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.

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°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-b-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°C (killed ×4·00 logs of E. coli O157 : H7 cells) without affecting cell fluorescence.

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 080. When the permeabilized cells were exposed to specific oligo(deoxynucleotides), prophage (480) 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 recBcells, the induction was not detected in recA-cells or in the cells lysogenic for induction-negative 480(ind-). Possible biological significance of the oligo(deoxynucleotide)-triggered prophage induction is discussed.Inhibition of DNA replication triggers a sequence ofevents that leads to the induction of various cellular functions in bacteria (1). Some of these inducible functions (SOS functions) include prophage induction, inducible mutagenesis and DNA repair, and filamentous cell growth. recA protein is involved in these processes, and the inhibition of DNA replication induces the synthesis of recA protein (2, 3). Roberts et al. (4) have demonstrated ATP-dependent cleavage ofA repressor by purified recA protein.By using a biochemical assay procedure (5) which is based on the phage-controlled transcription and translation of a bacterial operon (trp operon) integrated into an early transcribed region ofbacteriophage 4)80 genome, we characterized early events in vivo that lead to the inactivation of phage repressor molecules in Escherichia coli (6). We demonstrated that a specific DNase, recBC DNase, is involved in the process and suggested recBC DNase as the enzyme that provides an induction signal by its action on the DNA derived from damaged DNA. From in vitro studies of the enzymatic action of recBC DNase, one can conjecture that the signal is either double-stranded DNA with a single-stranded DNA region or acid-soluble products of DNA, namely, oligo(deoxynucleotides).In preliminary studies, we reported that phage (480) repressor molecules were inactivated when permeabilized cells were incubated with a micrococcal nuclease digest of DNA, and we subsequently identified the active component in the digest as d(A-G-G) (7). This result suggests that specific oligo(deoxynucleotides) or single-stranded DNA with specific base sequences is the likely signal for prophage induction, at least for 480. However, because the biochemical assay procedure used for that study was indirect and relatively insensitive, further studies on oligo(deoxynucleotide) induction had to wait until a more direct and sensitive biological assay procedure was developed. 080ind was kindly supplied by A. Matsushiro (Osaka University). 80ind-55s was isolated in this laboratory. Chemicals and Chemical Synthesi...

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 ()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.In previous studies, Smith and Oishi (9) analyzed the early molecular events leading to the induction of SOS functions by using prophage induction as a probe. After subjecting lysogens to various inducing conditions, we studied the kinetics of phage repressor inactivation by a biochemical assay procedure (8) which is based on the phage-controlled read-through transcription and translation of a bacterial operon (tip operon) integrated in the bacteriophage 480 genome. Smith and Oishi (9) found that various inducing agents exhibited distinctly different patterns in the kinetics of repressor inactivation, suggesting that the number of steps leading to repressor inactivation varies according to the primary DNA structure produced by the agent. Agents which cause strand scissions in DNA molecules exhibit early inactivation of the repressor molecules, whereas agents which modify DNA bases, such as UV light or mitomycin C, showed an intermediate type of inactivation. Inactivation due to inhibition of DNA replication by precursor deprivation (thymine deprivation) or by inactivation of the replication machinery (temperature-sensitive DNA mutants) was a distinctly slower process.A good correlation has also been observed between the timing of DNA degradation after the inducing treatment and the timing of repressor inactivation; furthermore, both DNA degradation and repressor inactivation triggered by these inducing treatments are delayed considerably when a recB (or recC) mutation is introduced in the lysogens, implicating an active role for recBC-DNase in prophage induction (9). The involvement of recBC-DNase in recA protein synthesis was demonstrated by Gudas and Par-dee (2). The dependence of repressor inactivation upon functional recBC allele is further demonstrated in permeabilized cells (5). A mixture of four deoxynucleoside triphosphates (dNTP's) and ATP destabilize active DNA replication forks in the permeabilized cells, and that degradation of nascent DNA by recBC-DNase triggers the induction process. Prophage induction by thymine starvation of a thymine-requiring strain is also completely dependent on a functional recBC gene product (6), suggesting a similar mechanism for induction. Based on these results, Oishi et al. (6) proposed the following sequence of events as the early molecular mechanisms which lead to the induction of prophage and of SOS function...

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