Christopher J. Doona

Microbial safety of fresh produce.

Section I: Microbial Contamination of Fresh Produce. Chapter 1. Enteric human pathogens associated with fresh produce: sources, transport and ecology (Robert E. Mandrell). Chapter 2. The origin and spread of human pathogens in fruit production systems (Susan Bach and Pescal Delaquis). Chapter 3. Internalization of Pathogens in Produce (Elliot T. Ryser, Jianjun Hao, and Zhinong Yan). Section II: Pre-harvest Strategies. Chapter 4. Produce safety in organic vs conventional crops (Francisco Diez-Gonzalez and Avik Mukherjee). Chapter 5. The Role of Good Agricultural Practices in Produce Safety (Robert B. Gravani). Chapter 6. Effective Managing through a Crisis (Will Daniels and Michael P. Doyle). Chapter 7. The Role of Water and Water testing in Produce Safety (Charles P. Gerba ). Chapter 8. Role of manure and compost in produce safety (Xiuping Jiang). Section III: Post-harvest Interventions. Chapter 9. Aqueous antimicrobial treatments to improve fresh and fresh-cut produce safety (Joy Herdt and Hao Feng). Chapter 10. Irradiation enhances quality and microbial safety of fresh and fresh-cut fruits and vegetables (Brendan A. Niemira and Xuetong Fan). Chapter 11. Biological control of human pathogens on produce (John Andrew Hudson, Craig Billington, and Lynn McIntyre). Chapter 12. Extension of Shelf-life and Control of Human Pathogens in Produce by Antimicrobial Edible Films and Coatings (Tara H. McHugh, Roberto J. Avena-Bustillos, and Wen-Xian Du). Chapter 13. Improving Microbial Safety of Fresh Produce Using Thermal Treatment (Xuetong Fan, Lihan Huang, Bassam Annous). Chapter 14. Enhanced Safety and Extended Shelf-life of Fresh Produce for the Military (Peter Setlow, Christopher J. Doona, Florence E. Feeherry, and Kenneth Kustin, Deborah Sisson, and Shubham Chandra). Section IV: Produce Safety during Processing and Handling. Chapter 15. Consumer and Food Service Handling of Fresh Produce (Christine M. Bruhn). Chapter 16. Plant Sanitation and Good Manufacturing Practices for Optimum Food Safety in Fresh-cut Produce (Edith Garrett). Chapter 17. Third party audit programs for the fresh produce industry (Kenneth S. Petersen). Chapter 18. Pathogen Detection in Produce using Applications of Immunomagnetic Beads and Biosensors (Shu-I Tu, Joseph Uknalis, Andrew Gehring, and Peter Irwin). Section V: Public, Legal, and Economic Perspectives. Chapter 19. Public Response to the 2006 Recall of Contaminated Spinach (William K. Hallman, Cara L. Cuite, Jocilyn E. Dellava, Mary L. Nucci, and Sarah C. Condry). Chapter 20. Produce in public: Spinach, safety and public policy (Douglas A. Powell, Casey J. Jacob and Benjamin Chapman). Chapter 21.Contaminated Fresh Produce and Product Liability: A Law-in-Action Perspective (Denis W. Stearns). Chapter 22. The Economics of Food Safety: The 2006 Foodborne Illness Outbreak Linked to Spinach (Linda Calvin, Helen H. Jensen and Jing Liang). Section VI: Research Challenges and Directions. Chapter 23. Research Needs and Future Directions (Brendan A. Niemira, Xuetong Fan, Christopher J. Doona, Florence E. Feeherry, Robert B. Gravani).

High Pressure Germination of Bacillus subtilis Spores with Alterations in Levels and Types of Germination Proteins

Examine effects of different levels and types of nutrient germinant receptors (GRs) and other germination proteins on Bacillus subtilis spore germination by a moderate high pressure (mHP) (150 megaPascals (MPa)) that triggers germination through GRs, and a very high pressure (vHP) (550 MPa) that triggers spore germination independent of GRs.

