A Study To Assess the Numbers and Prevalence of Bacillus cereus and Its Toxins in Pasteurized Fluid Milk.

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 >105 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 >105 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.

Complete Genome Sequences of 12 Isolates of Listeria monocytogenes Belonging to Serotypes 1/2a, 1/2b, and 4b Obtained from Food Products and Food-Processing Environments in Canada.

Listeria monocytogenes is the etiological agent for an often fatal foodborne illness known as listeriosis. Here, we present the complete genome sequences of 12 L. monocytogenes isolates representing the three most common serotypes of this pathogen (1/2a, 1/2b, and 4b), collected in Canada from different food products and environmental sources.

Multiple-locus variable number of tandem repeat analysis (MLVA) of Listeria monocytogenes directly in food samples.

Listeria monocytogenes is the etiologic agent of listeriosis responsible for severe and fatal infections in humans. Listeria contamination occurs quite often in a wide range of foods due to its ubiquitous nature. Isolates need to be characterized to a strain level for accurate diagnosis of Listeria infection, epidemiological studies, investigation of outbreaks and effective prevention and control of food-borne listeriosis. The purpose of this research was to evaluate the multiple-locus variable number of tandem repeat analysis (MLVA) for sub-typing L. monocytogenes isolates in pure cultures and in food matrices. Two multiplex PCR assays were formulated to amplify six specific loci using fluorescently-labeled primers; and the amplicons were analyzed by capillary electrophoresis. The MLVA method resulted in 34 unique DNA fingerprint patterns from 46 L. monocytogenes isolates of 10 serotypes which had 29 or 30 PFGE patterns with a single restriction enzyme and 34 AFLP patterns. The MLVA patterns of the 46 isolates remained unchanged in the presence of pre-enriched food matrices including sausage, ham, chicken, milk and lettuce. The MLVA method successfully typed L. monocytogenes strains spiked in cheese, roast beef, egg salad and vegetable samples after 48 h enrichment at the initial inoculation levels of 1-5 CFU per 25 g of food or higher. The limits of detection (typing) of the MLVA method were 10(3)-10(4)CFU/mL of pre-enriched food broth when evaluated using post-spiked sausage, ham, chicken, milk and lettuce samples. The MLVA method was simple, highly discriminatory, and easy to perform with portable (numerical) results. To our knowledge, this is the first report that describes the application of the MLVA method directly to food samples and demonstrates the possibility to obtain rapid and accurate subtyping results before an isolate is obtained.

Effect of nitrate and glucose addition on denitrification and nitric oxide reductase (cnorB) gene abundance and mRNA levels in Pseudomonas mandelii inoculated into anoxic soil.

The effect of glucose addition (0 and 500 µg C g(-1) soil) and nitrate (NO(3)) addition (0, 10, 50 and 500 µg NO(3)-N g(-1) soil) on nitric oxide reductase (cnorB) gene abundance and mRNA levels, and cumulative denitrification were quantified over 48 h in anoxic soils inoculated with Pseudomonas mandelii. Addition of glucose-C significantly increased cnorB(p) (P. mandelii and related species) mRNA levels and abundance compared with soil with no glucose added, averaged over time and NO(3) addition treatments. Without glucose addition, cnorB(p) mRNA levels were higher when 500 µg NO(3)-N g(-1) soil was added compared with other NO(3) additions. In treatments with glucose added, addition of 50 µg NO(3)-N g(-1) soil resulted in higher cnorB(p) mRNA levels than soil without NO(3) but was not different from the 10 and 500 µg NO(3)-N g(-1) treatments. cnorB(p) abundance in soils without glucose addition was significantly higher in soils with 500 µg NO(3)-N g(-1) soil compared to lower N-treated soils. Conversely, addition of 500 µg NO(3)-N g(-1) soil resulted in lower cnorB(p) abundance compared with soil without N-addition. Over 48 h, cumulative denitrification in soils with 500 µg glucose-C g(-1) soil, and 50 or 500 µg NO(3)-N g(-1) was higher than all other treatments. There was a positive correlation between cnorB(p) abundance and cumulative denitrification, but only in soils without glucose addition. Glucose-treated soils generally had higher cnorB(p) abundance and mRNA levels than soils without glucose added, however response of cnorB(p) abundance and mRNA levels to NO(3) supply depended on carbon availability.

Challenges in quantifying microbial gene expression in soil using quantitative reverse transcription real-time PCR.

The quantification of microbial gene expression in diverse soil samples via quantitative reverse transcription real time polymerase chain reaction (qRT-PCR) has numerous challenges including total RNA extraction, sample preparation, qRT-PCR optimization and the correlation of gene expression with function. Despite these challenges, microbial gene expression has been successfully quantified in soil microorganisms, and will yield valuable information on soil functions and expression of genes in soil samples. In this perspective we discuss challenges of measuring microbial gene expression and highlight recent applications using qRT-PCR to research gene function in soil.

Perspective: microfluidic applications in microbiology.

