Gilbert Shama

Listeria monocytogenes adheres to many materials found in food‐processing environments

M.R. BERESFORD, P.W. ANDREW AND G. SHAMA. 2001.

Atmospheric plasma inactivation of biofilm-forming bacteria for food safety control

The ability of atmospheric pressure glow discharges (APGD) to inactivate microorganisms has been demonstrated in a number of previous studies. However, most of this work has been performed using microorganisms that do not form biofilms and with the microorganisms supported on abiotic surfaces that discourage cell growth. When microorganisms attach to the surface of a food, they can extract nutrients from the food and proliferate at the surface. Often this growth takes the form of biofilms which comprise three-dimensional (3-D) networks of polysaccharides that attach microorganisms to surfaces and serve to protect them from external stresses; fresh foods, such as salad crops, frequently harbor biofilms. We believe that the use of APGD offers a potential for inactivating microorganisms on the surface of fresh foods that cannot be readily treated by other methods without inducing unacceptable changes to these foods. As a first step toward a full evaluation of the viability of the APGD technology for food safety control, we consider in this paper two key issues, namely: 1) whether atmospheric glow discharges can inactivate biofilm-forming microorganisms and 2) whether plasma treatment causes significant discoloration to food surfaces. Using the biofilm-forming bacterium Pantoea agglomerans and bell peppers (Capsicum annuum) as a typical example of plant tissue, we show that atmospheric He-O/sub 2/ plasmas can be effective inactivation agents without causing unacceptable levels of discoloration to the peppers, and that furthermore they are superior to the use of low-pressure ultraviolet sources.

Probing bactericidal mechanisms induced by cold atmospheric plasmas with Escherichia coli mutants

Mechanisms of plasma-induced microbial inactivation have commonly been studied with physicochemical techniques. In this letter, Escherichia coli K-12 and its Delta recA, Delta rpoS, and Delta soxS mutants are employed to discriminate effects of UV photons, OH radicals, and reactive oxygen species produced in atmospheric discharges. This microbiological approach exploits the fact that these E. coli mutants are defective in their resistance against various external stresses. By interplaying bacterial inactivation kinetics with optical emission spectroscopy, oxygen atoms are identified as a major contributor in plasma inactivation with minor contributions from UV photons, OH radicals, singlet oxygen metastables, and nitric oxide. (c) 2007 American Institute of Physics.

Listeria monocytogenes relA and hpt Mutants Are Impaired in Surface-Attached Growth and Virulence

We describe here the identification and characterization of two Listeria monocytogenes (Tn917-LTV3) relA and hpt transposon insertion mutants that were impaired in growth after attachment to a model surface. Both mutants were unable to accumulate (p)ppGpp in response to amino acid starvation, whereas the wild-type strain accumulated (p)ppGpp within 30 min of stress induction. The induction of transcription of the relA gene after adhesion was demonstrated, suggesting that the ability to mount a stringent response and undergo physiological adaptation to nutrient deprivation is essential for the subsequent growth of the adhered bacteria. The absence of (p)ppGpp in the hpt mutant, which is blocked in the purine salvage pathway, is curious and suggests that a functional purine salvage pathway is required for the biosynthesis of (p)ppGpp. Both mutants were avirulent in a murine model of listeriosis, indicating an essential role for the stringent response in the survival and growth of L. monocytogenes in the host. Taken as a whole, this study provides new information on the role of the stringent response and the physiological adaptation of L. monocytogenes for biofilm growth and pathogenesis.

Cold Atmospheric Plasma Decontamination of the Pericarps of Fruit

This investigation describes the inactivation by cold atmospheric plasmas of one pathogenic and three spoilage organisms on the pericarps of mangoes and melons. The operating voltage necessary for efficient microbial decontamination of fruit pericarps was first established using Escherichia coli at a concentration of 10(7) CFU/cm2 on the surface of mango. It was found that, when the plasma was sustained slightly above its breakdown voltage of 12 kV (peak to peak), no inactivation was detected when cells were plated onto tryptone soya extract agar (TSA). However, when plated onto eosin methylene blue agar, sublethal injury corresponding to approximately 1 log reduction was achieved, whereas on TSA supplemented with 4% NaCl a greater reduction of 1.5 log was revealed. When the voltage was increased by 33% to 16 kV, a reduction in cell counts of 3 log was achieved on all three plating media. Further investigations at these new operating conditions were conducted using a range of spoilage microorganisms (Saccharomyces cerevisae, Pantoea agglomerans, and Gluconacetobacter liquefaciens) all at a surface concentration of 106 CFU/cm2 on the pericarps of mango and melon. P. agglomerans and G. liquefaciens were reduced below the detection limit (corresponding to 3 log) after only 2.5 s on both fruits, whereas E. coli required 5 s to reach the same level of inactivation. S. cerevisae was the most resistant organism studied and was reduced in numbers below the detection limit after 10 s on mango and 30 s on melon. The optical emission spectra generated by the cold atmospheric plasma at both high and low operating voltages were compared in order to identify putative lethal species. It was shown that an increase in the applied voltage led to more efficient production of reactive plasma species, particularly oxygen atoms, and the production of oxygen atoms was related to the level of bacterial inactivation achieved. Production of atomic oxygen could be used as an indicator of inactivation efficiency for scaling up cold plasma systems for whole fruit.

