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We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log10 CFU reduction of bacteria when 2 x 10(2) cells were seeded on filter paper. Results showed > or = 3 log10 CFU reduction when polypropylene samples seeded with E. coli (5 x 10(4)) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect > or = 6 log10 CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated > or = 5 log10 CFU reduction of commercially prepared Bacillus subtilis spores (1 x 10(5)); 7 min OAUGDP exposures were required to generate a > or = 3 log10 CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed.

A study was conducted to determine the effect of one atmosphere uniform glow discharge plasma (OAUGDP) on inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on apples, cantaloupe, and lettuce, respectively. A five-strain mixture of cultured test organisms was washed, suspended in phosphate buffer, and spot inoculated onto produce (7 log CFU per sample). Samples were exposed inside a chamber affixed to the OAUGDP blower unit operated at a power of 9 kV and frequency of 6 kHz. This configuration allows the sample to be placed outside of the plasma generation unit while allowing airflow to carry the antimicrobial active species, including ozone and nitric oxide, onto the food sample. Cantaloupe and lettuce samples were exposed for 1, 3, and 5 min, while apple samples were exposed for 30 s, 1 min, and 2 min. After exposure, samples were pummeled in 0.1% peptone water-2% Tween 80, diluted, and plated in duplicate onto selective media and tryptic soy agar and incubated as follows: E. coli O157:H7 (modified eosin methylene blue) and Salmonella (xylose lysine tergitol-4) for 48 h at 37 degrees C, and L. monocytogenes (modified Oxford medium) at 48 h for 32 degrees C. E. coli O157:H7 populations were reduced by >1 log after 30-s and 1-min exposures and >2 log after a 2-min exposure. Salmonella populations were reduced by >2 log after 1 min. Three- and 5-min exposure times resulted in >3-log reduction. L. monocytogenes populations were reduced by 1 log after 1 min of exposure. Three- and 5-min exposure times resulted in >3- and >5-log reductions, respectively. This process has the capability of serving as a novel, nonthermal processing technology to be used for reducing microbial populations on produce surfaces.

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The medical, industrial, and food processing industries are constantly in search of new technologies to provide improved methods of sterilization and pasteurization. Proposed techniques must deal with such problems as thermal sensitivity and destruction by heat, formation of toxic by-products, cost, and inefficiency in performance. We report results from a newly invented plasma source, a one atmosphere uniform glow discharge plasma (OAUGDP), which is capable of operating at atmospheric pressure in air and providing antimicrobial active species at room temperature. OAUGDP exposures have reduced log numbers of bacteria (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa), bacterial endospores (Bacillus subtilis and Bacillus pumilus), and various yeast and bacterial viruses on a variety of surfaces. These surfaces included polypropylene, filter paper, paper strips, solid culture media, and glass. Experimental results showed at least a ⩾5 log10 colony forming units (CFU) reduction in bacteria within a range of 15–90 s of exposure, whether the samples were exposed in conventional sterilization bags or exposed directly to the plasma. An exception to these very short exposure times were experiments with solid culture media in which 5 min of plasma exposure was necessary to produce ⩾5 log10 CFU reduction in bacterial counts. The effects of plasma treatment on bacterial cell structures were investigated by exposing cells to plasma for various durations and examining them by transmission electron microscopy. The results showed that cell breakage (lysis) occurred with the release of cellular contents. These data were consistent with spectrophotometric results in which the release of cellular constituents was measured as a change in ultraviolet absorption at 260 nm. With all microorganisms tested, a biphasic survival curve (logarithmic number of survivors versus time) was observed in plots of dose-response data. Differences in susceptibilities of microorganisms observed on various surfaces suggested that the degree of lethality was dependent upon the time of diffusion of active species through the medium to the organism and the makeup of the microbial cellular surface.

