Glycine antagonism does not block ischemic spontaneous depolarization in the rat

Two enzyme-linked immunoassay (ELISA) systems for rapid screening of Listeria spp. were compared for their use in analysis of spiked foods regulated by the U.S. Food and Drug Administration. The Tecra Listeria kit is a 48 h visual ELISA that detects Listeria spp. through colorimetry. It has been approved for first action by AOAC INTERNATIONAL. The Vitek immunodiagnostic assay system for Listeria (VIDAS LIS) is a fully automated 48 h ELISA that detects Listeria spp. by immunofluorescence. Fifty-two food samples were artificially contaminated with high (11-42 colony-forming units [cfu]/25 g food) and low (2-8 cfu/25 g food) levels of L. monocytogenes and screened by the 2 protocols. Unspiked samples were also assayed as negative controls. Six unspiked samples were found positive for Listeria spp. by both methods: 3 were identified as L. monocytogenes and 3 as L. innocua by official methods. Both ELISA methods detected all spiked samples. One unspiked sample was assayed positive by Tecra and negative by VIDAS LIS. No Listeria spp. were recovered when the sample was tested by the conventional method. No interference due to background fluorescence of food matrixes was observed in the VIDAS LIS method. Results suggest a modified VIDAS LIS preenrichment medium may be used in place of the VIDAS standard medium in the protocol.     

Limited access to a dietary fat option affects ingestive behavior but not body composition in male rats

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes, Scott A and an environmental strain KM, on exposure to sea water at 12.8 or 20.8 degrees C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T90 values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0.6% yeast extract) at 12.8 and 20.8 degrees C were 61.7 and 69.2 h for L. monocytogenes Scott A, and 103.0 and 67.0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T90 values at 12.8 and 20.8 degrees C were 60.6 and 56.9 h for L. monocytogenes Scott A, and 83.0 and 65.9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12.8 and 20.8 degrees C was 1.7 and 17.7%, respectively, while for KM the values were 19.0 and 1.6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 degrees C showed an apparent increase in heat resistance after exposure to sea water at 20.8 degrees C for 7 d (D58 = 2.64 min) compared with before exposure (D58 = 1.24). This increase in thermal resistance was not apparent at temperatures greater than 63 degrees C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different (P > 0.05) over the temperature range tested (58-62 degrees C).     

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule

Listeria monocytogenes blood agar (LMBA) was compared to Listeria selective agar based on lithium chloride and ceftazidime (LA) and to the Oxford and Palcam media recommended by ISO and IDF for the detection and enumeration of L. monocytogenes from foodstuffs and food-processing environments. LMBA is based on trypticase soy agar with the following additions: sheep blood (5%) and as selective agents lithium chloride (10 g/l), polymyxin B sulphate (10 mg/l) and ceftazidime (20 mg/l), whereas the selectivity of LA is based on lithium chloride (15 g/l) and ceftazidime (15 g/l). The media were compared in the detection of L. monocytogenes after enrichment from naturally contaminated foodstuffs (n = 423) and from food-processing environments (n = 93), and in the enumeration of the species from naturally contaminated foodstuffs (n = 287). LMBA was superior both to the standard media and to LA in detection after enrichment and also in enumeration, except in the case of fresh broiler cut samples. The overall sensitivities of the Palcam, Oxford, LA and LMBA media were 68%, 67%, 74% and 96% in detection after enrichment and 64%, 73%, 76% and 80% in the enumeration of the species from ready to eat foods. The superiority of LMBA is based on distinguishing L. monocytogenes from other Listeria species by detection of beta-hemolysis, whereas the other media gave false-negative results because of the overgrowth of Listeria spp. other than L. monocytogenes, especially in detection after enrichment. A more selective medium than LMBA would have been required for the enumeration of the species from samples with high levels of competitive bacteria other than Listeria spp. The results indicate the need for a more specific isolation medium for L. monocytogenes in addition to those recommended by ISO and IDF for both detection and enumeration. LMBA offers an alternative to be used in combination with either Palcam or Oxford as well as with LA.     

Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100 degrees C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Stress proteins in Listeria monocytogenes

The proteins induced by the different stress conditions in Listeria monocytogenes were analyzed by two-dimensional (2-D) electrophoresis with the aid of a computerized 2-D gel analysis system. The stress conditions imposed were pH 4, pH 10, 0.015% sodium, dodecyl sulfate (SDS), 0.03% sodium deoxycholate and 4% ethanol. As previously seen for heat shock and cold shock, more than half of the proteins normally synthesized by Listeria cells were repressed under these stress conditions. Conversely, the synthesis of a great number of proteins was increased and novel proteins appeared upon stress. Each stress factor induced a specific set of proteins. These stress proteins were characterized by their apparent molecular mass and isoelectric point. No universal stress proteins were found to be common to all the stresses studied, while some proteins were commonly induced by two or three stress conditions. The degree of dissimilarity in stress responses was best illustrated by the induction of only two proteins common to exposure to the two detergents SDS and sodium deoxycholate. This work together with that on heat and cold shock, constitutes the basic step for the identification of stress proteins in Listeria.     

Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes

The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated. For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied. Death kinetics of heat-shocked cells is also discussed. For L. monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance. For L. monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance. The only survival curves of cells to have shoulders were those for cells that had been heat shocked. A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times. The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.     

Upregulation of urokinase-type plasminogen activator by endogenous and exogenous HIV-1 Tat protein in tumour cell lines derived from BK virus/tat-transgenic mice

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal culture medium and the same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli, E. coli-ETEC, E. coli-O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food sample was artificially inoculated with each individual bacterial species. Twelve different seafood samples and two soft cheese samples without artificial inoculation were examined by this protocol. Vibrio vulnificus, Salmonella spp., E. coli, Listeria monocytogenes and Bacillus cereus were detected in some foods. Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

Disinfection of eyelid speculums for retinopathy of prematurity examination

Depending on the growth medium used for enrichment of bacterial cells prior to assay, the monoclonal antibody (MAb) EM-6E11 recognizing Listeria genus-specific epitope on 43 and 94 to 97 kDa cell-surface antigens (A. K. Bhunia and M. G. Johnson, Appl. Environ. Microbiol. 58:1924-1929, 1992) exhibited extensive variability in the detection of Listeria species. MAb EM-6E11 strongly detected live cells of all Listeria species and all serotypes of L. monocytogenes by ELISA when cells were grown in nonselective brain heart infusion (BHI) broth, in selective Listeria enrichment broth (LEB), or in Listeria repair broth (LRB). In contrast, EM-6E11 detected only four of the thirteen serotypes of L. monocytogenes (serotypes 1/2c, 3b, 4ab, and 7) when cells were grown in the UVM1 formulation of Listeria enrichment broth (UVM1) or Fraser broth (FRB). This MAb failed to react with live cells of four other Listeria species, including L. ivanovii, L. welshimeri, L. grayi, and L. murrayi cells grown in UVM1 or FRB. Heating of Listeria cells at 100 degrees C for 20 min, irrespective of the enrichment media used, led to large losses of MAb EM-6E11 reactivity in ELISA, suggesting that the specific cell-surface epitopes involved may not be heat stable. Our results confirm that MAb EM-6E11 is suitable for detection of live cells but not heat-killed cells of Listeria spp. and can be used in conjunction with an enrichment step in BHI, LEB, or LRB but not in UVM1 or FRB.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Evaluation of universal preenrichment broth for the recovery of foodborne pathogens from milk and cheese

