Antimicrobial Activity of Cinnamaldehyde, Carvacrol, and Lauric Arginate against Salmonella Tennessee in a Glycerol-Sucrose Model and Peanut Paste at Different Fat Concentrations.

The objective of this study was to investigate the antimicrobial activities of carvacrol, cinnamaldehyde, and lauric arginate (LAE) against Salmonella in a low water activity (aw ) glycerol-sucrose model and in peanut paste with different fat concentrations. Salmonella Tennessee was inoculated into the model and the low fat (<5%) and high fat (50%) peanut paste adjusted to aw 1.0, 0.7, 0.5, and 0.3 and with or without cinnamaldehyde, carvacrol, or LAE. The survival of the bacterium over 3 or 5 days at 25°C was evaluated. Reduced aw alone decreased the viable population over time, with the highest reduction at the lowest aw. In the glycerol-sucrose model, all antimicrobial agents significantly reduced the population over time (P < 0.05) compared with the controls. LAE was more lethal than the essential oil components, reducing the population to undetectable levels by day 2 for all aw. Cinnamaldehyde was more effective than carvacrol at aw 0.5 and 0.3 (2.7- to 2.9-log versus 0.39- to 1.97-log reductions on day 3). In low-fat peanut paste, none of the antimicrobial agents inhibited growth of the pathogen at aw 1.0. However, inactivation was enhanced at reduced aw. Cinnamaldehyde and LAE both reduced the pathogen population to undetectable levels on day 5 at the highest concentration tested (ca. 10 times higher than that in the glycerol-sucrose model). Inactivation efficacy of all antimicrobial agents was greatly decreased but not eliminated in 50% fat peanut paste. Results suggest that the test antimicrobial agents were effective under low aw conditions, but significantly higher concentrations are needed for potential food applications, and fat concentration can negatively impact the efficacy of these antimicrobial agents.

Inactivation of Alicyclobacillus acidoterrestris using high pressure homogenization and dimethyl dicarbonate.

Vegetative cells and spores of five strains of Alicyclobacillus acidoterrestris (N-1100, N-1108, N-1096, SAC, and OS-CAJ) were screened for their sensitivity to high pressure homogenization (HPH, 0 to 300 MPa) in Bacillus acidoterrestris thermophilic broth. The most and least resistant strains, SAC and OS-CAJ, respectively, were further tested for their sensitivity to inactivation or growth inhibition by dimethyl dicarbonate (DMDC, 250 ppm). The combined effects of HPH and DMDC were then evaluated against SAC spores over a 24-h period after treatment. HPH alone significantly inactivated (P < 0.05) vegetative cells of all five strains. SAC vegetative cells were least affected by HPH, with only about a 0.5-log reduction after the 300-MPa treatment. Spores were not significantly reduced by HPH for any of the five strains. DMDC reduced the initial vegetative cell population by 2 log CFU/ml and significantly increased the time to reach stationary phase. For spores, a 0.5-log decrease from the initial spore population was achieved and growth was not significantly delayed. No significant difference was found between the two strains. Treatment with DMDC plus HPH slightly enhanced the inactivation effect over a 24-h period compared with treatment with HPH alone, but these differences were statistically inconsistent. Although HPH and DMDC treatments may help control vegetative cells of A. acidoterrestris, these treatments may not provide adequate overall control. Neither treatment, alone or in combination, is very effective against spores.

Sucrose monolaurate improves the efficacy of sodium hypochlorite against Escherichia coli O157:H7 on spinach.

It is well-recognized that chlorine has limited efficacy when applied to inactivate pathogens on fresh produce. One of the many factors limiting efficacy is the high interfacial tension of chlorine-based sanitizers that limits the access of chlorine to the microorganisms. In this work, we investigated the efficacy of sodium hypochlorite (200 ppm, pH 6.0) at 4 and 20 °C against Escherichia coli O157:H7 inoculated on baby spinach leaves as affected by the surfactant sucrose monolaurate (SML) at below (100 ppm), above (250 ppm), and well above (10,000 ppm) the critical micelle concentration (CMC) of ~200 ppm at 20 °C. The surfactant-containing chlorine treatments were compared to those with buffer only, surfactant only, and chlorine only. Significantly improved inactivation, as evidenced by survival of E. coli O157:H7 was achieved when 250 or 10,000 ppm SML was added with chlorine. This is attributed to the reduction of interfacial tension between the sanitizing solutions and spinach surface. Treatments at 20 °C resulted in greater mean inactivation than those at 4 °C but the difference was not significant. Comparisons of SML concentrations in treatment solutions before and after sanitization showed that SML decreased more at a lower temperature and when chlorine was present, resulting from adsorption of SML onto spinach matrix. Our work illustrates the importance of using surfactants at concentrations above the CMC to enhance the efficacy of chlorine sanitization.

Increased transcription of the phosphate-specific transport system of Escherichia coli O157:H7 after exposure to sodium benzoate.

Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (wt/vol) sodium benzoate. E. coli O157:H7 grown in 150 ml of Luria-Bertani broth was exposed to 0% (control) and 0.5% sodium benzoate. Each treatment was duplicated and sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted and analyzed with E. coli 2.0 Gene Chips. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. The phosphate-specific transport (Pst) system transports inorganic phosphate into bacterial cells, under phosphate-limited conditions. The Pst system was found to be highly upregulated. Increased expression of the Pst system was observed after the short 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were upregulated more than twofold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of Escherichia coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.

Real-time reverse-transcriptase polymerase chain reaction for the rapid detection of Salmonella using invA primers.

Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to help control the spread of disease. Reverse-transcriptase polymerase chain reaction (RT-PCR) can detect the presence of mRNA (shorter half-life than DNA) with greater potential for detecting viable pathogens. The chromosomally located invA gene required for host invasion by Salmonella is widely used for detection of this pathogen by PCR. Detection of Salmonella was undertaken by real-time RT-PCR (rt-RT-PCR) using newly designed invA gene primers to develop a sensitive and specific assay. Salmonella serovars Typhimurium and Enteritidis were grown (7.68 log(10) CFU/mL) in Luria-Bertani broth overnight at 37 degrees C, and RNA was extracted, followed by rt-RT-PCR with and without SYBR green I and agarose gel electrophoresis. All experiments were replicated at least thrice. Detection for both serovars using traditional RT-PCR was lower ( approximately 10(5) CFU/mL) than rt-RT-PCR (10(3) CFU/mL) by gel electrophoresis. Melt curve analysis showed melt temperatures at 87.5 degrees C with Ct values from 12 to 15 for up to 10(3) CFU/mL and improved to 10(2) CFU/mL after further optimization. Further, addition of RNA internal amplification control constructed using in vitro transcription with a T7 RNA polymerase promoter, to the RT-PCR assay also gave detection limits of 10(2) CFU/mL. Cross-reactivity was not observed against a panel of 21 non-Salmonella bacteria. Heat-inactivated (autoclaved) Salmonella showed faint or no detection by rt-RT-PCR or gel electrophoresis. This method has potential to be applied for the detection of Salmonella serovars in fresh produce and the simultaneous detection of foodborne viral (RNA viruses) and bacterial pathogens in a multiplex format.

Transcription analysis of stx1, marA, and eaeA genes in Escherichia coli O157:H7 treated with sodium benzoate.

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37 degrees C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate-treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

Nonthermal inactivation of Escherichia coli K-12 on spinach leaves, using dense phase carbon dioxide.

While the use of some chemical sanitizers is approved for inactivation of microbes on the surfaces of fruits and vegetables, these compounds often degrade product quality with limited improvement in product safety. The application of dense phase carbon dioxide (DPCD, or high-pressure CO2) is a nonthermal process for inactivation of foodborne pathogens inoculated into various juices and model solutions. In this work, DPCD was evaluated for its potential to inactivate Escherichia coli K-12 inoculated on fresh spinach leaves. Inoculated leaves were exposed for up to 40 min to DPCD at a subcritical condition (5 MPa, 40 degrees C) and two supercritical conditions (7.5 and 10 MPa, 40 degrees C) at a flow rate of 50 g of CO2/min. E. coli K-12 populations were reduced to nondetectable levels (approximately 5-log reduction) using supercritical treatment conditions at exposure times as short as 10 min; efficacy of DPCD at the subcritical state was limited. This research demonstrates that DPCD has potential as a pasteurization technology for application to leafy green vegetables, although issues with discoloration and other quality measures will need more extensive evaluations.

Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces.

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.

Inactivation of foodborne pathogens using a one atmosphere uniform glow discharge plasma.

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.

Survival of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice as affected by ozone and treatment temperature.

Inactivation of Escherichia coli O157:H7 and Salmonella in apple cider and orange juice treated with ozone was evaluated. A five-strain mixture of E. coli O157:H7 or a five-serovar mixture of Salmonella was inoculated (7 log CFU/ml) into apple cider and orange juice. Ozone (0.9 g/h) was pumped into juices maintained at 4 degrees C, ambient temperature (approximately 20 degrees C), and 50 degrees C for up to 240 min, depending on organism, juice, and treatment temperature. Samples were withdrawn, diluted in 0.1% peptone water, and surface plated onto recovery media. Recovery of E. coli O157:H7 was compared on tryptic soy agar (TSA), sorbitol MacConkey agar, hemorrhagic coli agar, and modified eosin methylene blue agar; recovery of Salmonella was compared on TSA, bismuth sulfite agar, and xylose lysine tergitol 4 (XLT4) agar. After treatment at 50 degrees C, E. coli O157:H7 populations were undetectable (limit of 1.0 log CFU/ml; a minimum 6.0-log CFU/ml reduction) after 45 min in apple cider and 75 min in orange juice. At 50 degrees C, Salmonella was reduced by 4.8 log CFU/ml (apple cider) and was undetectable in orange juice after 15 min. E. coli O157:H7 at 4 degrees C was reduced by 4.8 log CFU/ml in apple cider and by 5.4 log CFU/ml in orange juice. Salmonella was reduced by 4.5 log CFU/ml (apple cider) and 4.2 log CFU/ml (orange juice) at 4 degrees C. Treatment at ambient temperature resulted in population reductions of less than 5.0 log CFU/ml. Recovery of E. coli O157:H7 and Salmonella on selective media was substantially lower than recovery on TSA, indicating development of sublethal injury. Ozone treatment of apple cider and orange juice at 4 degrees C or in combination with mild heating (50 degrees C) may provide an alternative to thermal pasteurization for reduction of E. coli O157:H7 and Salmonella in apple cider and orange juice.

