Growth Of Vegetative Cells Research Articles

Inhibitory activity of reuterin, nisin, lysozyme and nitrite against vegetative cells and spores of dairy-related Clostridium species

The butyric acid fermentation, responsible for late blowing of cheese, is caused by the outgrowth in cheese of some species of Clostridium, resulting in texture and flavor defects and economical losses. The aim of this study was to evaluate the effectiveness of different antimicrobial compounds against vegetative cells and spores of C. tyrobutyricum, C. butyricum, C. beijerinckii and C. sporogenes strains isolated from cheeses with late blowing defect. Minimal inhibitory concentration (MIC) for reuterin, nisin, lysozyme and sodium nitrite were determined against Clostridium strains in milk and modified RCM (mRCM) after 7d exposure. Although the sensitivity of Clostridium to the tested antimicrobials was strain-dependent, C. sporogenes and C. beijerinckii generally had higher MIC values than the rest of Clostridium species. The majority of Clostridium strains were more resistant to antimicrobials in milk than in mRCM, and vegetative cells exhibited higher sensitivity than spores. Reuterin (MIC values 0.51–32.5mM) and nisin (MIC values 0.05–12.5μg/ml) were able to inhibit the growth of vegetative cells and spores of all assayed Clostridium strains in milk and mRCM. Strains of C. tyrobutyricum exhibited the highest sensitivity to lysozyme (MIC values<0.20–400μg/ml) and sodium nitrite (MIC values 18.75–150μg/ml). These results suggest that reuterin and nisin, with a broad inhibitory activity spectrum against Clostridium spp. spores and vegetative cells, may be the best options to control Clostridium growth in dairy products and to prevent associated spoilage, such as late blowing defect of cheese. However, further studies in cheese would be necessary to validate this hypothesis.

Muricholic Acids Inhibit Clostridium difficile Spore Germination and Growth

Infections caused by Clostridium difficile have increased steadily over the past several years. While studies on C. difficile virulence and physiology have been hindered, in the past, by lack of genetic approaches and suitable animal models, newly developed technologies and animal models allow these processes to be studied in detail. One such advance is the generation of a mouse-model of C. difficile infection. The development of this system is a major step forward in analyzing the genetic requirements for colonization and infection. While important, it is equally as important in understanding what differences exist between mice and humans. One of these differences is the natural bile acid composition. Bile acid-mediated spore germination is an important step in C. difficile colonization. Mice produce several different bile acids that are not found in humans. These muricholic acids have the potential to impact C. difficile spore germination. Here we find that the three muricholic acids (α-muricholic acid, β-muricholic acid and ω-muricholic acid) inhibit C. difficile spore germination and can impact the growth of vegetative cells. These results highlight an important difference between humans and mice and may have an impact on C. difficile virulence in the mouse-model of C. difficile infection.

Characterization of two resuscitation promoting factors of Listeria monocytogenes

In actinobacteria, resuscitation promoting factor (Rpf) proteins have been described as having the ability to increase the viable count of dormant cultures and stimulate growth of vegetative cells through lag phase reduction. Recently, it was suggested that proteins Lmo0186 and Lmo2522 of Listeria monocytogenes are equivalent to Rpf proteins based on their genomic context and conserved domain architecture. It was proposed that they have evolved through non-orthologous displacement of the Rpf domain found in actinobacteria. Here we present biological and biochemical data supporting a function of Lmo0186 and Lmo2522 as Rpfs. These proteins are collectively dispensable for growth but a lmo0186 lmo2522 double mutant exhibits an extended lag phase when diluted in minimal medium. This phenotype could be partially complemented by medium supplementation with fM to nM concentrations of purified hexahistidine-tagged versions of Lmo0186 and Lmo2522, showing that these proteins can stimulate growth. Gel filtration analysis and cross-linking experiments suggest that the recombinant proteins in solution are elongated monomers. Both proteins display muralytic activity against crude cell wall preparations and are active against an artificial lysozyme substrate. Our study thus supports the hypothesis that Lmo0186 and Lmo2522 are functional equivalents of actinobacteria Rpf proteins and represents the first characterization of two Rpf homologues from firmicutes.

