Isolation and detection of Listeria monocytogenes using fluorogenic and chromogenic substrates for phosphatidylinositol-specific phospholipase C.

The BCM Listeria monocytogenes detection system (LMDS) consists of a selective preenrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/differential plating medium (LMPM), and identification on a confirmatory plating medium (LMCM). The efficacy of the BCM LMDS was determined using pure cultures and naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed the growth of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphatidylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35 degrees C. An initial inoculum of 10 to 100 CFU/ml of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by either its fluorescence on BCM LMCM or acid production from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thuringiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge rehydrated in 10 ml of DE neutralizing broth. In an analysis of 162 environmental sponges from facilities inspected by the U.S. Department of Agriculture (USDA), the values for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% versus 40.0 and 66.1%, respectively. No false-positive organisms were isolated by BCM LMDS, whereas 26.5% of the sponges tested by the USDA method produced false-positive results.

A 5-h screening and 24-h confirmation procedure for detecting Escherichia coli O157:H7 in beef using direct epifluorescent microscopy and immunomagnetic separation.

An antibody-direct epifluorescent filter technique (Ab-DEFT) detected 100% of the raw ground beef samples inoculated with Escherichia coli O157:H7 cells (0.15 cells g-1) and incubated in a prewarmed, modified buffered peptone water (mBPW) non-selective enrichment broth for 5 h at 42 degrees C in an orbital shaking water bath (200 rev min-1). Over 50% of the microscopic fields viewed were positive (1-10 fluorescent cells field-1) in the Ab-DEFT. All positive screening results were confirmed within 24 h by subjecting 1 ml of the mBPW to the Dynabeads anti-E. coli O157 immunomagnetic separation procedure, followed by plating on MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. At this cell concentration, 41.7% of the inoculated samples were detected by the conventional method involving a 24-h selective enrichment. Exposure to viable cells before filtration was minimized by using a 0.58% formaldehyde concentration for 5 min at 50 degrees C (killed > 4.00 logs of E. coli O157:H7 cells) without affecting cell fluorescence.

Gram staining apparatus for space station applications.

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.

Prophage (phi 80) induction in Escherichia coli K-12 by specific deoxyoligonucleotides.

A cell preparation that is permeable to proteins and oligonucleotides yet produces infectious phage particles after induction treatments was obtained by plasmolysis of Escherichia coli cells lysogenic for phi 80. When the permeabilized cells were exposed to specific oligo(deoxynucleotides), prophage (phi 80) was induced during further incubation. Of the dinucleotides tested, only d(A-G), d(G-G), and d(I-G) induced prophage. The essential base sequence of the deoxydinucleotides for the induction was determined to be deoxy(purine-G). Among oligo(deoxynucleotides) with unique base composition examined, only oligo(deoxyguanylates) exhibited the inducing activity. Although this specific oligo(deoxynucleotide)-triggered induction occurred in recB- cells, the induction was not detected in recA- cells or in the cells lysogenic for induction-negative phi 80(ind-). Possible biological significance of the oligo(deoxynucleotide)-triggered prophage induction is discussed.

Prophage induction in a permeabilized cell system: induction by deoxyribonucleases and the role of recBC-deoxyribonuclease.

Permeabilized cells able to induce prophage were obtained by plasmolysis and preincubation of the cells in a reaction mixture which allows protein synthesis. These cells became permeable to low-molecular-weight proteins and oligonucleotides. We found that deoxyribonucleases (pancreatic deoxyribonuclease and micrococcal nuclease) triggered prophage (phi 80) induction. This deoxyribonuclease-triggered induction was completely dependent upon the presence of functional recBC genes in the lysogen, regardless of the recombination proficiency determined by recBC and sbcB genes. The possible role of recBC-deoxyribonuclease in prophage induction and recombination is discussed.