Survival of pathogenic Escherichia coli on basil, lettuce, and spinach.

The contamination of lettuce, spinach and basil with pathogenic E. coli has caused numerous illnesses over the past decade. E. coli O157:H7, E. coli O104:H4 and avian pathogenic E. coli (APECstx- and APECstx+) were inoculated on basil plants and in promix substrate using drip and overhead irrigation. When overhead inoculated with 7 log CFU/ml of each strain, E. coli populations were significantly (P = 0.03) higher on overhead-irrigated plants than on drip-irrigated plants. APECstx-, E. coli O104:H4 and APECstx+ populations were recovered on plants at 3.6, 2.3 and 3.1 log CFU/g at 10 dpi (days post-inoculation), respectively. E. coli O157:H7 was not detected on basil after 4 dpi. The persistence of E. coli O157:H7 and APECstx- were similar when co-inoculated on lettuce and spinach plants. On spinach and lettuce, E. coli O157:H7 and APEC populations declined from 5.7 to 6.1 log CFU/g and 4.5 log CFU/g, to undetectable at 3 dpi and 0.6-1.6 log CFU/g at 7 dpi, respectively. The detection of low populations of APEC and E. coli O104:H4 strains 10 dpi indicates these strains may be more adapted to environmental conditions than E. coli O157:H7. This is the first reported study of E. coli O104:H4 on a produce commodity.

Use of zero-valent iron biosand filters to reduce Escherichia coli O157:H12 in irrigation water applied to spinach plants in a field setting.

AIMS: Zero-valent iron (ZVI) filters may provide an efficient method to mitigate the contamination of produce crops through irrigation water. METHODS: A field-scale system was utilized to evaluate the effectiveness of a biosand filter (S), a biosand filter with ZVI incorporated (ZVI) and a control (C, no treatment) in decontaminating irrigation water. An inoculum of c.8·5log CFU100ml(-1) of Escherichia coli O157:H12 was introduced to all three column treatments in 20-l doses. Filtered waters were subsequently overhead irrigated to 'Tyee' spinach plants. Water, spinach plant and soil samples were obtained on days 0, 1, 4, 6, 8, 10, 13 and 15 and analysed for E. coli O157:H12 populations. RESULTS: ZVI filters inactivated c.6logCFU100ml(-1) E. coli O157:H12 during filtration on day 0, significantly (P<0·05) more than S filter (0·49CFU100ml(-1)) when compared to control on day 0 (8·3log CFU100ml(-1)). On day 0, spinach plants irrigated with ZVI-filtered water had significantly lower E. coli O157 counts (0·13logCFUg(-1)) than spinach irrigated with either S-filtered (4·37logCFUg(-1)) or control (5·23logCFUg(-1)) water. Soils irrigated with ZVI-filtered water contained E. coli O157:H12 populations below the detection limit (2logCFUg(-1)), while those irrigated with S-filtered water (3·56logCFUg(-1)) were significantly lower than those irrigated with control (4·64logCFUg(-1)). CONCLUSIONS: ZVI biosand filters were more effective in reducing E. coli O157:H12 populations in irrigation water than sand filters. SIGNIFICANCE AND IMPACT OF THE STUDY: Zero-valent ion treatment may be a cost-effective mitigation step to help small farmers reduce risk of foodborne E. coli infections associated with contamination of leafy greens.

Fate of Escherichia coli O157:H7 in ground beef following high-pressure processing and freezing.

AIMS: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. METHODS AND RESULTS: Fresh ground beef was inoculated with a five-strain cocktail of E. coli O157:H7 vacuum-packaged, pressure-treated at 400 MPa for 10 min at -5 or 20 degrees C and stored at -20 or 4 degrees C for 5-30 days. A 3-log CFU g(-1) reduction of E. coli O157:H7 in the initial inoculum of 1 x 10(6) CFU g(-1) was observed immediately after pressure treatment at 20 degrees C. During frozen storage, levels of E. coli O157:H7 declined to <1 x 10(2) CFU g(-1) after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55-65 degrees C. CONCLUSIONS: High-pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g(-1) and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. SIGNIFICANCE AND IMPACT OF THE STUDY: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.