Behavior of Vegetative Cells and Spores of Bacillus cereus in Two Food Systems

AbstractThe survival and growth of vegetative cells and spores of toxigenic Bacillus cereus CFR 1534 was investigated in a dehydrated (dry mix) blend of flour (roasted finger millet and Bengal gram) and chocolate milk. In the case of dry mix, there was a marginal initial increase of 2 logs in 2 days of storage at 20 and 30C, respectively. In chocolate milk, there was an increase of 1 log with certain inoculum levels introduced and storage temperatures. The survival pattern of vegetative cells/spores was primarily influenced by the initial numbers and, to a slight extent, by the water activity of food matrices. Although stored samples were negative for toxigenic trait of phosphatidylinositol phospholipase C in polymerase chain reaction, associated lecithinase activity was detected in plate assay. Response surface plots for vegetative cells and spores of B. cereus CFR 1534 did indicate the potential risk associated with post‐processing contamination of B. cereus.Practical ApplicationsThe ability of vegetative cells and spores of a toxigenic culture of Bacillus cereus to survive and grow in two food matrices was studied. Often, the bacterial species tend to remain in the product and increase in their numbers in a slow paced manner. The product with no visual changes, but harbors viable population of toxigenic Bacillus cereus can be of serious public health concern. The understanding of product profile would be of help in assessing the probable risk factors that could be associated with opportunistic foodborne pathogen like B. cereus in the food chain establishment.

Modelling the life cycle of dinoflagellates: a case study with Biecheleria baltica

The cold-water dinoflagellate Biecheleria baltica has increasingly dominated the phytoplankton spring bloom in the Baltic Sea during the past years. Life cycle transitions between bloom forming cells and resting cysts are assumed to regulate the bloom dynamics of this species. We investigate the seasonal cycle and succession of Biecheleria baltica's life cycle stages using a numerical model with four different stages, vegetative cells, gametes, resting cysts and germinating cells. The transitions among the stages are functions of environmental conditions and endogenous factors. Coupled to a water column model, the model is able to represent the seasonal cycle of Biecheleria baltica with two blooms in spring. The first bloom can be explained by germination of resting cysts in winter, the second by growth of vegetative cells. Sensitivity experiments indicate that temperature is an important factor regulating the composition of Biecheleria baltica life cycle stages; increased or decreased temperature leads to fewer growing cells and more resting cysts during spring. Our newly developed life cycle model can be used to study in more detail cyst formation, cyst distribution and consequences for biogeochemical cycling in the past and future.

Effects of monolaurin and lactic acid bacteria starter culture on growth of vegetative cells of Bacillus cereus in Iranian white fresh cheese

The harmful effects of many chemical food preservatives are well established, so this has triggered interest in natural methods of preservation. Monolaurin, a monoester of lauric acid, founds naturally in some foods and has various antiviral and antibacterial activities. Evaluation of the effects of monolaurin separately and in combination with lactic acid bacteria (LAB) starter culture on growth of vegetative cells of Bacillus cereus ATCC11778 in manufactured cheeses was the purpose of this research. In this study, the number of B. cereus in four groups of cheese (C1: without starter culture and monolaurin, T1: without starter culture; with monolaurin, C2: with starter culture; without monolaurin, T2: with starter culture and monolaurin) was counted on days 0, 1, 3, 5, and 7 of manufacture. In T1 group, monolaurinin concentrations of 800, 1200, 1600 and 2000 ppm decreased the number of B. cereus by 1.2, 2.1, 3 and 3.4 logs, respectively in comparison with C1 group. InT2 group with the same concentrations of monolaurin, the number of B. cereus in comparison with C2 group was not significantly affected (p>0.05). In C2 group, starter culture decreased the number of B. cereus by 2.9 logs in comparison with C1 group. In contrary, the combination of starter culture with monolaurin in T2 group increased the number of B. cereus by 0.6 logs in comparison with C2 group. Furthermore, in C2 and T2 groups by increasing the storage time, the number of B. cereus decreased. According to these results, it can be concluded that in cheese samples of T1 group, monolaurin separately showed the inhibitory effects on the growth of B. cereus cells while in cheese samples of T2 group, the combination of monolaurin with starter culture did not demonstrate the synergistic inhibitory effects on the growth of this bacterium. Therefore, simultaneous use of monolaurin with starter culture is not recommended for improving the microbial shelf-life of Iranian white fresh cheese.

