Response of spheroplasts and chelator-permeabilized cells of gram-negative bacteria to the action of the bacteriocins pediocin SJ-1 and nisin.

We have attempted to bypass the outer membrane (OM) barrier of Escherichia coli and Salmonella typhimurium with pediocin SJ-1 (as compared with nisin) using chelating agents as OM permeabilizers. EDTA, and to less extent EGTA, enabled nisin, but not pediocin SJ-1, to permeate the cell OM of E. coli, to have access to the cytoplasmic membrane and to cause subsequent permeability changes, indicated by an increase in ANS fluorescence intensity and a shift of its emission maximum. Such spectral changes did not occur when, prior to addition, EDTA was saturated with Ca2+ and Mg2+. ANS fluorescence data indicated that, in spite of the fact that pediocin SJ-1 did traverse the EDTA-permeabilized OM of E. coli, it did not cause perturbation of its cytoplasmic membrane and was, therefore, unable to cause cell death. Spheroplasts prepared from E. coli were lysed when treated with nisin but not with pediocin SJ-1. We suggest that the resistance of Gram-negative bacteria to pediocin SJ-1 is due not only to this material's inability to permeate the OM but also (in contrast to nisin) to its inability to interact with the cytoplasmic membrane.

Growth and survival of Listeria monocytogenes in vacuum-packaged ground beef inoculated with Lactobacillus alimentarius FloraCarn L-2.

A culture of the psychotrophic strain FloraCarn L-2 of Lactobacillus alimentarius was added to ground beef (pH 5.4) inoculated with two isolates of Listeria monocytogenes able to grow in refrigerated ground beef. The ground beef was vacuum-packaged and stored for 9 weeks at 4 degrees C. Populations of inoculated L. monocytogenes initially were 6.3 to 6.4 log10 CFU/g and increased to 7.4 log10 CFU/g in ground beef with no added lactobacilli. Addition of L. alimentarius L-2 or its antibiotic-resistant mutant SRL-2 reduced the final populations of L. monocytogenes to 4.3 or 4.1 log10 CFU/g, respectively. L. alimentarius L-2 did not produce bacteriocins or hydrogen peroxide in vitro. The antilisterial effect of L. alimentarius observed in laboratory media and ground beef is attributed to lactic acid (ca. 50 mM) produced by growing cultures.

The susceptibility of chicks to Salmonella montevideo in artificially contaminated poultry feed.

Feed artificially contaminated with various levels of nalidixic-acid-resistant Salmonella montevideo was fed to newly hatched chicks for 7 days. Cloacal and cecal swabs were obtained from the chicks at 7, 14, and 21 days of age to monitor Salmonella colonization relative to the feed contamination level. In one of three trials, less than one Salmonella montevideo per gram of feed was sufficient to establish colonization in 1-to-7-day-old chicks.

Salmonella and other Enterobacteriaceae found in commercial poultry feed.

Poultry feed (mash and pelleted) and meat and bone meal samples were collected from commercial mills. All samples were analyzed for Enterobacteriaceae count (ENT) and Salmonella. The genus and species of the various Enterobacteriaceae present were also determined. The average ENT for mash, pelleted, and meal samples was log 4.1, .8, and 1.8/g, respectively. Enterobacteriaceae were present in 100, 60, and 92% and Salmonella in 58, 0, and 92% of the mash, pelleted, and meal samples, respectively. Overall, the Enterobacteriaceae most frequently isolated from all samples were Enterobacter agglomerans, Enterobacter cloacae, and Klebsiella pneumoniae. Although no Salmonella were found in the pelleted samples, the presence of other Enterobacteriaceae suggests that commercial pelleting may not totally destroy Salmonella since their heat resistance is similar to the other organisms found.

Recovery of inoculated Salmonella from poultry feed containing furazolidone.

It was determined that the presence of furazolidone, a common feed additive, prevented detection of Salmonella in feed samples. Artificially inoculated Salmonella were not recovered from feed samples containing furazolidone when buffered peptone broth (BP) was used as an enrichment medium, but Salmonella were recovered from all feed samples containing furazolidone when thiol broth was used as a substitute for BP.