Effects of moderately high pressure plus heat on the germination and inactivation of Bacillus cereus spores lacking proteins involved in germination.

AIMS: To determine the germination and inactivation of Bacillus cereus spores lacking various germination proteins using moderately high pressure (MHP) and heat. METHODS: The inactivation and germination of wild-type B. cereus spores in buffer by MHP (150 MPa) at various temperatures, as well as the MHP inactivation and germination of B. cereus spores lacking individual germinant receptors and monovalent cation antiporters, was determined. RESULTS: Loss of individual germinant receptors had no large effects on spore inactivation or germination, although germination of receptor-deficient spores was generally slightly decreased. Loss of the GerN in particular the GerN and GerT antiporters also decreased spore germination by MHP, especially at 40 and 50 degrees C. CONCLUSIONS: Both inactivation and germination of B. cereus spores by MHP increased with rise of temperature; however, mutant strains lacking individual germinant receptor had similar levels of germination as compared to wild-type spores. To evaluate the role of germinant receptors in MHP, a strain lacking a large number of germinant receptors is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this work may lead to a better understanding of how MHP causes germination of spores of B. cereus.

Studies of the release of small molecules during pressure germination of spores of Bacillus subtilis.

AIMS: To measure rates of release of small molecules during pressure germination of Bacillus subtilis spores, and the role of SpoVA proteins in dipicolinic acid (DPA) release. METHODS AND RESULTS: Rates of DPA release during B. subtilis spore germination with pressures of 150 or 500 megaPascals were much higher in spores with elevated levels of SpoVA proteins, and spores with a temperature-sensitive mutation in the spoVA operon were temperature-sensitive in DPA release during pressure germination. Spores also released arginine and glutamic acid, but not AMP, during pressure germination. CONCLUSIONS: Pressure germination of B. subtilis spores causes release of many small molecules including DPA. SpoVA proteins are involved in the release of DPA, perhaps because SpoVA proteins are a component of a DPA channel in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new insight into the mechanism of pressure germination of spores of Bacillus species, a process that has significant potential for usage in the food industry.

Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages.

AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.

Analysis of factors influencing the rate of germination of spores of Bacillus subtilis by very high pressure.

AIMS: To elucidate the factors that determine the rate of germination of Bacillus subtilis spores with very high pressure (VHP) and the mechanism of VHP germination. METHODS AND RESULTS: Spores of B. subtilis were germinated rapidly with a VHP of 500 MPa at 50 degrees C. This VHP germination did not require the spore's nutrient-germinant receptors, as found previously, and did not require diacylglycerylation of membrane proteins. However, the spore's pool of dipicolinic acid (DPA) was essential. Either of the two redundant enzymes that degrade the spore's peptidoglycan cortex, and thus allow completion of spore germination, was essential for completion of VHP germination. However, neither of these enzymes was needed for DPA release triggered by VHP treatment. Completion of spore germination as well as DPA release with VHP had an optimum temperature of approx. 60 degrees C, in contrast to an optimum temperature of 40 degrees C for germination with the moderately high pressure of 150 MPa. The rate of spore germination by VHP decreased approx. fourfold when the sporulation temperature increased from 23 degrees C to 44 degrees C, and decreased twofold when 1 mol l(-1) salt was present in sporulation. However, large variations in levels of unsaturated fatty acids in the spore's inner membranes did not affect rates of VHP germination. Complete germination of spores by VHP was not inhibited significantly by killing of spores with several oxidizing agents, and was not inhibited by ethanol, octanol or o-chlorophenol at concentrations that abolish nutrient germination. Completion of spore germination by VHP was also inhibited by Hg(2+), but this ion did not inhibit DPA release caused by VHP. In contrast, dodecylamine, a surfactant that can trigger spore germination, strongly inhibited DPA release caused by VHP treatment. CONCLUSIONS: VHP does not cause spore germination by acting upon the spore's nutrient-germinant receptors, but by directly causing DPA release. This DPA release then leads to subsequent completion of germination. VHP likely acts on the spore's inner membrane to cause DPA release, targeting either a membrane protein or the membrane itself. However, the precise identity of this target is not yet clear. SIGNIFICANCE AND IMPACT OF THE STUDY: There is significant interest in the use of VHP to eliminate or reduce levels of bacterial spores in foods. As at least partial spore germination by pressure is almost certainly essential for subsequent spore killing, knowledge of factors involved and the mechanism of VHP germination are crucial to the understanding of spore killing by VHP. This work provides new insight into factors that can affect the rate of B. subtilis spore germination by VHP, and into the mechanism of VHP germination itself.