Function of the SpoVAEa and SpoVAF Proteins of Bacillus subtilis Spores

The Bacillus subtilis spoVAEa and spoVAF genes are expressed in developing spores as members of the spoVA operon, which encodes proteins essential for the uptake and release of dipicolinic acid (DPA) during spore formation and germination. SpoVAF is likely an integral inner spore membrane protein and exhibits sequence identity to A subunits of the spores nutrient germinant receptors (GRs), while SpoVAEa is a soluble protein with no obvious signals to allow its passage across a membrane. However, like SpoVAD, SpoVAEa is present on the outer surface of the spores inner membrane, as SpoVAEa was accessible to an external biotinylation agent in spores and SpoVAEa disappeared in parallel with SpoVAD during proteinase K treatment of germinated spores. SpoVAEa and SpoVAD were also distributed similarly in fractions of disrupted dormant spores. Unlike spoVAD, spoVAEa is absent from the genomes of some spore-forming members of the Bacillales and Clostridiales orders, although SpoVAEas amino acid sequence is conserved in species containing spoVAEa. B. subtilis strains lacking SpoVAF or SpoVAEa and SpoVAF sporulated normally, and the spores had normal DPA levels. Spores lacking SpoVAF or SpoVAEa and SpoVAF also germinated normally with non-GR-dependent germinants but more slowly than wild-type spores with GR-dependent germinants, and this germination defect was complemented by ectopic expression of the missing proteins.

Use of Raman Spectroscopy and Phase-Contrast Microscopy To Characterize Cold Atmospheric Plasma Inactivation of Individual Bacterial Spores

ABSTRACT Raman spectroscopy and phase-contrast microscopy were used to examine calcium dipicolinate (CaDPA) levels and rates of nutrient and nonnutrient germination of multiple individual Bacillus subtilis spores treated with cold atmospheric plasma (CAP). Major results for this work include the following: (i) >5 logs of spores deposited on glass surfaces were inactivated by CAP treatment for 3 min, while deposited spores placed inside an impermeable plastic bag were inactivated only ∼2 logs in 30 min; (ii) >80% of the spores treated for 1 to 3 min with CAP were nonculturable and retained CaDPA in their core, while >95% of spores treated with CAP for 5 to 10 min lost all CaDPA; (iii) Raman measurements of individual CAP-treated spores without CaDPA showed differences from spores that germinated with l-valine in terms of nucleic acids, lipids, and proteins; and (iv) 1 to 2 min of CAP treatment killed 99% of spores, but these spores still germinated with nutrients or exogenous CaDPA, albeit more slowly and to a lesser extent than untreated spores, while spores CAP treated for >3 min that retained CaDPA did not germinate via nutrients or CaDPA. However, even after 1 to 3 min of CAP treatment, spores germinated normally with dodecylamine. These results suggest that exposure to the present CAP configuration severely damages a spores inner membrane and key germination proteins, such that the treated spores either lose CaDPA or can neither initiate nor complete germination with nutrients or CaDPA. Analysis of the various CAP components indicated that UV photons contributed minimally to spore inactivation, while charged particles and reactive oxygen species contributed significantly. IMPORTANCE Much research has shown that cold atmospheric plasma (CAP) is a promising tool for the inactivation of spores in the medical and food industries. However, knowledge about the effects of plasma treatment on spore properties is limited, especially at the single-cell level. In this study, Raman spectroscopy and phase-contrast microscopy were used to analyze CaDPA levels and kinetics of nutrient- and non-nutrient-germinant-induced germination of multiple individual spores of Bacillus subtilis that were treated by a planar CAP device. The roles of different plasma species involved in spore inactivation were also investigated. The knowledge obtained in this study will aid in understanding the mechanism(s) of spore inactivation by CAP and potentially facilitate the development of more effective and efficient plasma sterilization techniques in various applications.