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This study was conducted to determine the efficacy of a one atmosphere uniform glow discharge plasma (OAUGDP) for inactivation of foodborne pathogens and to evaluate the influence of growth temperature, pH, and culture age on their inactivation. Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Salmonella Enteritidis, Vibrio parahaemolyticus, Yersinia enterocolitica, and Shigella flexneri were evaluated. Three-strain mixtures of each bacterium were inoculated (6-7 log CFU/cm(2)) onto microscope slides containing nonselective agar media adjusted to pH 5 or 7. Samples were exposed to plasma for 0-240 sec immediately, or after incubation for 24 h at 10 degrees C or 35 degrees C. After exposure, the agar was removed from the slides and pummeled in 0.1% peptone water with a stomacher, serially diluted, surface plated onto nonselective media, and incubated at 35 degrees C. Exposure time, pH, incubation temperature, and culture age affected survival of all pathogens exposed to plasma (P < 0.05). The greatest reduction of pathogens generally occurred during the initial exposure time of 30 or 90 sec. Pathogens incubated for 24 h before exposure were more resistant than those exposed immediately after inoculation. Incubation at 35 degrees C before exposure resulted in greater resistance to plasma inactivation than incubation at 10 degrees C. No appreciable differences between gram-positive and gram-negative pathogens were observed, although the spore-forming B. cereus was more resistant to plasma than non-spore-formers. These findings support the potential for plasma treatment of foods or surfaces for pathogen reduction. Increased sensitivity of pathogens to plasma at reduced pH and temperature is encouraging, since these conditions are applicable to many foods during processing, handling, and storage.

(12)(13)(14)16) and pili (20,25), are posttranslationally modified. The biological significance of these modifications remains unknown. Although thought to be of little significance until recently, posttranslational phosphorylation in bacteria is now recognized as playing a vital role in individual-enzyme regulation (11, 21), as well as in global control mechanisms (5, 9, 23, 24).Tyrosine phosphorylation, although somewhat less common than modifications of serine and threonine, contributes to specific and highly important regulatory events in eukaryotic cells (4, 6). Tyrosine phosphorylation in prokaryotes remains controversial (10); however, evidence demonstrating the presence of tyrosine kinases and phosphorylation of several bacterial proteins at tyrosine has now accumulated (3,8,17).In an initial report, we demonstrated the presence of phosphotyrosine (P-Tyr) in b-type flagellin (15). In research presented here, we have extended these findings (i) to show that P-Tyr is present in a-type flagellins, (ii) to locate and compare distribution of phosphate in phosphopeptides of both flagellin types, and (iii) to substantiate the presence of unmodified tyrosine phosphate. Several lines of evidence presented support the conclusion that P. aeruginosa native flagellar filaments, as well as isolated flagellin, contain unmodified tyrosine phosphate.Isolation and purification of 32P-labeled a-and b-type flagellins. P. aeruginosa strains used in this study were common laboratory strains. The b-type strains used included M-2 and PAO1 (both containing seven tyrosines; Mr, * Corresponding author. 53,000). The a-type strains used were PAK (Mr 43,000; two Tyr), 170018 (Mr, 45,000; seven Tyr), 5940 (Mr 47,000; five Tyr), and 2993 (Mr, 50,000; two Tyr). Bacteria were grown in modified sodium succinate mineral salts medium (MSM) (1, 15). To achieve maximum radiolabeling, the total phosphate concentration was decreased from that in MSM to approximately twofold above growth-limiting levels (0.62 mM phosphate).[32P]phosphoric acid was added at a concentration of either 5 or 10 ,uCi/ml, and the cells were grown at 30°C for 22 h. Labeled flagellar preparations from the a-and b-type cells were isolated by mechanical shearing and differential centrifugation and were further purified by several steps of molecular sieving, as previously described (15). Since cells were not broken, flagellar purification procedures were designed primarily to remove excessive lipopolysaccharide (LPS). Labeling of flagellar protein was then demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The presence of phosphate-labeled b-type flagellins has previously been shown (15). A purified preparation of a representative a-type flagellin (PAK) was separated by electrophoresis in 10% homogeneous gels and stained with Coomassie brilliant blue (Fig. 1A). As expected, the Mr of strain PAK (Fig. 1A, lane 1) was 43,000 to 45,000 (26). The autoradiogram shown in Fig. 1B showed radioactivity associated with a-type flagellin, as previously shown wi...