Response of four types of coliphages to high hydrostatic pressure.

Pressure inactivation of four types of coliphages, varphiX 174 (ssDNA virus), MS2 (ssRNA virus), lambda imm434 (dsDNA virus) and T4 (dsDNA virus), was studied to evaluate their potential as human enteric viral surrogates for use in validation of commercial pressure processing treatments. Phage varphiX 174 demonstrated an unexpected high resistance to pressure with no more than 1-log(10) reduction observed following exposures to 350-600 MPa. There was no greater than 1-log(10) reduction below 500 MPa for MS2 in modified phosphate-buffered saline, but a 3.3-log(10) reduction was observed for MS2 pressurized at 600 MPa. Coliphages lambda imm434 and T4 were relatively sensitive to pressure in demonstrating inactivation at 350 MPa. At 21 degrees C, lambda imm434 was inactivated in modified phosphate-buffered saline or Dulbecco's Modified Eagle's Medium plus 5% fetal bovine sera by at least 7.5-log(10) when exposed to 400 MPa for 5 min. Treatment at 450 MPa for 5 min was necessary to obtain a log(10) reduction of 6-7 for T4.

Inactivation of Campylobacter jejuni by high hydrostatic pressure.

AIMS: To investigate the response of Campylobacter jejuni ATCC 35919 and 35921 to high pressure processing (HPP) while suspended in microbiological media and various food systems. METHODS AND RESULTS: Campylobacter jejuni 35919 and 35921 were subjected to 10-min pressure treatments between 100 and 400 MPa at 25 degrees C suspended in Bolton broth, phosphate buffer (0.2 m, pH 7.3), ultra-high temperature (UHT) whole milk, UHT skim milk, soya milk and chicken pureé. The survivability of C. jejuni was further investigated by inoculated pack studies. HPP at 300-325 MPa for 10 min at 25 degrees C was sufficient to reduce viable numbers of both strains to below detectable levels when cells were pressurized in Bolton broth or phosphate buffer. All food products examined offered a protective effect in that an additional 50-75 MPa was required to achieve similar levels of inactivation when compared with broth and buffer. Inoculated pack studies showed that the survivability of C. jejuni following pressurization improved with decreasing post-treatment storage temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: These data demonstrated that HPP at levels of

Bacteriocins and their Food Applications.

Over the last 2 decades, a variety of bacteriocins, produced by bacteria that kill or inhibit the growth of other bacteria, have been identified and characterized biochemically and genetically. This review article focuses on the ecology of bacteriocins, determination of bacteriocin activity, biosynthesis of bacteriocins, and mode of action. Bacteriocin production and modeling are discussed in the article. Nisin is discussed in some detail in this article since it is currently the only purified bacteriocin approved for food use in the U.S. and has been successfully used for several decades as a food preservative in more than 50 countries. For activity spectra and food applications, the review article focuses primarily on class I and class IIa bacteriocins produced by lactic acid bacteria (LAB) given their development as food preservatives.

Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat.

Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp. in a model agar system. The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods. The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media. The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates. A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp. N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp. N1098 in apple juice. The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods. The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores. The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

Potential of an antibacterial ultraviolet-irradiated nylon film.