The use of universal preenrichment broth for the recovery of verotoxigenic Escherichia coli, Salmonella spp., and Listeria monocytogenes from milk and cheese was examined. Universal preenrichment broth supported the growth of low inoculum levels (10 cfu/ml) of these organisms in pure cultures and in mixed cultures containing higher levels of other pathogens or bacterial flora from raw milk. This medium also supported the recovery and growth of heat-injured Salmonella spp., L. monocytogenes, and verotoxigenic E. coli at inoculum levels of 10(2) cfu/ml to yield cell levels of 10(8) cfu/ml in pure cultures and at least 10(5) cfu/ml in the presence of high levels of known competitive pathogens or microflora of cheese samples after 24 h of incubation. Universal preenrichment broth performed better than Listeria enrichment broth in supporting the recovery and growth of heat-injured L. monocytogenes and equally as well as buffered peptone water or trypticase soy broth in supporting the growth of uninjured L. monocytogenes, Salmonella spp., and verotoxigenic E. coli. Coenrichment of these pathogens in universal preenrichment broth reduced the quantity of milk or cheese samples that were required for analysis and also reduced the cost and labor involved in preparing and processing separate preenrichment media.     

Effects of above-optimum growth temperature and cell morphology on thermotolerance of Listeria monocytogenes cells suspended in bovine milk

The thermotolerances of two different cell forms of Listeria monocytogenes (serotype 4b) grown at 37 and 42.8 degrees C in commercially pasteurized and laboratory-tyndallized whole milk (WM) were investigated. Test strains, after growth at 37 or 42.8 degreesC, were suspended in WM at concentrations of approximately 1.5 x 10(8) to 3.0 x 10(8) cells/ml and were then heated at 56, 60, and 63 degrees C for various exposure times. Survival was determined by enumeration on tryptone-soya-yeast extract agar and Listeria selective agar, and D values (decimal reduction times) and Z values (numbers of degrees Celsius required to cause a 10-fold change in the D value) were calculated. Higher average recovery and higher D values (i.e., seen as a 2.5- to 3-fold increase in thermotolerance) were obtained when cells were grown at 42.8 degrees C prior to heat treatment. A relationship was observed between thermotolerance and cell morphology of L. monocytogenes. Atypical Listeria cell types (consisting predominantly of long cell chains measuring up to 60 micron in length) associated with rough (R) culture variants were shown to be 1.2-fold more thermotolerant than the typical dispersed cell form associated with normal smooth (S) cultures (P 相似文献   

Entry of Listeria monocytogenes into neurons occurs by cell-to-cell spread: an in vitro study

Listeria monocytogenes is an intracellular pathogen that causes severe central nervous system infection in humans and animals. The ability of this bacterium to penetrate nerve cells was investigated by using rat spinal cell cultures. Entry into distinct cell types, i. e., glial cells and neurons, was monitored by a differential immunofluorescence technique with antibodies against cell type-specific markers and the bacterial pathogen. L. monocytogenes was detected predominantly within macrophages constituting the microglia. Astrocytes and oligodendrocytes, the major components of macroglia, were infected to a lesser extent. Surprisingly, Listeria innocua, a noninvasive and nonpathogenic species, also has the capacity to enter into these three types of glial cells. Entry into neurons was a very rare event. In contrast, we found that L. monocytogenes could efficiently invade neurons when these latter cells were cocultivated with Listeria-infected mouse macrophages. In this case, infection of neurons occurs by cell-to-cell spread via an actA-dependent mechanism. These data support the notion that infected phagocytes can be vectors by which L. monocytogenes gains access to privileged niches such as the central nervous system.     

