Guy E. Skinner

Food safety objective approach for controlling Clostridium botulinum growth and toxin production in commercially sterile foods.

As existing technologies are refined and novel microbial inactivation technologies are developed, there is a growing need for a metric that can be used to judge equivalent levels of hazard control stringency to ensure food safety of commercially sterile foods. A food safety objective (FSO) is an output-oriented metric that designates the maximum level of a hazard (e.g., the pathogenic microorganism or toxin) tolerated in a food at the end of the food supply chain at the moment of consumption without specifying by which measures the hazard level is controlled. Using a risk-based approach, when the total outcome of controlling initial levels (H(0)), reducing levels (ΣR), and preventing an increase in levels (ΣI) is less than or equal to the target FSO, the product is considered safe. A cross-disciplinary international consortium of specialists from industry, academia, and government was organized with the objective of developing a document to illustrate the FSO approach for controlling Clostridium botulinum toxin in commercially sterile foods. This article outlines the general principles of an FSO risk management framework for controlling C. botulinum growth and toxin production in commercially sterile foods. Topics include historical approaches to establishing commercial sterility; a perspective on the establishment of an appropriate target FSO; a discussion of control of initial levels, reduction of levels, and prevention of an increase in levels of the hazard; and deterministic and stochastic examples that illustrate the impact that various control measure combinations have on the safety of well-established commercially sterile products and the ways in which variability all levels of control can heavily influence estimates in the FSO risk management framework. This risk-based framework should encourage development of innovative technologies that result in microbial safety levels equivalent to those achieved with traditional processing methods.

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 degrees C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80 degrees 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.

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 degrees 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 microg/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 microg/ml) incubated for 4 months versus that recovered from peptone-yeast extract-glucose-starch (PYGS) plating agar containing lysozyme (10 microg/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 microg/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 microg/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.

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.

Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor

Botulinum neurotoxin (BoNT) is the most potent toxin known. The ingestion of food contaminated with biologically active BoNT causes foodborne botulism, which can lead to respiratory paralysis, coma, and death after ingestion of as little as 70 μg for a 70 kg human. Because of its lethality and challenges associated with current detection methods, there is an urgent need for highly sensitive rapid screening techniques capable of detecting biologically active BoNT. Here, we describe a Förster resonance energy transfer-based nanobiosensor that uses quantum dots (QDs) and two specific quencher-labeled peptide probes to detect and differentiate two biologically active forms of BoNT, serotypes A and B, which were responsible for 80% of human foodborne botulism cases in the U.S. from 2012 to 2015. Each peptide probe contains an enzymatic cleavage site specific to only one serotype. QDs were selected based on the spectral overlap with the quenchers. In the presence of the target BoNT serotype, the peptide probe is cleaved and the quenching of QD photoluminescence (PL) is reduced, giving a signal that is easily detected by a PL spectrophotometer. This sensor performance was evaluated with light chains of BoNT/A and BoNT/B (LcA and LcB), catalytic domains of the respective serotypes. LcA and LcB were detected in 3 h with limits of detection of 0.2 and 2 ng/mL, respectively. The specificity of the sensor was evaluated, and no cross-reactivity from nontarget serotypes was observed with 2 h of incubation. Because each serotype-specific peptide is conjugated to a QD with a unique emission wavelength, multiple biologically active BoNT serotypes could be detected in one PL spectrum. The sensor was also shown to be responsive to BoNT/A and BoNT/B holotoxins. Good performance of this sensor implies its potential application as a rapid screening method for biologically active BoNT/A and BoNT/B in the laboratory and in the field.

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.