Efficacy of decontaminants and disinfectants against Clostridium difficile

Clostridium difficile is a common nosocomial pathogen transmitted mainly via its spores. These spores can remain viable on contaminated surfaces for several months and are resistant to most commonly used cleaning agents. Thus, effective decontamination of the environment is essential in preventing the transmission of C. difficile in health-care establishments. However, this emphasis on decontamination must also be extended to laboratories due to risk of exposure of staff to potentially virulent strains. Though few cases of laboratory-acquired infection have been reported, the threat of infection by C. difficile in the laboratory is real. Our aim was to test the efficacy of four disinfectants, Actichlor, MicroSol 3+, TriGene Advance and Virkon, and one laboratory decontaminant, Decon 90, against vegetative cells and spores of C. difficile. Five strains were selected for the study: the three most commonly encountered epidemic strains in Scotland, PCR ribotypes 106, 001 and 027, and control strains 630 and VPI 10463. MICs were determined by agar dilution and broth microdilution. All the agents tested inhibited the growth of vegetative cells of the selected strains at concentrations below the recommended working concentrations. Additionally, their effect on spores was determined by exposing the spores of these strains to different concentrations of the agents for different periods of time. For some of the agents, an exposure of 10 min was required for sporicidal activity. Further, only Actichlor was able to bring about a 3 log(10) reduction in spore numbers under clean and dirty conditions. It was also the only agent that decontaminated different hard, non-porous surfaces artificially contaminated with C. difficile spores. However, this too required an exposure time of more than 2 min and up to 10 min. In conclusion, only the chlorine-releasing agent Actichlor was found to be suitable for the elimination of C. difficile spores from the environment, making it the agent of choice for the decontamination of laboratory surfaces.

Psychrophilic and psychrotrophic clostridia: sporulation and germination processes and their role in the spoilage of chilled, vacuum‐packaged beef, lamb and venison

SummarySpoilage of beef, lamb and venison by psychrophilic and psychrotrophic clostridial species renders meat unacceptable resulting in financial losses and reduced consumer confidence. A number of clostridial strains, including Clostridium algidicarnis, Clostridium algidixylanolyticum, Clostridium estertheticum, Clostridium frigidicarnis and Clostridium gasigenes, have been implicated in red meat spoilage. Unlike other spoilers, these clostridia are able to grow in anaerobic conditions and at chilled temperatures (some at −1.5 °C the optimal storage temperature for chilled red meat). The spoilage they cause is characterised by softening of the meat, production of large amounts of drip (exudates), offensive odours and in the case of C. estertheticum and C. gasigenes production of gas. Spoilage occurs following the introduction of clostridial spores into vacuum packages during processing. Germination of spores is necessary for the growth of vegetative cells, which cause spoilage. Current mitigation strategies focus on good management practice within meat processing plants. However, this is not always sufficient to prevent spoilage. This review summarises the issues associated with meat spoilage because of psychrotolerant clostridia and discusses areas that require further study.

