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The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

United States Army-Natick Soldier RD&E Center, Warfighter Directorate

Stopped-flow spectrophotometric measurements identify and determine equilibrium data for thiourea (tu) complexes of copper(II) formed in aqueous solution. In excess Cu(II), the complex ion [Cu(tu)](2+) has a stability constant beta(1) = 2.3 +/- 0.1 M(-)(1) and molar absorptivity at 340 nm of epsilon(1) = (4.0 +/- 0.2) x 10(3) M(-)(1) cm(-)(1) at 25.0 degrees C, 2.48 mM HClO(4), and &mgr; = 464 mM (NaClO(4)). The fast reduction of Cu(II) by excess tu obeys the rate law -d[Cu(II)]/dt = k'[Cu(II)](2)[tu](7) with a value for the ninth-order rate constant k' = (1.60 +/- 0.18) x 10(14) M(-)(8) s(-)(1), which derives from a rate-determining step involving the bimolecular decomposition of two complexed Cu(II) species. Copper(II) catalyzes the reduction of hexachloroiridate(IV) by tu according to the rate law -d[IrCl(6)(2)(-)]/dt = (k(2,unc)[tu](2) + k(1,cat) [tu](5)[Cu(II)])[IrCl(6)(2)(-)]. Least-squares analysis yields values of k(2,unc) and k(1,cat) equaling 385 +/- 4 M(-)(2) s(-)(1) and (3.7 +/- 0.1) x 10(13) M(-)(6) s(-)(1), respectively, at &mgr; = 115 mM (NaClO(4)). The corresponding mechanism has a rate-determining step that involves the oxidation of [Cu(II)(tu)(5)](2+) by [IrCl(6)](2)(-) rather than the bimolecular reaction of two cupric-tu complexes.

Equilibrium and Redox Kinetics of Copper(II)-Thiourea Complexes.

Predictive microbial models generally rely on the growth of bacteria in laboratory broth to approximate the microbial growth kinetics expected to take place in actual foods under identical environmental conditions. Sigmoidal functions such as the Gompertz or logistics equation accurately model the typical microbial growth curve from the lag to the stationary phase and provide the mathematical basis for estimating parameters such as the maximum growth rate (MGR). Stationary phase data can begin to show a decline and make it difficult to discern which data to include in the analysis of the growth curve, a factor that influences the calculated values of the growth parameters. In contradistinction, the quasi-chemical kinetics model provides additional capabilities in microbial modelling and fits growth-death kinetics (all four phases of the microbial lifecycle continuously) for a general set of microorganisms in a variety of actual food substrates. The quasi-chemical model is differential equations (ODEs) that derives from a hypothetical four-step chemical mechanism involving an antagonistic metabolite (quorum sensing) and successfully fits the kinetics of pathogens (Staphylococcus aureus, Escherichia coli and Listeria monocytogenes) in various foods (bread, turkey meat, ham and cheese) as functions of different hurdles (a(w), pH, temperature and anti-microbial lactate). The calculated value of the MGR depends on whether growth-death data or only growth data are used in the fitting procedure. The quasi-chemical kinetics model is also exploited for use with the novel food processing technology of high-pressure processing. The high-pressure inactivation kinetics of E. coli are explored in a model food system over the pressure (P) range of 207-345 MPa (30,000-50,000 psi) and the temperature (T) range of 30-50 degrees C. In relatively low combinations of P and T, the inactivation curves are non-linear and exhibit a shoulder prior to a more rapid rate of microbial destruction. In the higher P, T regime, the inactivation plots tend to be linear. In all cases, the quasi-chemical model successfully fit the linear and curvi-linear inactivation plots for E. coli in model food systems. The experimental data and the quasi-chemical mathematical model described herein are candidates for inclusion in ComBase, the developing database that combines data and models from the USDA Pathogen Modeling Program and the UK Food MicroModel.

A quasi-chemical model for the growth and death of microorganisms in foods by non-thermal and high-pressure processing.

The water dynamics and retrogradation kinetics behavior of gelatinized wheat starch by either ultrahigh pressure (UHP) processing or heat are investigated. Wheat starch completely gelatinized in the condition of 90, 000 psi at 25 degrees C for 30 min (pressurized gel) or 100 degrees C for 30 min (heated gel). The physical properties of the wheat starches were characterized in terms of proton relaxation times (T2 times) measured using time-domain nuclear magnetic resonance spectroscopy and evaluated using commercially available continuous distribution modeling software. Different T2 distributions in both micro- and millisecond ranges between pressurized and heated wheat starch gels suggest distinctively different water dynamics between pressurized and heated wheat starch gels. Smaller water self-diffusion coefficients were observed for pressurized wheat starch gels and are indicative of more restricted translational proton mobility than is observed with heated wheat starch gels. The physical characteristics associated with changes taking place during retrogradation were evaluated using melting curves obtained with differential scanning calorimetry. Less retrogradation was observed in pressurized wheat starch, and it may be related to a smaller quantity of freezable water in pressurized wheat starch. Starches comprise a major constituent of many foods proposed for commercial potential using UHP, and the present results furnish insight into the effect of UHP on starch gelatinization and the mechanism of retrogradation during storage.