Fate of Spoilage Microorganisms in Frozen and Chilled Concentrated Tomato Juices 1.

Survival of four potential spoilage organisms was studied in tomato concentrates (22°, 28° and 34° Brix) stored at +7. 0 and -18 C for 4 months. In concentrates stored at -18 C, viable counts of Lactobacillus plantarum remained practically stable whereas those of Leuconostoc mesenteroides declined rapidly during the storage period, its death rate being reduced as the juice concentration was increased. Viable counts of Candida krusei and even more so of Torulopsis holmii , decreased progressively during frozen storage. In concentrates held at 0 C, viable counts of the four test organisms decreased during storage, regardless of the juice concentration. but at +7 C, for three out of the four test organisms, the ability to spoil the concentrates was dependent on the juice concentration.

Incidence of Campylobacter jejuni and Campylobacter coli Serogroups in a Chicken Processing Factory.

Chicken carcasses and water samples were tested for contamination with Campylobacter jejuni or Campylobacter coli at a water-immersion stage in a kosher poultry processing factory. Among the wide variety of serogroups found on carcasses and water samples, only serogroups #2, #11 and #12 ranked among those most frequently isolated in Israel from humans.

Spoilage of Soft Drinks Caused by Bacterial Flocculation 1.

Fruit-flavored soft drinks (pH 3.0) spoiled due to flocculation caused by strains of Acetobacter spp. The floc consisted of bacterial cells attached to cellulose microfibrils. Floc production was inhibited at 4°C; it was not prevented by addition of 200 ppm benzoate, 200 ppm sorbate or 100 ppm sulfur dioxide.

Destruction of coliforms in water and sewage water by dye-sensitized photooxidation.

A new approach to disinfection of water and sewage water, by oxidative destruction of microorganisms by photosensitization, is described. Samples of water and sewage water, to which an inoculum of fecal Escherichia coli had been added, were exposed to solar radiation in the presence of a dye sensitizer, under continuous aeration. The effects of the sensitizer methylene blue at concentrations of 0 to 10 mg/liter, different radiation times from 0 to 2 h, and sunlight intensities of 0, 68, and 2,030 muE/m2 per s were investigated. In laboratory-scale experiments, 1.3 X 10(9) coliforms in 100 ml of oxidation pond municipal sewage water containing 0.5 mg of methylene blue were destroyed in about 30 min. Similar results were obtained with nonchlorinated potable water. These results demonstrate the possibilities available for the disinfection of water and sewage water by this method.

Effect of ascorbic, isoascorbic and dehydroascorbic acids on the growth and survival of Campylobacter jejuni.

Ascorbic acid (AsA), added to nutrient broth at a concentration of 5 mmol/l, was bactericidal towards Campylobacter jejuni grown at 42 degrees C in a micro-aerobic atmosphere. Specific enzymes, radical scavengers, metal chelators and reducing agents were tested as possible antagonists to the cytotoxicity of AsA. The addition of catalase or of the metal chelators ceruloplasmin or Desferal did not prevent the cytotoxic effect of AsA. The addition of the hydroxyl radical scavengers mannitol, formate, histidine or DMSO also failed to counteract the toxicity of AsA. On the other hand, thiourea or cysteamine and the reducing agents cysteine or dithionite significantly increased the recovery of C. jejuni in the presence of AsA. Although the possibility of the involvement of hydroxyl radicals in AsA cytotoxicity cannot be ruled out, it appears that the toxic effect of AsA is due mostly to the formation of products of oxidation of AsA and particularly to dehydroascorbic acid (DHA). Dehydroascorbic acid was also bactericidal to C. jejuni at a concentration of 5 mmol/l. Of all the compounds tested, only cysteamine was effective in preventing (partially) the toxic effect of DHA. The growth of C. jejuni was not inhibited by the addition of 5 mmol/l of isoascorbic acid or sodium isoascorbate.

Effect of free-radical and oxygen scavengers on photochemically generated oxygen toxicity and on the aerotolerance of Campylobacter jejuni.

