Samuel A. Palumbo

Construction and Characterization of Escherichia coli O157:H7 Strains Expressing Firefly Luciferase and Green Fluorescent Protein and Their Use in Survival Studies‡

The firefly ( Photinus pyralis ) luciferase (luc) gene on plasmid vector pBESTluc and the Aequorea victoria green fluorescent protein (gfp) gene on plasmid vector pGFP were introduced into strains of Escherichia coli O157:H7. The recombinant E. coli strains were indistinguishable from their parent strains in biochemical and immunological assays and in a multiplex PCR reaction. There was no significant difference in the growth kinetics of the luc-bearing recombinants and the parent strains. At 37°C all of the recombinant strains maintained the vectors and expressed luciferase and the green fluorescent protein when grown both with and without antibiotic selection. Individual colonies of luc-bearing E. coli strains were readily luminescent in the dark after being sprayed with a solution of 1 mM beetle luciferin. The recombinants containing pGFP emitted bright green fluorescence when excited with UV light and the addition of any other proteins, substrates, or cofactors was not required. The green fluorescent protein-expressing E. coli O157:H7 strains were used in studies examining the survival of the organism in apple cider and in orange juice. In apple cider the organism declined to undetectable levels in 24 days at refrigeration temperature while in orange juice the strains survived with only small decreases in number during the 24-day sampling period. These recombinant E. coli O157:H7 strains, containing readily identifiable and stable markers, could be useful as positive controls in microbial assays as well as in studies monitoring bacterial survival and the behavior of E. coli O157:H7 in foods and in a food processing environment.

Thermal destruction ofListeria monocytogenes in liver sausage slurry

Abstract Thermal destruction ofListeria monocytogenes was determined in a liver sausage slurry (1:1, liver sausage batter and water) using a submerged ampule technique.D-values forL. monocytogenes Scott A grown at 37°C were 8·9 min at 57·2°C, 2·4 min at 60·0°C, and 1·1 min at 62·8°C (Z=6·2°C) based on analysis of the linear portion of the survivor curves.D-values of 6·6, 1·6, and 0·4 min (Z=4·65°C) were obtained when the data were analyzed using non-linear techniques.L. monocytogenes strain V7 (D60=1.0min) was more thermosensitive than Scott A (D60=1·6min) or HO-VJ-S (D60=1·6min). When Scott A was grown at 19°C, there was a decrease in thermal resistance (D60=0·8min). These data indicate thatL. monocytogenes has a thermal resistance in liver sausage comparable to that observed in other food systems.

Model for the aerobic growth of Aeromonas hydrophila K144

The combined effects of temperature (5 to 42°C), NaCl (0.5 to 4.5%), pH (5.3 to 7.3), and NaN02 (0 to 200 μg/ml) on the aerobic growth of Aeromonas hydrophila K144 were studied in brain heart infusion (BHI) broth using a modified central composite design. Variable combinations were tested in triplicate aerobic flasks; viable cell counts were made at intervals during incubation by surface plating on tryptic soy agar. Growth curves were generated using the Gompertz equation in conjunction with a nonlinear iterative regression analysis. Values for the four Gompertz parameters (A, C, B, and M) were obtained for the variable combinations tested. Using response surface techniques, quadratic and cubic equations containing the four variables of temperature, pH, NaCl, and NaN02 were developed to yield predictive values for the B and M Gompertz values. Goodness of fit evaluation of the models was by R2 values. Comparison of predicted and observed values of B and M and evaluation of predicted lag times and generation times indicated that the quadratic model gave a better fit. Overall, the variable combinations interacted to decrease the generation time and increase the lag time. The results indicate that pH, salt, and nitrite can decrease the growth of A. hydrophila when combined with low temperature incubation.

Use of Starter Cultures in Meats

Use of starter cultures in meat products is reviewed, with emphasis on the types of microorganisms employed for production of various products, and the effect of starter cultures on food safety. Desirable starter culture characteristics are identified, and the effect of fermentation on the nutritive quality of meats is considered. Food safety aspects of starter culture use discussed include the effects on survival of viruses, trichinae, and pathogenic bacteria, and on the control of mycotoxin, nitrosamine, and pressor amine contamination.

Effect of Food Environment on Staphylococcal Enterotoxin Synthesis: A Review

Effects of various nutritional and environmental factors on growth and enterotoxin synthesis by Staphylococcus aureus in model systems and foods are reviewed. Factors discussed include effects of inoculum size, competing microflora, gaseous atmosphere, carbon source, temperature, pH, sodium chloride, water activity, mineral ions and sublethal stress. Areas where additional research is needed are also discussed.

Minimum and Maximum Temperatures for Growth and Verotoxin Production by Hemorrhagic Strains of Escherichia coli

The influence of temperature on growth and verotoxin production by Escherichia coli strains was studied in brain heart infusion (BHI) broth both in shake cultures at various temperatures and in a temperature-gradient incubator. All strains of E. coli surveyed grew from at least 10 to 45°C, with some strains growing at 8° C. Verotoxin production (determined using the Vero cell-assay system) was a function of both temperature and time, with the highest titers produced at temperatures supporting the fastest growth (based on days to visible turbidity) and highest viable cell counts. However, for strains producing verotoxin, toxin production was detected at any temperature supporting growth. Three strains (of 16 tested) increased 1000-fold in viable count in 4 to 6 days at 10°C. The data presented here indicate that most E. coli strains surveyed can easily grow at ca. 10°C and thus suggest the potential for growth in temperature-abused refrigerated foods.

