Pseudomonas mandelii liquid cultures were studied to determine the effect of pH and temperature on denitrification gene expression, which was quantified by quantitative reverse transcription-PCR. Denitrification was measured by the accumulation of nitrous oxide (N 2 O) in the headspace in the presence of acetylene. Levels of gene expression of nirS and cnorB at pH 5 were 539-fold and 6,190-fold lower, respectively, than the levels of gene expression for cells grown at pH 6, 7, and 8 between 4 h and 8 h. Cumulative denitrification levels were 28 mol, 63 mol, and 22 mol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20°C and 30°C exhibited 9-fold and 94-fold increases in levels of cnorB expression between 0 h and 2 h, respectively, and an average 17-fold increase in levels of nirS gene expression. In contrast, induction of cnorB and nirS gene expression for P. mandelii cells grown at 10°C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 mol for P. mandelii cells grown at 10°C and 20°C and 30 mol for cells grown at 30°C. Overall, levels of cnorB and nirS expression were relatively insensitive to pH values over the range of pH 6 to 8 but were substantially reduced at pH 5, whereas gene expression was sensitive to temperature, with induction and time to achieve maximum gene expression delayed as the temperature decreased from 30°C. Low pH and temperature negatively affected denitrification activity.Denitrification is a respiratory microbiological process in which nitrate (NO 3 Ϫ ) or nitrite (NO 2 Ϫ ) is reduced to gaseous nitric oxide (NO), nitrous oxide (N 2 O), or molecular nitrogen (N 2 ) under oxygen-limited conditions (33). Denitrification can result in substantial gaseous losses of N, an important plant nutrient, from agricultural fields (7,14). N 2 O depletes stratospheric ozone and contributes to global warming (28). An understanding of the environmental controls on denitrifier activity is essential for comprehending the spatial and temporal regulation of denitrification within agricultural production systems.Denitrification is carried out by various microorganisms belonging to several genera and species of bacteria (3,4,27,33). The strain of Pseudomonas mandelii used in this study was a dominant culturable denitrifier isolated from an agricultural field in a potato production system in New Brunswick, Canada (4).Several environmental factors control the process of denitrification. These include oxygen availability, substrate availability (i.e., NO 2 Ϫ and NO 3 Ϫ ), pH, temperature, and the abundance and species of denitrifiers. The availability of a substrate, the absence of oxygen, and the presence of active denitrifiers are the main controlling factors (24). However, pH and temperature also play a role in influencing denitrifier growth, metabolism, denitrification gene expression, and, subsequently, denitrification rate. A recent review of environmental controls on denitrifying communities and...
Bacillus cereus is a pathogenic adulterant of raw milk and can persist as spores and grow in pasteurized milk. The objective of this study was to determine the prevalence of B. cereus and its enterotoxins in pasteurized milk at its best-before date when stored at 4, 7, and 10°C. More than 5.5% of moderately temperature-abused products (stored at 7°C) were found to contain >10 CFU/mL B. cereus , and about 4% of them contained enterotoxins at a level that may result in foodborne illness; in addition, more than 31% of the products contained >10 CFU/mL B. cereus and associated enterotoxins when stored at 10°C. Results from a growth kinetic study demonstrated that enterotoxin production by B. cereus in pasteurized milk can occur in as short as 7 to 8 days of storage at 7°C. The higher B. cereus counts were associated with products containing higher butterfat content or with those produced using the conventional high-temperature, short-time pasteurization process. Traditional indicators, aerobic colony counts and psychrotrophic counts, were found to have no correlation with level of B. cereus in milk. The characterization of 17 representative B. cereus isolates from pasteurized milk revealed five toxigenic gene patterns, with all the strains carrying genes encoding for diarrheal toxins but not for an emetic toxin, and with one strain containing all four diarrheal enterotoxin genes (nheA, entFM, hblC, and cytK). The results of this study demonstrate the risks associated even with moderately temperature-abused pasteurized milk and the necessity of a controlled cold chain throughout the shelf life of fluid milk to enhance product safety and minimize foodborne illness.
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