Exposure of a nutrient agar medium to the combined action of fluorescent light and air produced toxic factors in the medium which affected the growth of Campylobacter jejuni. Sodium dithionite (5-10 mM), a powerful reducing agent, and catalase were effective in counteracting the injurious action of light and air. Among the quenchers of singlet oxygen tested, only histidine had a beneficial effect on the recovery of C. jejuni in the photo-oxidized medium, while the addition of superoxide dismutase, a hydroxyl radical scavenger such as cysteamine, or the free radical antioxidants tocopherol and butylated hydroxy toluene, did not increase the recovery rate of photochemically injured cells. Histidine (40 mM) and dithionite (5-10 mM) also assisted recovery of C. jejuni inoculated on nutrient agar stored in air in the dark. Cysteamine and dithionite were toxic to Campylobacter when added at concentrations of greater than or equal to 10 mM and greater than or equal to 20 mM, respectively. A high inoculum of C. jejuni could not be recovered in unsupplemented nutrient agar incubated in air but was recovered in atmospheres containing 17 or 21% oxygen plus 10% carbon dioxide. The addition of dithionite, catalase or histidine resulted some colony formation on nutrient agar incubated in air. Among the scavengers tested, only dithionite was consistently able to maintain the viability of C. jejuni on nutrient agar stored in air for longer than 4 weeks. In view of the ability of catalase, dithionite and histidine to enhance the aerotolerance of C. jejuni, it is concluded that various oxygen species might be involved in the toxicity of high levels of oxygen.

Antagonistic Compounds Produced by a Chicken Intestinal Strain of Lactobacillus acidophilus.

The production of antagonistic compounds was studied with a strain of Lactobacillus acidophilus isolated from chicken intestinal tract. Accumulation of lactic acid (0.15 mol/l), hydrogen peroxide (280 nmol/h/mg cell dry weight), and a bacteriocin (ca. 10,000 arbitrary activity units per ml) was observed in cultures of this strain. In conditions eliminating the effects of organic acids and hydrogen peroxide, the bacteriocin, designated LA-147, showed inhibitory activity against strains of Lactobacillus leichmannii but not against several other species of Lactobacillus , or other selected gram-positive and gram-negative species. LA-147 (MW ca. 38.5 kDa) was bactericidal against sensitive cells; it was inactivated by heating for 15 min at 100°C, and by the action of protease and alpha-chymotrysin.

Sensitization of Escherichia coli to nisin by maltol and ethyl maltol.

When used separately, 20 mmol l-1 maltol or 1600 AU ml-1 nisin resulted in a 0-0.6 log10 reduction in viable counts of Escherichia coli in a buffer system. However, when added in combination they yielded a 1.8-5.5-log-cycle reduction in viable counts of E. coli at pH 5.0 and 6.8 respectively. It is postulated that maltol (and ethyl maltol) destabilizes the cell outer membrane by chelation of Mg2+ and/or Ca2+, thus permeabilizing the E. coli cell to nisin.

Effect of Ascorbic and Isoascorbic acids on Survival of Campylobacter jejuni in Poultry Meat 1.

Samples of radiation-sterilized mechanically deboned turkey meat were inoculated with a strain of Campylobacter jejuni , stored at 5°C, and viable counts of the test organism determined during a 7-week period. As compared to results obtained with unsupplemented samples, addition of ascorbic acid or sodium isoascorbate (erythorbate) to the meat, at a concentration of 5 mmol/kg, caused an increase in the death rate of C. jejuni . Autooxidation of these compounds, during storage of the meat, supports the view that their toxic effect is mainly due to their oxidation products.

Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents.

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.

Factors that interact with the antibacterial action of thyme essential oil and its active constituents.

