The effectiveness of treating irrigation water using ultraviolet radiation or sulphuric acid fertilizer for reducing generic Escherichia coli on fresh produce-a controlled intervention trial.

AIMS: The aims of this study were to: (i) estimate the effectiveness of ultraviolet radiation (UV) and sulphuric acid-based fertilizer (SA), at reducing levels of generic Escherichia coli in surface irrigation water and on produce and surface soil in open produce fields; and (ii) describe the population dynamics of generic E. coli in produce fields. METHODS AND RESULTS: Spinach and cantaloupe plots were randomly assigned to control, UV or SA treatment groups. Irrigation water was inoculated with Rifampicin-resistant E. coli prior to treatment. More than 75% of UV- and SA-treated tank water samples had counts below the detection limit, compared to a mean count of 3·3 Log10 CFU per ml before treatment. Levels of Rifampicin-resistant E. coli in soil and produce both increased and decreased over 10-15 days after irrigation, depending on the plot and time-period. CONCLUSIONS: UV and SA treatments effectively reduce the levels of E. coli in surface irrigation water. Their effectiveness at reducing contamination on produce was dependent on environmental conditions. Applying wait-times after irrigation and prior to harvest is not a reliable means of mitigating against contaminated produce. SIGNIFICANCE AND IMPACT OF THE STUDY: The results are of timely importance for the agricultural industry as new FSMA guidelines require producers to demonstrate a low microbial load in irrigation water or allow producers to apply a wait-time to mitigate the risk of contaminated produce.

Short communication: Homologous expression of recombinant and native thurincin H in an engineered natural producer.

The Bacillus bacteriocin thurincin H exhibits a wide inhibitory spectrum of activity against various foodborne pathogens, such as Listeria monocytogenes, and dairy spoilage bacteria, especially different Bacillus species commonly existing in dairy products. Previously, we constructed 3 plasmids to express native thurincin H homologously in an engineered natural producer, Bacillus thuringiensis SF361thnH(-). This host is deficient in thurincin H production because of an in-frame deletion of structural genes thnA1, thnA2, and thnA3 from the chromosome of the natural producer B. thuringiensis SF361. The previously constructed expression vectors were constructed by cloning the native thurincin H promoter, 3 (or 1) copies of structural genes, and the native (or Cry protein) terminator into an Escherichia coli-B. thuringiensis shuttle vector pHT315. In this study, 3 corresponding expression vectors (pGW134, pGW135, and pGW136) were constructed to express recombinant thurincin H-His6 in the same host, in which a 6-histidine tag was fused to the C terminus of each structural gene. The resulting low level of bacteriocin production indicated that the His tag might negatively interfere with subsequent posttranslational modification or exportation processes after the thurincin H-His6 prepeptide was translated. Additionally, in order to overexpress native thurincin H, 2 additional plasmids (pGW137 and pGW138) were constructed, consisting of the sporulation-dependent Cry protein dual promoter BtI and BtII, the thnA1 structural gene, and the thurincin H native or Cry protein terminator. However, production was low on Luria broth plates and absent on sporulation plates. It is possible that the resulting thurincin H prepeptide was not correctly modified or exported to the extracellular environment, due to the undesired biochemical and physiological changes during the sporulation phase.

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

Shelf-life evaluation of natural antimicrobials for Concord and Niagara grape juices.