The antibacterial effectiveness of an ultraviolet-irradiated nylon 6, 6 film was investigated for potential use as a food-packaging material to reduce the surface microbial contamination of foods. The film-surface analyses showed that UV irradiation induced conversion of surface amide groups to amines. Irradiation also increased the dimensional scale of the film surface topography (depth of valleys) approximately 5-fold on the scale of nanometers. The irradiated nylon demonstrated antagonistic activity against Staphylococcus aureus 25923 and Escherichia coli TV1058 with 4.5 and 6 log reductions, respectively, of an initial population of 10(6) cfu mL(-1). The irradiated nylon was ineffective against Pseudomonas fluorescens 13525 and Enterococcus faecalis 19433 under similar conditions. The film demonstrated increased antimicrobial activity against S. aureus 25923 with increasing temperatures up to 45 degrees C, the highest temperature tested. Protein and salt inhibited the antibacterial nature of the irradiated film. Amines in solution (4.31 x 10(-8) M; the calculated equivalent of amines on the film) killed at least 1 x 10(4) cfu mL(-1) E. coli TV1058, and 4. 31 x 10(-7) M amines killed up to 1 x 10(7) cfu mL(-1) E. coli TV1058. The amines in solution required similar exposure time to the bacteria for population reduction as was observed with the irradiated film.

Response of pathogenic Vibrio species to high hydrostatic pressure.

Vibrio parahaemolyticus ATCC 17802, Vibrio vulnificus ATCC 27562, Vibrio cholerae O:1 ATCC 14035, Vibrio cholerae non-O:1 ATCC 14547, Vibrio hollisae ATCC 33564, and Vibrio mimicus ATCC 33653 were treated with 200 to 300 MPa for 5 to 15 min at 25 degrees C. High hydrostatic pressure inactivated all strains of pathogenic Vibrio without triggering a viable but nonculturable (VBNC) state; however, cells already existing in a VBNC state appeared to possess greater pressure resistance.

Viability and enzymatic activity of bifidobacteria in milk.

Bifidobacterium breve NCFB 2258, Bifidobacterium bifidum NCFB 2715, Bifidobacterium longum ATCC 15707, Bifidobacterium angulatum ATCC 27535, and Lactobacillus acidophilus N2 were evaluated for viability and beta-galactosidase and alpha-galactosidase activities under conditions of refrigerated and frozen storage in reconstituted NDM. beta- and alpha-Galactosidase activities were variable. Bifidobacterium angulatum 27535 exhibited much greater enzymatic activities than those of the other strains. All strains demonstrated culture and enzyme stability upon refrigerated storage in fermented and unfermented reconstituted NDM. Bifidobacteria were significantly less tolerant of low temperature storage than was L. acidophilus N2.

Anomalies in mineralization of low concentrations of organic compounds in lake water and sewage.

The rates of mineralization of nitrilotriacetic acid (NTA), 2,4-dichlorophenoxyacetic acid (2,4-D), p-nitrophenol, aniline, and isopropyl N-phenylcarbamate (IPC) at one or more concentrations ranging from 100 pg/ml to 1.0 microgram/ml were proportional to chemical concentrations in samples of three lakes. The rates at 100 pg of NTA, 2,4-D, p-nitrophenol, and aniline per ml in samples of one or more lakes were less than predicted, assuming the rates were linearly related to the concentration. Neither NTA nor 2,4-dichlorophenol at 2.0 ng/ml was mineralized in some lake waters, but higher levels of the two chemicals were converted to CO2 in samples of the same waters. In samples from two lakes, little or no mineralization of IPC or 2,4-D occurred at 1.0 microgram/ml, but 10 ng/ml or lower levels of the herbicides were mineralized. The mineralization in sewage of 1.0 microgram of NTA per ml was biphasic; about 20% of the substrate was mineralized in 20 h, and mineralization was only reinitiated after a period of 130 h. The biphasic transformation was not a result of the accumulation of organic products, and it was still evident if protozoan activity was inhibited. NTA also underwent a biphasic mineralization in lake waters, and the biphasic pattern was not altered by additions of growth factors and inorganic nutrients. From 40 to 60% of the carbon of aniline added to lake water at levels of 100 pg/ml to 1.0 microgram/ml was mineralized, but more than 90% of the carbon of NTA, 2,4-D, or p-nitrophenol added to lake water at 10 ng/ml or 1.0 microgram/ml was mineralized.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of enterotoxin(s) by Staphylococcus hyicus.