Green synthesis of silver and copper nanoparticles using ascorbic acid and chitosan for antimicrobial applications

Silver and copper nanoparticles were produced by chemical reduction of their respective nitrates by ascorbic acid in the presence of chitosan using microwave heating. Particle size was shown to increase by increasing the concentration of nitrate and reducing the chitosan concentration. Surface zeta potentials were positive for all nanoparticles produced and these varied from 27.8 to 33.8 mV. Antibacterial activities of Ag, Cu, mixtures of Ag and Cu, and Ag/Cu bimetallic nanoparticles were tested using Bacillus subtilis and Escherichia coli. Of the two, B. subtilis proved more susceptible under all conditions investigated. Silver nanoparticles displayed higher activity than copper nanoparticles and mixtures of nanoparticles of the same mean particle size. However when compared on an equal concentration basis Cu nanoparticles proved more lethal to the bacteria due to a higher surface area. The highest antibacterial activity was obtained with bimetallic Ag/Cu nanoparticles with minimum inhibitory concentrations (MIC) of 0.054 and 0.076 mg/L against B. subtilis and E. coli, respectively.

Cold atmospheric plasma disinfection of cut fruit surfaces contaminated with migrating microorganisms

The efficacy of cold atmospheric gas plasmas against Escherichia coli type 1, Saccharomyces cerevisiae, Gluconobacter liquefaciens, and Listeria monocytogenes Scott A was examined on inoculated membrane filters and inoculated fruit surfaces. Inoculated samples were exposed to a cold atmospheric plasma plume generated by an AC voltage of 8 kV at 30 kHz. The cold atmospheric plasma used in this study was very efficient in reducing the microbial load on the surfaces of filter membranes. However, its efficacy was markedly reduced for microorganisms on the cut surfaces. This lack of effect was not the result of quenching of reactive plasma species responsible for microbial inactivation but principally the result of the migration of microorganisms from the exterior of the fruit tissue to its interior. The velocity of migration through melon tissues was estimated to be around 300 microm min(-1) for E. coli and S. cerevisiae and through mango tissues to be 75 to 150 microm min(-1). These data can serve as operational targets for optimizing the performance of gas plasma inactivation processes. The current capabilities of cold atmospheric plasmas are reviewed and ways to improve their bactericidal efficacy are identified and discussed. Considerable scope exists to enhance significantly the efficacy of cold atmospheric plasmas for decontaminating fresh cut fruits.

Effects of cell surface loading and phase of growth in cold atmospheric gas plasma inactivation of Escherichia coli K12

Aims:  To determine the effects of surface cell concentration and phase of growth on the inactivation of Escherichia coli cells using an atmospheric nonthermal plasma.

Effects of microbial loading and sporulation temperature on atmospheric plasma inactivation of Bacillus subtilis spores

Current inactivation studies of Bacillus subtilis spores using atmospheric-pressure glow discharges (APGD) do not consider two important factors, namely microbial loading at the surface of a substrate and sporulation temperature. Yet these are known to affect significantly microbial resistance to heat and hydrogen peroxide. This letter investigates effects of microbial loading and sporulation temperature on spore resistance to APGD. It is shown that microbial loading can lead to a stacking structure as a protective shield against APGD treatment and that high sporulation temperature increases spore resistance by altering core water content and cross-linked muramic acid content of B. subtilis spores.

The uses and abuses of rapid bioluminescence-based ATP assays.

Bioluminescence-based ATP testing of solid surfaces has become well established in the food processing industry as part of general hazard analysis and critical control points (HACCP) measures. The rise in healthcare associated infections (HAIs) at the turn of the century focussed attention on the environment as a potential reservoir of the agents responsible for such infections. In response to the need for objective methods of assessing the efficiency of cleaning in healthcare establishments and for rapid methods for detecting the presence of the pathogens responsible for HAIs, it was proposed that ATP testing of environmental surfaces be introduced. We examine the basis behind the assumptions inherent in these proposals. Intracellular ATP levels are shown to vary between microbial taxa and according to environmental conditions. Good correlations between microbial numbers and ATP levels have been obtained under certain specific conditions, but never within healthcare settings. Notwithstanding, ATP testing may still have a role in providing reassurance that cleaning regimes are being carried out satisfactorily. However, ATP results should not be interpreted as surrogate indicators for the presence of microbial pathogens.