This study was conducted to evaluate the effectiveness of natural antimicrobials for shelf-life extension of cold-filled still and carbonated Concord and Niagara grape juices, which have traditionally been preserved with chemical preservatives. Commercial juices were inoculated with a spoilage yeast cocktail of Dekkera, Kluveromyces, Brettanomyces, and Zygosaccharomyces at 10(2) and 10(4) CFU/ml. The following agents were added to still juices: no preservative (negative control), 0.05% potassium sorbate plus 0.05% sodium benzoate (positive control), 0.1 or 0.2% cultured dextrose, 250 ppm of dimethyldicarbonate (DMDC), 10 or 20 ppm of natamycin, and 250 ppm of DMDC plus 5 or 10 ppm of natamycin. Carbonated juice was treated with the negative control, positive control, and 250 ppm of DMDC plus 10 ppm of natamycin. Microbial stability of samples was assessed every 2 weeks during 6 months of storage at 21°C by yeast enumeration and measurement of turbidity, pH, and °Brix. Juices were deemed spoiled when yeast counts exceeded 10(6) CFU/ml. Cultured dextrose was not effective at levels tested in both types of juice. The most promising results were obtained with DMDC and natamycin combination treatments in still Niagara juice and in carbonated Concord and Niagara juices. In these treatments, shelf-life extension similar to that of the positive control (153 to 161 days) was achieved while maintaining similar turbidity, pH, and °Brix. Spoiled juices had lower pH and °Brix values and higher turbidity due to microbial activity and increased in microbial levels.

Inactivation of different strains of Escherichia coli O157:H7 in various apple ciders treated with dimethyl dicarbonate (DMDC) and sulfur dioxide (SO2) as an alternative method.

Escherichia coli has been identified as the causative agent in numerous foodborne illness outbreaks associated with the consumption of fresh apple cider. Apple cider has a pH which is normally below 4.0 and would not be considered a medium capable of supporting the growth of foodborne pathogens. The association of unpasteurized apple cider with foodborne illness due to E. coli O157:H7 has however, led to increased interest in potential alternative methods to produce pathogen free cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6)-10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895) and tested to determine the effectiveness of sulfur dioxide (SO(2)) and dimethyl dicarbonate (DMDC). Bacterial populations for treated and untreated samples were then enumerated by using non-selective media. Eight different ciders were treated with DMDC (125 and 250 ppm) and SO(2) (25, 50, 75, 100 ppm). Greater than a 5-log reduction was achieved at room temperature with 250 ppm of DMDC and 50 ppm of SO(2) after the incubation time of 6h and 24h, respectively. Addition of DMDC and/or SO(2) may offer an inexpensive alternative to thermal pasteurization for the production of safe apple cider for small apple cider producers.

Purification and structural characterization of bacillomycin F produced by a bacterial honey isolate active against Byssochlamys fulva H25.

AIMS: Isolate and characterize antifungal peptides exhibiting activity against Byssochlamys fulva H25, a spoilage mould associated with juices and beverages. METHODS AND RESULTS: A bacterium (H215) isolated from honey showed high antifungal activity against B. fulva H25. The antifungal producer strain was identified as Bacillus subtilis using 16S rDNA sequencing. The antifungal peptide was purified by 20% ammonium sulfate precipitation of the bacterial culture supernatant, followed by Octyl-Sepharose CL-4B and reverse phase-high performance liquid chromatography. The five active fractions were lyophilized and subjected to mass, tandem mass spectrometry and amino acid analysis to deduce their corresponding molecular masses and structural characteristics. The five peaks were determined to be identical to bacillomycin F, varying in the length of the fatty acid chain moiety from C14 to C16. CONCLUSIONS: The broad-spectrum antifungal activity produced by a bacterium from honey was determined to be due to the production of bacillomycin F. SIGNIFICANCE AND IMPACT OF THE STUDY: The antifungal compound produced by a bacterial strain isolated from honey was determined to be stable over a broad pH range and was stable to heat treatments up to 100 degrees C. This is the first report of honey microflora producing bacillomycin F or any antifungal compound.

Efficacy of UV light for the reduction of Listeria monocytogenes in goat's milk.

Certain types of goat's cheeses are produced using unpasteurized milk, which increases the food safety concerns for these types of products. Popularity and consumption of goat's milk products have increased, and the niche market includes gourmet goat's cheeses. The U.S. Code of Federal Regulations and the Pasteurized Milk Ordinance both address the possibility for processing alternatives to heat treatment, and the use of UV light treatment may be a viable alternative that still ensures the safety of the product. Fresh goat's milk was inoculated with Listeria monocytogenes (L-2289) at 10(7) CFU/ml and exposed to UV light using the CiderSure 3500 apparatus (FPE Inc., Macedon, NY). Inoculated milk was exposed to a UV dose range between 0 and 20 mJ/cm2 to determine the optimal UV dose. A greater than 5-log reduction was achieved (P < 0.0001) when the milk received a cumulative UV dose of 15.8 +/- 1.6 mJ/cm2. The results of this study indicate that UV irradiation could be used for the reduction of L. monocytogenes in goat's milk.