Fighting Ebola with novel spore decontamination technologies for the military

Recently, global public health organizations such as Doctors without Borders (MSF), the World Health Organization (WHO), Public Health Canada, National Institutes of Health (NIH), and the U.S. government developed and deployed Field Decontamination Kits (FDKs), a novel, lightweight, compact, reusable decontamination technology to sterilize Ebola-contaminated medical devices at remote clinical sites lacking infra-structure in crisis-stricken regions of West Africa (medical waste materials are placed in bags and burned). The basis for effectuating sterilization with FDKs is chlorine dioxide (ClO2) produced from a patented invention developed by researchers at the US Army Natick Soldier RD&E Center (NSRDEC) and commercialized as a dry mixed-chemical for bacterial spore decontamination. In fact, the NSRDEC research scientists developed an ensemble of ClO2 technologies designed for different applications in decontaminating fresh produce; food contact and handling surfaces; personal protective equipment; textiles used in clothing, uniforms, tents, and shelters; graywater recycling; airplanes; surgical instruments; and hard surfaces in latrines, laundries, and deployable medical facilities. These examples demonstrate the far-reaching impact, adaptability, and versatility of these innovative technologies. We present herein the unique attributes of NSRDEC’s novel decontamination technologies and a Case Study of the development of FDKs that were deployed in West Africa by international public health organizations to sterilize Ebola-contaminated medical equipment. FDKs use bacterial spores as indicators of sterility. We review the properties and structures of spores and the mechanisms of bacterial spore inactivation by ClO2. We also review mechanisms of bacterial spore inactivation by novel, emerging, and established non-thermal technologies for food preservation, such as high pressure processing, irradiation, cold plasma, and chemical sanitizers, using an array of Bacillus subtilis mutants to probe mechanisms of spore germination and inactivation. We employ techniques of high-resolution atomic force microscopy and phase contrast microscopy to examine the effects of γ-irradiation on bacterial spores of Bacillus anthracis, Bacillus thuringiensis, and Bacillus atrophaeus spp. and of ClO2 on B. subtilis spores, and present in detail assays using spore bio-indicators to ensure sterility when decontaminating with ClO2.

Modeling moisture migration in a multi-domain food system: Application to storage of a sandwich system

Moisture transport in a food system involving two different materials of unequal moisture content was modeled with water activity as the driving force using a porous media framework. This model was applied to a bread-barbecue chicken pocket sandwich stored in isothermal conditions. The model successfully predicted the equilibrium condition, where the two materials, bread and chicken, reached the same water activity, but not the same water content. The transient changes in the moisture content in the bread and chicken were predicted and shown to be significantly affected by air gap between the bread and chicken. The prediction process was also sensitive to the Darcy permeability values for the materials. The modeling framework presented for a sandwich system is very general and can easily be extended to other multicomponent food systems.

Ascorbate-induced oxidation of formate by peroxodisulfate: product yields, kinetics and mechanism

The slow reaction between peroxodisulfate and formate is significantly accelerated by ascorbate at room temperature. The products of this induced oxidation, CO2 and oxalate (C2O2–4), were analyzed by several methods and the kinetics of this reaction were measured. The overall mechanism involves free radical species. Ascorbate reacts with peroxodisulfate to initiate production of the sulfate radical ion (SO•–4), which reacts with formate to produce carbon dioxide radical ion (CO•–2) and sulfate. The carbon dioxide radical reacts with peroxodisulfate to form CO2 or self-combines to form oxalate. Competition occurring between these two processes determines the overall fate of the carbon dioxide radical species. As pH decreases, protonation of the carbon dioxide radical ion tends to favor production of CO2.

Mathematical Models Based on Transition State Theory for the Microbial Safety of Foods by High Pressure