The influence of anthropogenic nitrogen loading and meteorological conditions on the dynamics and toxicity of Alexandrium fundyense blooms in a New York (USA) estuary

The goal of this two-year study was to explore the role of nutrients and climatic conditions in promoting reoccurring Alexandrium fundyense blooms in the Northport-Huntington Bay complex, NY, USA. A bloom in 2007 was short and small (3 weeks, 10 3 cells L −1 maximal density) compared to 2008 when the A. fundyense bloom, which persisted for 6 weeks, achieved cell densities >10 6 cells L −1 and water column saxitoxin concentrations >2.4 × 10 4 pmol STX eq. L −1. During the 2008 bloom, both deployed mussels (used as indicator species) and wild soft shell clams became highly toxic (1400 and 600 μg STX eq./100 g shellfish tissue, respectively) resulting in the closure of shellfish beds. The densities of benthic A. fundyense cysts at the onset of this bloom were four orders of magnitude lower than levels needed to account for observed cell densities, indicating in situ growth of vegetative cells was responsible for elevated bloom densities. Experimental enrichment of bloom water with nitrogenous compounds, particularly ammonium, significantly increased A. fundyense densities and particulate saxitoxin concentrations relative to unamended control treatments. The δ 15N signatures (12–23‰) of particulate organic matter (POM) during blooms were similar to those of sewage (10–30‰) and both toxin and A. fundyense densities were significantly correlated with POM δ 15N ( p < 0.001). These findings suggest A. fundyense growth was supported by a source of wastewater such as the sewage treatment plant which discharges into Northport Harbor. Warmer than average atmospheric temperatures in the late winter and spring of 2008 and a cooler May contributed to an extended period of water column temperatures optimal for A. fundyense growth (12–20 °C), and thus may have also contributed toward the larger and longer bloom in 2008. Together this evidence suggests sewage-derived N loading and above average spring temperatures can promote intense and toxic A. fundyense blooms in estuaries.

Procurement of spore-freeBacillus anthracisfor molecular typing outside of BSL3 environment

To (i) develop a protocol that would eliminate or greatly reduce sporulation within Bacillus anthracis vegetative cells, and (ii) harvest an adequate number of cells and sufficient DNA suitable for molecular methods including Riboprint analysis and pulse field gel electrophoresis (PFGE). Seven strains of B. anthracis (Ames, French B2, Heluky, Kruger, Pasteur, Sterne, and Vollum) were grown at 37, 42 and 45 degrees C under normal air, enhanced CO(2), microaerophilic, and anaerobic conditions on solid media and subcultured in two broths with and without supplements. The bacterial cells were centrifuged and washed. Slides made from the cell pellets were stained with Malachite Green and observed for the presence of spores. Cell preparations were subjected to 80 degrees C for 30 min and processed for and analysed by either Riboprinte or PFGE. Multiple pellets of each strain were processed, stained, placed onto solid culture media, incubated for 7 days and observed for growth. The cell preparations yielded clear and reproducible results with both molecular methods. None of the cell preparations yielded growth on the culture media. This method eliminated viable spores in cell preparations of B. anthracis, yet still allowed the growth of vegetative cells to provide sufficient DNA suitable for analysis by Riboprinter and PFGE. This method will provide safe cell preparations, prevent instrument contamination, and may be useful for other aerobic and anaerobic spore-formers.

Life‐cycle kinetic model for endospore‐forming bacteria, including germination and sporulation

We develop a mechanistic life-cycle model for endospore-forming bacteria (EFB) and test the model with experiments with a Bacillus mixed culture. The model integrates and quantifies how sporulation and germination are triggered by depletion or presence of a limiting substrate, while both substrates affect the rate of vegetative growth by a multiplicative model. Kinetic experiments show the accumulation of small spherical spores after the triggering substrate is depleted, substantially more rapid decay during sporulation than for normal decay of vegetative cells, and a higher specific substrate utilization rate for the germinating cells than that for growth of vegetative cells. Model simulations capture all of these experimental trends. According to model predictions, when a batch reactor is started, seeding with EFB spores instead of active EFB delays the onset of rapid chemical oxygen demand (COD) utilization and biomass growth, but the end points are the same. Simulated results with low aeration intensity show that germination can consume some substrate without dissolved oxygen (DO) depletion.