Influence of apple cultivars on inactivation of different strains of Escherichia coli O157:H7 in apple cider by UV irradiation.

This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10(6) to 10(7) CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm(2). Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (alpha < or = 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (alpha < or = 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of (o)Brix, pH, and malic acid content failed to show any statistically significant relationship (R(2) > or = 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested.

Modeling of Escherichia coli inactivation by UV irradiation at different pH values in apple cider.

This study examined the effects and interactions of UV light dose (1,800 to 20,331 microJ/cm2) and apple cider pH (2.99 to 4.41) on the inactivation of Escherichia coli ATCC 25922, a surrogate for E. coli O157:H7. A predictive model was developed to relate the log reduction factor of E. coli ATCC 25922 to the UV dose. Bacterial populations for treated and untreated samples were enumerated with the use of nonselective media. The results revealed that UV dose was highly significant in the inactivation of E. coli, whereas pH showed no significant effect at higher UV doses. Doses of 6,500 microJ/cm2 or more were sufficient to achieve a greater than 5-log reduction of E. coli. Experimental inactivation data were fitted adequately by a logistic regression model. UV irradiation is an attractive alternative to conventional methods for reducing bacteria in unpasteurized apple cider.

Inactivation of Cryptosporidium parvum oocysts in fresh apple cider by UV irradiation.

This study evaluated the efficacy of UV irradiation on the inactivation of Cryptosporidium parvum oocysts in fresh apple cider. Cider was inoculated with oocysts and exposed to 14.32 mJ of UV irradiation/cm(2). Oocyst viability was assessed with the gamma interferon gene knockout (GKO) mouse and infant BALB/cByJ mouse models. All GKO mice challenged with UV-treated cider demonstrated no morbidity or mortality, and infant BALB/c mice challenged with treated cider were negative for the presence of C. parvum. In contrast, the GKO mice challenged with non-UV-treated inoculated cider died and the parasite was detected in the ileums of all challenged infant mice. This study shows that UV irradiation can be used to inactivate C. parvum in fresh apple cider.

Heat resistance of Escherichia coli O157:H7 in apple juice.

The objective was to determine the effect of cider composition on the heat resistance of Escherichia coli O157:H7. The average D52 value in a model Empire apple juice was 18 min with a z value of 4.8 degrees C. Increasing the Brix from 11.8 to 16.5 degrees had no effect on thermal resistance, while increasing L-malic acid from 0.2 to 0.8%, or reducing the pH from 4.4 to 3.6 sensitized the cells to heat. The greatest effect on heat resistance was afforded by the preservatives benzoic and sorbic acids: D50 values in ciders containing 1,000 mg/l were 5.2 min in the presence of sorbic acid and only 0.64 min in the presence of benzoic acid. Commercial apple juice concentrates yielded lower numbers of survivors than single-strength juices even though their higher sugar concentrations of about 46 degrees Brix increased heat resistance.

Growth Characteristics of Aciduric Sporeforming Bacilli Isolated from Fruit Juices.

Two aciduric, aerobic, sporeforming bacteria were isolated from pasteurized juices. The gram-positive, catalase-positive rods produced spores that were located subterminally in a swollen sporangium. The cultures had an optimal pH of 3.5-4.0 for growth and preferred potato dextrose agar over many of the rich media usually used for cultivating sporeforming bacteria. Spore inocula grew well in apple juice and white grape juice. Red grape juice was inhibitory, perhaps because of the concentrations of certain phenolic compounds. The spores were sufficiently heat resistant (D90 values of 16 to 23 min and z-values of 7.2 to 7.7°C) to survive commercial pasteurization processes.