The application of microfluidics technology to microbiology research is an excellent platform for the analysis of microorganisms and their nucleic acids. This technology combines engineering, physics, chemistry, biology and computing to control the devices. In this perspective we discuss how microfluidics can be applied to microbiological research and used in diagnostic applications. We also summarize advantages and limitations of this technology, as well as highlight some recent microbiological applications.

Effect of nitrate and acetylene on nirS, cnorB, and nosZ expression and denitrification activity in Pseudomonas mandelii.

Nitrate acts as an electron acceptor in the denitrification process. The effect of nitrate in the range of 0 to 1,000 mg/liter on Pseudomonas mandelii nirS, cnorB, and nosZ gene expression was studied, using quantitative reverse transcription-quantitative PCR. Denitrification activity was measured by using the acetylene blockage method and gas chromatography. The effect of acetylene on gene expression was assessed by comparing denitrification gene expression in P. mandelii culture grown in the presence or absence of acetylene. The higher the amount of NO(3)(-) present, the greater the induction and the longer the denitrification genes remained expressed. nirS gene expression reached a maximum at 2, 4, 4, and 6 h in cultures grown in the presence of 0, 10, 100, and 1,000 mg of KNO(3)/liter, respectively, while induction of nirS gene ranged from 12- to 225-fold compared to time zero. cnorB gene expression also followed a similar trend. nosZ gene expression did not respond to NO(3)(-) treatment under the conditions tested. Acetylene decreased nosZ gene expression but did not affect nirS or cnorB gene expression. These results showed that nirS and cnorB responded to nitrate concentrations; however, significant denitrification activity was only observed in culture with 1,000 mg of KNO(3)/liter, indicating that there was no relationship between gene expression and denitrification activity under the conditions tested.

Effect of pH and temperature on denitrification gene expression and activity in Pseudomonas mandelii.

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 micromol, 63 micromol, and 22 micromol at pH 6, 7, and 8, respectively, at 8 h, whereas negligible denitrification was measured at pH 5. P. mandelii cells grown at 20 degrees C and 30 degrees 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 degrees C did not occur in the first 4 h. Levels of cumulative denitrification at 10 h were 6.6 micromol for P. mandelii cells grown at 10 degrees C and 20 degrees C and 30 micromol for cells grown at 30 degrees 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 degrees C. Low pH and temperature negatively affected denitrification activity.

Nitric oxide reductase gene expression and nitrous oxide production in nitrate-grown Pseudomonas mandelii.

Pure cultures of Pseudomonas mandelii were incubated with or without nitrate, which acts as a substrate and an electron acceptor for denitrification. Nitric oxide reductase (cnorB) gene expression was measured using a quantitative reverse transcription-PCR, and nitrous oxide emissions were measured by gas chromatography. P. mandelii cells in either the presence or absence of nitrate demonstrated an increase in cnorB gene expression during the first 3 h of growth. The level of expression of cnorB in nitrate-amended cells remained high (average, 2.06 x 10(8) transcripts/microg of RNA), while in untreated cells it decreased to an average of 3.63 x 10(6) transcripts/microg of RNA from 4 to 6 h. Nitrous oxide accumulation in the headspace was detected at 2 h, and cumulative emissions continued to increase over a 24-h period to 101 mumol in nitrate-amended cells. P. mandelii cnorB gene expression was not detected under aerobic conditions. These results demonstrate that P. mandelii cnorB gene expression was induced 203-fold at 4 h when nitrate was present in the medium. Accumulations of N(2)O indicated that the cNorB enzyme was synthesized and active.

The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings.

Carnation cuttings treated with non-transformed and 1-aminocyclopropane (ACC) deaminase-containing Azospirillum brasilense Cd1843 produced significantly more roots than untreated controls and fewer roots than cuttings treated with 0.1% indolebutyric acid (IBA). The roots produced by cuttings treated with ACC deaminase-containing Azospirillum brasilense Cd1843 were the longest roots resulting from any of the treatments, followed by non-transformed Azospirillum brasilense Cd1843, 0.1% IBA, and treatment with water. The results are interpreted in terms of a previously proposed model of bacterial promotion of plant growth by ACC deaminase and indoleacetic acid, and may have implications for the use of plant growth-promoting bacteria in the flower industry.

Microbial gene expression in soil: methods, applications and challenges.

About 99% of soil microorganisms are unculturable. However, advances in molecular biology techniques allow for the analysis of living microorganisms. With the advent of new technologies and the optimization of previous methods, various approaches to studying gene expression are expanding the field of microbiology and molecular biology. Methods used for RNA extraction, DNA microarrays, real-time PCR, competitive RT-PCR, stable isotope probing and the use of reporter genes provide methods for detecting and quantifying gene expression. Through the use of these methods, researchers can study the influence of soil environmental factors such as nutrients, oxygen status, pH, pollutants, agro-chemicals, moisture and temperature on gene expression and some of the mechanisms involved in the responses of cells to their environment. This review will also address information gaps in bacterial gene expression in soil and possible future research to develop an understanding of microbial activities in soil environments.