69-A, 1.91 to 1.33 min and PA3679, 2.35 to 1.29 min). Some C. botulinum type A strains were more resistant than C. sporogenes PA3679 and B. amyloliquefaciens to combined high pressure and heat, based on D-values determined at 105°C. Pulsed-field gel electrophoresis (PFGE) was also performed to establish whether strains with a similar restriction banding pattern also exhibited similar D-values. However, no correlation between the genomic background of a strain and its resistance to high pressure processing was observed, based on PFGE analysis. Spores of proteolytic type B strains of C. botulinum were less resistant to combined high pressure and heat (700 MPa and 105°C) treatment when compared with spores of type A strains.

Several studies have been published on the inactivation of bacterial spores by using high pressure processing in combination with heat. None of the studies investigated the effect of the packaging system or the pressurizing fluid on spore inactivation. The objective of this study was to select and validate an appropriate packaging system and pressure transfer fluid for inactivation of Clostridium botulinum spores by using high pressure processing in combination with thermal processing. Inactivation of spores packaged in three packaging systems (plastic pouches, cryovials, and transfer pipettes) was measured in two pressure test systems (laboratory-scale and pilot-scale) at 700 MPa and >105°C. Total destruction (>6.6-log reduction) of the spores packaged in the graduated tube part of transfer pipettes was obtained after processing for up to 10 min at 118°C and 700 MPa in both pressure test systems, compared with the spores packaged either in plastic pouches or cryovials. Reduction of spores packaged in plastic pouches was lowest (