Inactivation of Escherichia coli O157:H7 by high-intensity ultrasonication in the presence of salts.

Inactivation of Escherichia coli O157:H7 suspended in salt solutions (Na(2)PO(4), NaCl, NaNO(3), NH(4)Cl, CaCl(2), and AlCl(3)) at concentrations ranging from 0 to 5% (w/v) by high-intensity ultrasound at two different temperature conditions (ice water bath, 40 degrees C water bath) and three ultrasonic intensity levels (9.5, 21.8, 49.2 W/cm(2)) was determined. Increases in sonication treatment time, intensity, and temperature led to increased lethality of E. coli O157:H7. However, cell lethality, as a function of solute concentration, varied depending upon type of salt. CaCl(2), NaCl, NaNO(3), Na(2)PO(4), and NH(4)Cl had little or no effect on survival of E. coli O157:H7 (<1-log reduction) regardless of other treatment conditions at 9.5 W/cm(2). Concentrations of >0.5% of CaCl(2), NaNO(3), Na(2)PO(4), and NH(4)Cl adversely affected survival (1.0- to 1.6-log reduction) with treatment of 21.8 W/cm(2) in an ice bath and showed greater inactivation (1.2-4.0-log reduction) in the 40 degrees C water bath. Treatment of 49.2 W/cm(2) showed the greatest impact regardless of other parameters. In the 40 degrees C water bath, treatment for 10 min at 49.22 W/cm(2) led to total inactivation for cells suspended in 0.5% NH(4)Cl, 1% CaCl(2), 2% NaCl, 5% Na(2)PO(4), and all concentrations of AlCl(3). Complete inactivation also occurred in an ice bath for 5% AlCl(3), which was shown to be the most effective salt. The most efficient treatment combination was in the 40 degrees C water bath for 10 min at 49.2 W/cm(2) with 5% AlCl(3). Inactivation of E. coli O157:H7 is attributed to cavitation-induced shear forces, reaction of cavitation-generated hydrogen peroxide with microbial cell wall constituents and electrostatic interactions of dissociated salts with cell membranes.

Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices.

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

Survival of Escherichia coli O157:H7 during Fermentation of Apple Cider.

Survival of Escherichia coli O157:H7 in fermenting and nonfermenting fresh apple cider was determined. Populations of E. coli O157:H7 were reduced from 6.4 log CFU/ml to undetectable levels (detection limit of 0.5 log CFU/ml) in fermenting cider after 3 days at 20°C and from 6.5 log CFU/ml to 2.9 log CFU/m1 after 10 days at 20°C in nonfermenting cider. After 1 day of incubation, recovery of E. coli O157:H7 from fermenting and nonfermenting cider was statistically (P < 0.01) lower on sorbitol MacConkey agar than on tryptone soya agar supplemented with cycloheximide. These results suggest that substantial portions of the surviving E. coli O157:H7 populations were sublethally injured by cider components (i.e., acid and ethanol). The pH of fermenting cider was not significantly different (P > 0.05) from that of nonfermenting cider throughout the 10-day test period. Final ethanol concentrations in fermenting cider reached 6.01% (vol/vol) after 10 days at 20°C. Inactivation of E. coli O157:H7 in fermenting cider is attributed to the combined effects of pH and ethanol. Results of this study indicate that E. coli O157:H7 is capable of survival in fresh apple cider at 20°C, while alcoholic fermentation of fresh cider is an effective means of destroying this pathogen.

Growth of Salmonella spp. in Cantaloupe, Watermelon, and Honeydew Melons.

The ability of Salmonella spp. to grow on the interior tissues of cantaloupe, watermelon, and honeydew melons was investigated. Pieces of rind-free melons (pH 5.90-6.67) and tryptic soy broth (TSB, pH 5.90) were inoculated with a mixed culture (approximately 100 CFU/g or ml) containing equal proportions of five species of Salmonella ( S. anatum , S. Chester , S. havana , S. poona , and S. senftenberg ). Inoculated melon pieces and TSB were incubated for 24 h at 5 or 23°C. Viable populations of salmonellae were determined by surface plating test portions on Hektoen enteric agar. Results indicated that Salmonella growth was rapid and prolific on the melons and in TSB at 23°C incubation. Final populations on watermelons were approximately 1.0 log10 greater than populations on cantaloupe and honeydew and in TSB. Although viable Salmonella populations on melons and in TSB did not increase during the 24-h incubation at 5°C, little or no decrease in viable populations was observed.