The viable counts of Salmonella typhimurium on nutrient agar (NA) decreased upon the addition of either the essential oil of thyme or its constituent thymol, especially under anaerobic conditions. Antagonistic effects of thymol against Staphylococcus aureus were also greater under anaerobic conditions. In contrast to the phenolic constituents of the oil, thymol and carvacrol, the chemically related terpenes p-cymene and gamma-terpinene had no antagonistic effects against Salm. typhimurium. The addition of Desferal to NA counteracted the antibacterial effects of both thyme oil and thymol. No support was obtained, however, for a possible role of iron in the oxygen-related antibacterial action of the thyme oil and thymol or for the observed effect of Desferal. In the presence of thymol, the viable counts of Salm. typhimurium obtained on a minimal medium (MM) were lower than those obtained on NA. Addition of bovine serum albumin (BSA) neutralized the antibacterial action of thymol. It is suggested that the effects of BSA or Desferal are due to their ability to bind phenolic compounds through their amino and hydroxylamine groups, respectively, thus preventing complexation reactions between the oil phenolic constituents and bacterial membrane proteins. This hypothesis is supported by the marked decrease in the viable counts of Salm. typhimurium caused by either thyme oil or thymol when the pH of the medium was changed from 6.5 to 5.5 or the concentration of Tween 80 in the medium was reduced.

Identification of chemical constituents of tomato juice which affect the heat resistance of Lactobacillus fermentum.

Pectins appear to be the main factors in tomato juice which are associated with the protection of Lactobacillus fermentum against destruction or injury by heat. Hydrolysis of pectins by enzymic action makes the cells more vulnerable to heat.

Significance of Psychrotrophic Strains of Serratia liquefaciens in Milk 1.

Enterobacter cloacae , Citrobacter freundii , Serratia liquefaciens and Escherichia coli were the predominant Enterobacteriaceae species isolated from raw milk samples collected from refrigerated bulk tank trucks at the entry of a milk processing plant. About half of the 181 Enterobacteriaceae isolated were psychrotrophs and these included 80% of E. cloacae , 80% of Klebsiella ozaenae and 62% of S. liquefaciens . S. liquefaciens grew in raw and in pasteurized milk at refrigeration temperatures, causing significant pH reduction but only slight lipolytic changes. The significant proteolytic activity of S. liquefaciens in refrigerated milk samples is assumed to have been the cause of the unclean flavor detected.

Purification, partial characterization and plasmid-linkage of pediocin SJ-1, a bacteriocin produced by Pediococcus acidilactici.

Pediococcus acidilactici SJ-1, isolated from a naturally-fermented meat product, produced an antibacterial agent active against selected strains of Lactobacillus spp., Clostridium perfringens and Listeria monocytogenes. The agent was bactericidal against sensitive indicators, and sensitive to proteolytic enzymes; it was identified as a bacteriocin, and was designated as pediocin SJ-1. It was stable over a wide pH range (3-9), and apparently most stable in the lower part of that range. At pH 3.6, pediocin SJ-1 was stable at heat-processing temperatures within the range 65-121 degrees C; its activity decreased significantly, however, when it was heated at pH 7.0. The activity of pediocin SJ-1 on sensitive indicator cells was lost in the presence of alpha-amylase, suggesting that it contains a glyco moiety, necessary for its antibacterial action. Native pediocin SJ-1 exists in the form of monomers and aggregates (with molecular weights in the range 80-150 kDa). Pediocin SJ-1 was purified 262-fold by direct application of cell-free supernatant fluids to a cation-exchange chromatography column, and was resolved by SDS-PAGE as a single peptide band with a MW of ca 4 kDa. The original pediocin SJ-1-producing strain (bac+) harbours three plasmids of 4.6, 23.5, and 45.7 MDa. Production of pediocin SJ-1, but not immunity to SJ-1, is associated with the 4.6 MDa plasmid.

High-performance liquid chromatography of biogenic amines, derivatized with 9-fluorenylmethyl chloroformate.

A procedure was elaborated for the analysis of three biogenic amines posing a considerable health hazard. The method takes advantage of the characteristics of the 9-fluorenylmethyl chloroformate (FMOC) derivative, namely specificity, stability and compatibility for either fluorescence or UV-absorbance detection. The FMOC-tyramine derivative was probably adsorbed to labware when acetone served as the solvent for FMOC. Methanol, substituted for acetone, removed this problem. Excellent linearity was obtained with standard solutions of tyramine, tryptamine and phenylethylamine. Meat samples, spiked with the mentioned amines, also showed good linearity. Perchloric acid was chosen for deproteinization, as potassium perchlorate may be eliminated on neutralization. Histamine failed to react with FMOC or was not detected under the test conditions.