Five strains of Staphylococcus hyicus (3 of subspecies hyicus and 2 of chromogenes) were tested serologically for their ability to produce known and unknown enterotoxin(s) and were also examined using monkey bioassays. None of the 5 strains produced any detectable level of the known enterotoxins A--E. However, all of them produced emetic responses in 2 or more of 6 cynomologus monkeys when culture growth was fed intragastrically. One of the 5 strains (S. hyicus subspecies hyicus, VII 76) produced emetic responses in 3 of 6 monkeys with 50 ml of culture growth. The other 4 strains required 1 l of culture broth (concentrated 20-fold) to produce an emetic response in at least 2 of 6 monkeys. Enterotoxin production is, therefore, not unique to S. aureus species. Since some of these organisms do not produce coagulase and thermonuclease, they would be ignored in food hazard evaluation.

Caffeine inhibition of aflatoxin production: mode of action.

Evaluation of caffeine and a number of related methylxanthines indicated that the ability of the compound to inhibit growth and aflatoxin production by Aspergillus parasiticus is highly specific and does not involve an inhibition of cyclic AMP phosphodiesterase. Supplementation of the culture medium with purine bases, nucleosides, and nucleotides suggested that the inhibition of fungal growth could be partially overcome by adenine or guanine but that the purines had little effect on the inhibition of aflatoxin production. Likewise, increasing the levels of trace minerals did not overcome the inhibition of toxin production. Electron microscopic evaluation of caffeine-treated and -untreated cultures indicated that the compound produced observable changes in the ultrastructure of the fungus.

Characterization of staphylococci.

A total of 158 Staphylococcus strains from various sources were characterized by biochemical, physiological, and morphological tests. Numerical taxonomy was applied by using these features. Taxonomic analysis was done with programs run under the MVS-TSO system of the IBM 370 complex and PDP-10 system of the National Institutes of Health. DNA-DNA hybridization with nitrocellulose filters was done to compare selected atypical cultures with American Type Culture Collection reference strains. We found that the use of the nomenclature of Bergey's Manual (8th edition) to identify these strains by species was not adequate. DNA homology values supported the formation of Staphylococcus hyicus subsp. hyicus separate from Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus. The three tests that best separated these strains into four species were (i) tube coagulase (6-h or 24-h porcine plasma or 24-h Difco rabbit plasma), (ii) production of acetoin or acid aerobically from ribose, maltose, or trehalose, and (iii) growth in the presence of novobiocin. Four strains of S. hyicus subsp. hyicus (VII76, VII113, VII131, and VA519) gave typical enterotoxigenic responses in monkey-feeding tests but were negative for enterotoxins A through E, suggesting the presence of one or more new enterotoxins. Two coagulase-negative, heat-stable DNase-positive strains (D143 and ARM) could not be classified by either DNA-DNA hybridization or numerical taxonomy, and D143 was enterotoxigenic as measured by the monkey-feeding bioassay. DNA homology showed that strain FRI-698M was more closely related to S. epidermidis than to S. aureus, yet it produced enterotoxin D. These data suggest the occurrence of coagulase-negative enterotoxigenic strains that are not S. aureus; nonetheless, a positive tube coagulase test and heat-stable DNase test should together be useful for routine screening of most potentially enterotoxigenic staphylococci in foods.

Attenuation of Microbial Growth on Modified Atmosphere-Packaged Fish.

Four species of fish from Atlantic waters, Meronia americanus (perch), Cynoscion regalis (seatrout), Micropogon undulatis (croaker) and Pomatomus saltatrix (bluefish), were processed (gutted or filleted), packaged under carbon dioxide and refrigerated. Stability of the fish under the modified atmosphere preservation (MAP) system was compared to that of fish stored conventionally. Use of the MAP system resulted in a 45 to 55% increase in stability, primarily due to an extension in the lag phase of psychrotrophic organisms and to their reduced growth rate in the logarithmic phase. By the 10th day of storage, the conventionally packed fish always exhibited a 100-fold higher psychrotroph count than the CO2-packed fish. Levels of Vibrio parahaemolyticus were negligible in this MAP system and no Salmonella spp. or Staphylococcus aureus was detected, even at an abuse temperature (10°C) of storage. Positive evidence for preformed Clostridium botulinum toxin was lacking.

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.