Antibodies to Listeria monocytogenes

Listeria monocytogenes is one of the leading foodborne pathogens and has been implicated in numerous outbreaks in the last 2 decades. Immunocompromised populations are usually the most susceptible to Listeria infections. Although the pathogenic mechanism is a complex process, significant progress has been made in unravelling the mechanism in recent years. It is now clear that numerous extracellular and cell-associated proteins, such as internalin, listeriolysin, actin polymerization protein, phospholipase, metalloprotease, and possibly p60 proteins, are essential for L. monocytogenes entry into mammalian cells, survival inside the phagosome, escape into the cytoplasm, and cell-to-cell spread. Other proteins may be responsible for growth and physiology or to maintain the structural integrity of the bacteria. Monoclonal and polyclonal antibodies have been developed against many of those antigens or their synthetic derivatives that have helped greatly to determine the structure and function of these antigens. The antibodies were also used for the diagnosis and detection, immunocytochemical staining, and serotyping of Listeria. Humoral immune response to live L. monocytogenes cells was examined in naturally or experimentally infected hosts. Studies revealed that only extracellular antigens induced the humoral response, whereas cell-associated antigens had apparently no response. It is speculated that during the occasional bacteremic phase, L. monocytogenes releases extracellular antigens that are then processed by the immune system for antibody production. As L. monocytogenes is an intracellular pathogen, the cell-associated antigens are not persistent in the blood circulation and thus fail to stimulate the humoral immune response.     

Further data on the characters of Listeria strains

The composition of a minimal medium suitable for the demands of modern genetical work is described. The nutritional requirements of some 15 strains of L. monocytogenes were studied on this medium. DL-6,8-thioctic acid was not essential. The organisms also grew without thiamine but for a convenient colony size this vitamin had to be used. The presence of Fe++, Mg++, and Ca++ ions as well as a low redox potential supplied by thioglycollate was necessary. There was some difference in the amino acid requirements of the strains. The most common demand was for leucine, iso-leucine, valine, and cysteine. The serotype, agglutinability, hemolytic effect, virulence, and nutritional requirements of the Listeria were compared. It seems that these properties are not consistently linked. Two 'EMS' mutants were also studied. The arginine, glutamine, and methionine metabolism of the auxotrophic mutant 025/4/4 was damaged and its virulence was lost. Data connected with the virulence markers of Listeria are discussed.     

The chemical stability of enkephalin-like tetrapeptides in a cannula implanted in the rat neostriatum for multiple microinjections

A method for identification of Listeria in food samples was developed. It consisted of cultivating of suspected specimen on standard agar medium, direct absorption of grown colonies onto nitrocellulose membrane and processing of the latter with rabbit serum raised against purified cell wall protein Lm79/39 of L. monocytogenes. Analysis using anti-rabbit peroxidase conjugate and 4-chloro-1-naphthol and H2O2 solutions allowed direct detection of Listeria colonies which remained readily available for subsequent isolation.     

Internalin A can mediate phagocytosis of Listeria monocytogenes by mouse macrophage cell lines

Listeria monocytogenes internalin A (InlA) is a surface protein that mediates the attachment of Listeria to, and invasion of, hepatocytes, epithelial, and endothelial cells. In this study, we tested whether InlA could also mediate phagocytosis of L. monocytogenes by the non-listericidal mouse macrophage cell lines J774A.1 and H36.12j. Recombinant InlA (rInlA) was used to derive mouse monoclonal anti-InlA antibodies (mAb) and rabbit anti-InlA antibodies. Fluorescence microscopy demonstrated that these anti-InlA antibodies reacted with wild-type L. monocytogenes, L. ivanovii, and L. innocua+, a mutant transformed with the inlAB operon that expresses surface InlA but failed to react with Bug 8, an InlA/InlB-negative transposon mutant of L. monocytogenes or with noninvasive Listeria sp. Fluorescence microscopy, radiolabeling, and flow cytometry showed that rInlA bound specifically to both macrophage cell lines. Incubation of macrophages and wild-type L. monocytogenes in the presence of rInlA or pretreatment of Listeria with anti-InlA antibodies specifically inhibited, by at least 50%, the phagocytosis of Listeria by both of these cells. By comparison, treatment with these reagents failed to affect the phagocytosis of Streptococcus pyogenes by either macrophage cell line nor did these reagents alter the ability of macrophages to internalize wild-type L. monocytogenes. We found that Bug 8, but not wild-type L. monocytogenes, failed to grow within both of these non-listericidal macrophage cell lines. In contrast to infection by wild-type L. monocytogenes, Bug 8 was rapidly eliminated from the spleens of both C57Bl/6 and DBA/2 mice. Data presented here show that only invasive Listeria sp. have surface InlA and that L. monocytogenes can enter non-listericidal macrophage cell lines by binding of bacterial InlA to the macrophage cell surface.     