Thermal resistance of Salmonella spp. and Listeria monocytogenes in liquid egg yolk and egg yolk products

The effectiveness of various pasteurization procedures in destroying Listeria monocytogenes and Salmonella enteritidis in liquid egg products was evaluated. Survivor studies were perfonned on individual strains of L. monocytogenes and L. innocua in commercially broken raw egg yolk samples after heating at 61.1, 63.3, and 64.4°C using submerged vials, and on Salmonella spp. at 60.0, 61.1, and 62.2°C. Surviving bacteria were enumerated on TSA and results expressed as D-values. The influence of aw -lowering ingredients such as salt and sugar on thermal resistance in yolk was investigated using a five-strain mixture of L. monocytogenes or a mixture of Salmonella spp. (four strains of S. enteritidis , one stain each of S. senftenberg and S. typhimurium ) at 61.1°C to 66.7°C. At 61.1°C (present minimum temperature for pasteurization of plain egg yolk), a 7-log-unit reduction of Salmonella took 1.4 to 2.4 min, whereas a 7-log-unit reduction of L. monocytogenes took 4.9 to 16.1 min. The D-value for L. monocytogenes at 64.4°C increased from 0.44 min in plain yolk to 8.26 min after a 21.5-min lag (total time to achieve 1-log-unit reduction was 30.7 min) in yolk with 10% salt and 5% sugar, and 27.3 min after a 10.5-min lag (total time 37.8 min for 1-log-unit reduction) in yolk with 20% salt. The D-value for Salmonella in egg yolk at 64.4°C was < 0.2 min, but when 10% salt was added, the D-value was 6.4 min. Aw -lowering solutes in liquid egg yolk increased the thermal resistance of Salmonella and L. monocytogenes .

Resistance ofListeria monocytogenes to freezing in foods

Abstract The ability of L. monocytogenes to survive freezing and frozen storage at −18°C was studied in ground beef, ground turkey, frankfurters, canned corn, ice-cream mix, and tomato soup. Injury of L. monocytogenes as a result of freezing and frozen storage, as well as the ability of various Listeria -selective media to quantitatively recover the organism after freezing was also investigated. The responses of the organism, i.e. survival, injury, and quantitative on selective media, were related to the pH (acidity) of the food. Five of the examined foods had pH values of 5·8 or above, while tomato soup had a pH of 4·74. L. monocytogenes survived freezing and frozen storage well in five of the examined foods, was not injured, and was quantitatively recovered on Listeria -selective media. In contrast, the organism showed a decline in viable count after extended frozen storage in tomato soup, was injured, and could not be quantitatively recovered on Listeria -selective media. These results indicate that for most foods, freezing prior to analysis for L. monocytogenes should not hamper quantitative determination of the organism.

Population changes and verotoxin production of enterohemorrhagic Escherichia coli strains inoculated in milk and ground beef held at low temperatures

This study investigated the influence of low temperature and background flora on growth and verotoxin production by strains of enterohemorrhagic Escherichia coli in milk and ground beef. In the presence of no or low background flora, there was growth of the strains at 8°C. High background flora in ground beef inhibited growth at this temperature. In the foods held at low temperatures, only small amounts of verotoxin were detected; however, even at the optimum 37°C, there was still relatively little verotoxin formed compared to that in broth cultures. Even under nongrowth conditions (high background flora or 5°C holding temperature), the strains remained viable. These data suggest any food contaminated by these bacteria and held at the recommended temperature of 5°C will remain hazardous, and under certain conditions, holding at temperatures :≥8°Cwould increase the hazard.

Microorganisms as food additives

Microorganisms, both bacteria and fungi, are used as additives in meats. milk, cereals, vegetables and fruits to produce fermented products. The fermented foods differ from the starting material in texture, flavor and keeping quality. Fermentation causes changes in the nutritional content of foods; vitamin and amino acid levels may increase, decrease or remain static, depending on the type of microorganism used and the product fermented. Microorganisms also impart desirable flavors, improve texture and enhance digestibility of foods. Fermentation destroys food spoilage organisms and permits preservation of food. Lactobacilli in cultured milks are used to supplement the normal intestinal flora in individuals suffering from digestive ailments or enteric diseases. Cultured milks are tolerated by lactose-intolerant individuals because of lactose utilization in the gastrointestinal tract by ingested lactobacilli. If sufficient acid is formed, foods which have undergone a lactic acid fermentation, such as fermented sausages or cheese. do not support growth of food poisoning microorganisms. Products which undergo controlled commercial fungal fermentations have been shown not to contain mycotoxins. Histamines and other biogenic amines are present in cheese and other fermented products. Fermentation offers a means of producing safe, nutritious foods with desirable organoleptic qualities and extended storage stability.