Detection of Viable Ascospores of Neosartorya.

Studies on 29 Neosartorya cultures showed that many ascospores, as enumerated in a haemocytometer, failed to produce colonies when cultured on agar media. Although the percent recoveries differed greatly between spore crops, germination and outgrowth of most were stimulated by heat activation in grape juice for 30-60 min at 70°C. Most probable number and CFU counts were usually similar which indicated that low recoveries were not due to an inadequate incubation period or to the production of self-inhibitory compounds. Low percent recoveries did not affect the D values obtained in heat-resistance trials.

Effect of Processing Conditions on the Microflora of Fresh-Cut Vegetables.

Surveys were made of commercial processing lines used to prepare fresh-cut vegetables such as chopped salad ingredients, carrot sticks, and cauliflower florets. Washing and chlorinated water dips only partially removed the microorganisms that were intrinsic to the vegetables. Major sources of in-plant contamination were the shredders used to prepare chopped lettuce and coleslaw. Gram-negative rods were the predominant microflora with species of Pseudomonas being most numerous; many were psychrotrophic. Only low numbers of lactic acid bacteria and fungi were recovered.

Effect of Low Concentrations of Sorbic Acid on the Heat Resistance and Viable Recovery of Neosartorya fischeri Ascospores.

Concentrations of sorbic acid as high as 1000 mg/L had little effect on the heat resistance of ascospores. Growth of surviving spores that had been exposed to a lethal temperature, however, was greatly inhibited by concentrations as low as 70 mg/L. The results suggest that spoilage of thermally processed fruit products can be prevented by the incorporation of low amounts of sorbic acid.

Normal liver chromatin contains a firmly bound and larger protein related to the principal cytosolic target polypeptide of a hepatic carcinogen.

A 14,000-dalton polypeptide was previously reported to be the principal protein target of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene) in liver cytosol at the start of hepatocarcinogenesis in rats. The 14,000-dalton polypeptide was purified to homogeneity according to gel electrophoreses in both NaDodSO4-containing medium and acetic acid/urea and also by immunogenicity. An immunologically related form of the cytosolic target polypeptide has now been found to be present in the nuclei of normal rat liver as a 17,500-dalton polypeptide that is firmly and ionically bound to chromatin. Serial salt extractions of isolated liver nuclei or chromatin at 0.15 and 0.35 ionic strengths fail to dissolve the bound polypeptide, according to electrophoretic transfer immunoblot analyses. Most of the 17,500-dalton polypeptide is extracted at 0.65 ionic strength, the remainder at 1.2, and none at 2.0, nor thereafter in 8 M urea. In addition, short-term digestion of purified liver nuclei with micrococcal nuclease solubilizes the 17,500-dalton polypeptide. All three protocols also solubilize low levels of intermediate 17,500- to 14,000-dalton species, the latter size being the same as that of the cytosolic protein target of the carcinogen. The presence of protease inhibitors during the isolations and extractions of the nuclei and chromatin reduces the amounts of these smaller polypeptides. In normal rat liver only nuclei and cytoplasm of hepatocytes contain reactive antigen according to peroxidase-antiperoxidase immunohistochemistry, staining most intensely perilobularly, less in the lobular midzone, and least centrilobularly. The nuclei of the perilobular hepatocytes constitute the strongest staining compartment within all of normal liver. Of 22 nonhepatic tissues of normal rats, 16 contain relatively few cells with immunoreactive cytoplasm. Nonhepatic nuclear antigen is present only in villar crest cells of duodenum (which are normally exposed to liver bile), also having cytoplasmic antigen as well. Five kinds of evidence appear to connect the chromatin-bound 17,500-dalton polypeptide of normal liver nuclei to the cytosolic 14,000-dalton polypeptide that is the principal target of the carcinogen early during hepatocarcinogenesis in rats. The present findings indicate a direct connection between a chromosomal protein and the immediate principal cytosolic protein target of a carcinogen.