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.     

Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system

The intestinal stage of listeriosis was studied in a rat ligated ileal loop system. Listeria monocytogenes translocated to deep organs with similar efficiencies after inoculation of loops with or without Peyer's patches. Bacterial seeding of deep organs was demonstrated as early as 15 min after inoculation. It was dose dependent and nonspecific, as the delta inlAB, the delta hly, and the delta actA L. monocytogenes mutants and the nonpathogenic species, Listeria innocua, translocated similarly to wild-type L. monocytogenes strains. The levels of uptake of listeriae by Peyer's patches and villous intestine were similar and low, 50 to 250 CFU per cm2 of tissue. No listeria cells crossing the epithelial sheet of Peyer's patches and villous intestine were observed by transmission electron microscopy. The lack of significant interaction of listeriae and the follicle-associated epithelium of Peyer's patches was confirmed by scanning electron microscopy. The follicular tissue of Peyer's patches was a preferential site of Listeria replication. With all doses tested, the rate of bacterial growth was 10 to 20 times higher in Peyer's patches than in villous intestine. At early stages of Peyer's patch infection, listeriae were observed inside mononuclear cells of the dome area. Listeriae then disseminated throughout the follicular tissue except for the germinal center. The virulence determinants hly and, to a lesser extent, actA, but not inlAB, were required for the completion of this process. This study suggests that Peyer's patches are preferential sites for replication rather than for entry of L. monocytogenes, due to the presence of highly permissive mononuclear cells whose nature remains to be defined.     

Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua

Listeria monocytogenes is a facultative intracellular organism that is capable of replicating within macrophage and macrophage-like cells. The species secretes a phosphatidylinositol-specific phospholipase C (PI-PLC) encoded by the plcA gene. A plcA gene from L. monocytogenes was cloned downstream of a gram-positive promoter in the plasmid pWS2-2. To determine what effect plcA would have on intracellular survival when introduced into Listeria innocua, a species that does not growth intracellularly or contain plcA, transformation with the recombinant pWS2-2 plasmid was performed. Phospholipase C activity in Listeria innocua/pWS2-2 was confirmed on a brain heart infusion-phosphatidylinositol agar plate, whereas wild-type L. innocua did not produce PI-PLC activity. Intracellular growth of L. innocua/pWS2-2 was subsequently measured in the macrophage-like cell line J774 by Giemsa staining and viable count determinations at specific time points following infection. The J774 cells infected with wild-type L. innocua showed a falling viable count through 8 h postinfection. Although J774 cells infected with L. innocua/pWS2-2 also initially displayed reduced viable counts, the viable count rose after 6 h postinfection and increased further at 8 h postinfection before a subsequent decline again at 16 h postinfection. Giemsa staining revealed fewer than 6 bacteria in individual macrophage cells at 2 h postinfection, and yet approximately 15% of the J774 cells had 6 to 12 bacteria localized to one area of the macrophage cell after 6 h; moreover, electron micrographs showed that the L. innocua/pWS2-2 cells were replicating inside the phagosome of the host cell. Furthermore, Thoria Sol labeling demonstrated that lysosomes had fused with these phagosomes, and acridine orange staining revealed that the compartments were acidified. These results demonstrate that L. innocua cells transformed with the plasmid-borne plcA gene, and expressing functional PI-PLC, are able to grow intracellularly in what appear to be phagolysosomes, although between 3 and 6 h is needed for this to manifest itself. Intracellular growth specifically in L. innocua may be a secondary function associated with the plcA gene product. The addition of this one gene, plcA, to a species of Listeria that in the wild-type state does not replicate intracellularly apparently can now allow some of the bacteria to transiently multiply inside the phagosomes of host macrophage cells.