Prior to the development of transition state theory, the Arrhenius equation was the principal relationship used in describing the temperature dependence of chemical reaction rates. Research into determining the theoretical basis for the Arrhenius parameters A (pre-exponential factor) and Ea (activation energy) led to the development of transition state theory and the Eyring equation, whose central postulate is a hypothetical transient state called the activated complex that forms through interactions between reactants before they can become products during the process of a chemical reaction. It is from the perspective of transition state theory that we develop two secondary models to reflect the effects of temperature and of high pressure on microbial inactivation by the emerging nonthermal technology of high pressure processing (HPP), and we designate these as transition state (TS) models TST and TSP, respectively. These secondary models are applied to data obtained with two primary models, the enhanced quasi-chemical kinetics (EQCK) differential equation model and the Weibull distribution empirical model, that were used to evaluate nonlinear inactivation kinetics for baro-resistant Listeria monocytogenes in a surrogate protein food system by HPP for various combinations of pressure (207–414 MPa) and temperature (20–50 °C). The mathematical relationships of TST and TSP involve primarily the unique model parameter called “processing time parameter” (t p ), which was developed to evaluate inactivation kinetics data showing tailing. These detailed secondary models, as applied to the parameters of the EQCK and Weibull primary models, have important ramifications for ensuring food safety and the shelf life of food products and support the growing uses of HPP for the safe preservation of foodstuffs.

Chemical Kinetics for the Microbial Safety of Foods Treated with High Pressure Processing or Hurdles

The application of chemical kinetics is well known in food engineering, such as the use of Arrhenius plots and D- and z-values to characterize linear microbial inactivation kinetics by thermal processing. The emergence and growing commercialization of nonthermal processing technologies in the past decade provided impetus for the development of nonlinear models to describe nonlinear inactivation kinetics of foodborne microbes. One such model, the enhanced quasi-chemical kinetics (EQCK) model, postulates a mechanistic sequence of reaction steps and uses a chemical kinetics approach to developing a system of rate equations (ordinary differential equations) that provide the mathematical basis for describing an array of complex nonlinear dynamics exhibited by microbes in foods. Specifically, the EQCK model characterizes continuous growth–death–tailing dynamics (or subsets thereof) for pathogens such as Staphylococcus aureus, Listeria monocytogenes, or Escherichia coli in various food matrices (bread, turkey, ham, cheese) controlled by “hurdles” (water activity, pH, temperature, antimicrobials). The EQCK model is also used with high pressure processing (HPP), to characterize nonlinear inactivation kinetics for E. coli (inactivation plots show lag times), baro-resistant L. monocytogenes (inactivation plots show slight lag times and protracted tailing), and Bacillus amyloliquefaciens spores (inactivation plots show protracted tailing; HPP also induces spore activation and spore germination). We invoke further chemical kinetics principles by applying transition-state theory (TST) to the HPP inactivation of L. monocytogenes and develop novel dimensionless secondary models for temperature and pressure (TST temperature and TST pressure) to estimate kinetics parameters (activation energy Ea and activation volume ∆V‡), thereby offering new insights into the inactivation mechanisms of pathogenic organisms by HPP.

A Quasi-chemical Model for Bacterial Spore Germination Kinetics by High Pressure

High pressure processing (HPP) is an emerging non-thermal technology that is growing exponentially in use worldwide for the pasteurization of commercial foodstuffs. At combinations of elevated pressures and temperatures, HPP inactivates bacterial spores, but HPP has not yet been implemented commercially for food sterilization. Studies of the mechanisms of bacterial spore inactivation by HPP using primarily spores of Bacillus species have shown that spore germination precedes inactivation, with the release of dipicolinic acid from the spore core as the rate-determining step. Investigations probing spore resistance to and germination by HPP using Bacillus subtilis, a number of selected B. subtilis mutants, Bacillus amyloliquefaciens, and Clostridium difficile spores have compiled a wealth of detailed mechanistic information, while also accumulating abundant germination kinetics data that has not previously been analyzed by predictive models. Presently, we devise a “quasi-chemical” model for bacterial spore germination dynamics by HPP. This quasi-chemical germination model (QCGM) hypothesizes a three-step mechanism and derives a set of ordinary differential equations to model the observed germination dynamics. The results with this model are viewed in the context of historical studies of spore activation, germination, and inactivation, with an eye toward potentially integrating differential equation models for germination and inactivation into a single, comprehensive model for spore dynamics by HPP. With the increasing use of high hydrostatic pressure to investigate mechanisms of bacterial spore resistance and physiology, the QCGM results help promote the efficient control of bacterial spores, whether for the inactivation of Clostridium botulinum spores in low-acid foods or aerosolized Bacillus anthracis spores on textiles used in protective clothing, tents, or shelters.