N. Rukma Reddy

Amplified Immunoassay ELISA-ELCA for Measuring Clostridium botulinum Type E Neurotoxin in Fish Fillets

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.

Botulism: the causative agent and its control in foods

Abstract Clostridium botulinum is the causative agent in four types of botulism: foodborne, infant, wound and those classified as undetermined. The types of C. botulinum and food products involved in various foodborne botulism outbreaks are discussed in this review. Most foodborne botulism outbreaks result from consumption of home-processed or home-canned foods; relatively few are caused by commercial products. Various physical and chemical treatments that can be used in foods either to destroy C. botulinum spores or control their outgrowth and toxin production are presented, and concerns about potential foodborne botulism outbreaks from new generation foods are discussed.

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.

Genetic Diversity of Clostridium sporogenes PA 3679 Isolates Obtained from Different Sources as Resolved by Pulsed-Field Gel Electrophoresis and High-Throughput Sequencing

ABSTRACT 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 Campbells 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.

Effect of Packaging Systems and Pressure Fluids on Inactivation of Clostridium botulinum Spores by Combined High Pressure and Thermal Processing

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 (<4.8 log) for both pressure test systems when processed at the same conditions (i.e., 700 MPa and 118°C). Within the pilot-scale pressure system, increasing the process temperature from 118 to 121°C at 700 MPa for 10 min resulted in only a small increase in spore reduction (<5.1 log) for spores packaged in plastic pouches, whereas there were no recoverable spores for either of the other two packaging systems. Use of plastic pouches for packaging spores in inactivation kinetic studies could lead to erroneous conclusions about the effect of high pressure in combination with heat. BioGlycol is the pressure-heat transfer fluid of choice, as compared with Duratherm oil, to maximize the temperature response rate during pressurization within the laboratory-scale pressure test system.

Effect of sporulation temperature on the resistance of Clostridium botulinum type A spores to thermal and high pressure processing.

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 organisms 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.

Thermal and Pressure-Assisted Thermal Destruction Kinetics for Spores of Type A Clostridium botulinum and Clostridium sporogenes PA3679.

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) for <1 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 a >4.5-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.

Effect of Fill Temperature on Clostridium botulinum Type A Toxin Activity during the Hot Filling of Juice Bottles

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 bottles 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.