Water dynamics and retrogradation of ultrahigh pressurized wheat starch.

The enhanced quasi-chemical kinetics (EQCK) model is presented as a methodology to evaluate the nonlinear inactivation kinetics of baro-resistant Listeria monocytogenes in a surrogate protein food system by high-pressure processing (HPP) for various combinations of pressure (P= 207 to 414 MPa) and temperature (T= 20 to 50 °C). The EQCK model is based on ordinary differential equations derived from 6 "quasi-chemical reaction" steps. The EQCK model continuously fits the conventional stages of the microbial lifecycle: lag, growth, stationary phase, and death; and tailing. Depending on the conditions, the inactivation kinetics of L. monocytogenes by HPP show a lag, inactivation, and tailing. Accordingly, we developed a customized, 4-step subset version of the EQCK model sufficient to evaluate the HPP inactivation kinetics of L. monocytogenes and obtain values for the model parameters of lag (λ), inactivation rate (μ), rate constants (k), and "processing time" (tp). This latter parameter was developed uniquely to evaluate kinetics data showing tailing. Secondary models are developed by interrelating the fitting parameters with experimental parameters, and Monte Carlo simulations are used to evaluate parameter reproducibility. This 4-step model is also compared with the empirical Weibull and Polylog models. The success of the EQCK model (as its 4-step subset) for the HPP inactivation kinetics of baro-resistant L. monocytogenes showing tailing establishes several advantages of the EQCK modeling approach for investigating nonlinear microbial inactivation kinetics, and it has implications for determining mechanisms of bacterial spore inactivation by HPP.

Inactivation kinetics of Listeria monocytogenes by high-pressure processing: pressure and temperature variation.

As previously reported, gerP Bacillus subtilis spores were defective in nutrient germination triggered via various germinant receptors (GRs), and the defect was eliminated by severe spore coat defects. The gerP spores' GR-dependent germination had a longer lag time between addition of germinants and initiation of rapid release of spores' dipicolinic acid (DPA), but times for release of >90% of DPA from individual spores were identical for wild-type and gerP spores. The gerP spores were also defective in GR-independent germination by DPA with its associated Ca(2+) divalent cation (CaDPA) but germinated better than wild-type spores with the GR-independent germinant dodecylamine. The gerP spores exhibited no increased sensitivity to hypochlorite, suggesting that these spores have no significant coat defect. Overexpression of GRs in gerP spores did lead to faster germination via the overexpressed GR, but this was still slower than germination of comparable gerP(+) spores. Unlike wild-type spores, for which maximal nutrient germinant concentrations were between 500 μM and 2 mM for l-alanine and ≤10 mM for l-valine, rates of gerP spore germination increased up to between 200 mM and 1 M l-alanine and 100 mM l-valine, and at 1 M l-alanine, the rates of germination of wild-type and gerP spores with or without all alanine racemases were almost identical. A high pressure of 150 MPa that triggers spore germination by activating GRs also triggered germination of wild-type and gerP spores identically. All these results support the suggestion that GerP proteins facilitate access of nutrient germinants to their cognate GRs in spores' inner membrane.

Analysis of the effects of a gerP mutation on the germination of spores of Bacillus subtilis.

Germination of Bacillus spores with a high pressure (HP) of ∼150 MPa is via activation of spores' germinant receptors (GRs). The HP germination of multiple individual Bacillus subtilis spores in a diamond anvil cell (DAC) was monitored with phase-contrast microscopy. Major conclusions were that (i) >95% of wild-type spores germinated in 40 min in a DAC at ∼150 MPa and 37°C but individual spores' germination kinetics were heterogeneous; (ii) individual spores' HP germination kinetic parameters were similar to those of nutrient-triggered germination with a variable lag time (Tlag) prior to a period of the rapid release (ΔTrelease) of the spores' dipicolinic acid in a 1:1 chelate with Ca(2+) (CaDPA); (iii) spore germination at 50 MPa had longer average Tlag values than that at ∼150 MPa, but the ΔTrelease values at the two pressures were identical and HPs of <10 MPa did not induce germination; (iv) B. subtilis spores that lacked the cortex-lytic enzyme CwlJ and that were germinated with an HP of 150 MPa exhibited average ΔTrelease values ∼15-fold longer than those for wild-type spores, but the two types of spores exhibited similar average Tlag values; and (v) the germination of wild-type spores given a ≥30-s 140-MPa HP pulse followed by a constant pressure of 1 MPa was the same as that of spores exposed to a constant pressure of 140 MPa that was continued for ≥35 min; (vi) however, after short 150-MPa HP pulses and incubation at 0.1 MPa (ambient pressure), spore germination stopped 5 to 10 min after the HP was released. These results suggest that an HP of ∼150 MPa for ≤30 s is sufficient to fully activate spores' GRs, which remain activated at 1 MPa but can deactivate at ambient pressure.

Monitoring rates and heterogeneity of high-pressure germination of bacillus spores by phase-contrast microscopy of individual 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 spore's 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 spore's 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 SpoVAEa's 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.

Christopher J. Doona

United States Army-Natick Soldier RD&E Center, Warfighter Directorate

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military