Factors influencing the initiation of blooms of the raphidophyte Heterosigma akashiwo and the diatom Skeletonema costatum in a port in Japan

We investigated how environmental factors initiate Heterosigma akashiwo and Skeletonema costatum blooms from resting stages in bottom sediments in a shallow port over 2 yr. Using field‐collected sediments, we also conducted laboratory experiments on how light intensity affects germination of resting stages and growth of the germinated cells. Both phytoplankton species bloomed only in summer, when water temperature and solar radiation were high enough for growth. All three blooms of H. akashiwo and the earliest bloom of S. costatum in a year occurred right after transmission of strong light (&gt;200 [mol quanta m−2 s−1) to the bottom layer and a peak occurred in dissolved inorganic phosphorus (DIP). In the laboratory, resting stages of H. akashiwo and S. costatum germinated even in dim light (20 and 65 [mol quanta m−2 s−1, respectively), but germinated cells required stronger light of &gt;130 and 280 [mol quanta m−2 s−1, respectively, for rapid growth. This value is much higher than the threshold for survival, and is higher than the half‐saturating light intensity for growth of vegetative cells. Abundance of the resting stages of both species in the sediments rapidly increased during blooms and logarithmically decreased during nonbloom periods, suggesting that resting stages are continuously consumed. For both species, our results suggest that blooms initiate when transmission of sufficient light permits: first, germination of cells from the sediment; second, rapid growth of these germinated cells. Temperature and DIP must also exceed a facilitating threshold.

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats

To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.

EFFECT OF YOGHURT STARTER LACTIC ACID BACTERIA ON

The growth of lactic acid bacteria of yoghurt culture Str.salivarius sub-sp thermophilus (Str. thermophilus) and Lacto bacillus delrueckeii sub-sp bulgaricus (Lacto. bulgaricus)in milk inhibited the growth of vegetative cells of B .cereus and spores that resisted the high heat treatment which was isolated from raw milk and prevented the B .cereus from producing the enterotoxins in yoghurt . The B .cereus did not affect the growth of yoghurt starter culture, also the rate of acid development in yoghurt was not affected in the presence of B. cereus. The poisoning by this bacteria did not happen in dairy product when number decreased to less than 106 c.f.u. /ml .

Modeling Culture Profiles of the Heterocystous N2‐Fixing Cyanobacterium Anabaenaflos‐aquae

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.

Validation of Bacon Processing Conditions To Verify Control of Clostridium perfringens and Staphylococcus aureus

It is unclear how rapidly meat products, such as bacon, that have been heat treated but not fully cooked should be cooled to prevent the outgrowth of spore-forming bacterial pathogens and limit the growth of vegetative cells. Clostridium perfringens spores and vegetative cells and Staphylococcus aureus cells were inoculated into ground cured pork bellies with and without 1.25% liquid smoke. Bellies were subjected to the thermal profiles of industrial smoking to 48.9°C (120°F) and normal cooling of bacon (3 h) as well as a cooling phase of 15 h until the meat reached 7.2°C (45°F). A laboratory-scale bacon smoking and cooling operation was also performed. Under normal smoking and cooling thermal conditions, growth of C. perfringens in ground pork bellies was <1 log regardless of smoke. Increase of S. aureus was 2.38 log CFU/g but only 0.68 log CFU/g with smoke. When cooling spanned 15 h, both C. perfringens and S. aureus grew by a total of about 4 log. The addition of liquid smoke inhibited C. perfringens, but S. aureus still achieved a 3.97-log increase. Staphylococcal enterotoxins were detected in five of six samples cooled for 15 h without smoke but in none of the six samples of smoked bellies. In laboratory-scale smoking of whole belly pieces, initial C. perfringens populations of 2.23±0.25 log CFU/g were reduced during smoking to 0.99±0.50 log CFU/g and were 0.65±0.21 log CFU/g after 15 h of cooling. Populations of S. aureus were reduced from 2.00±0.74 to a final concentration of 0.74±0.53 log CFU/g after cooling. Contrary to findings in the ground pork belly system, the 15-h cooling of whole belly pieces did not permit growth of either pathogen. This study demonstrates that if smoked bacon is cooled from 48.9 to 7.2°C (120 to 45°F) within 15 h, a food safety hazard from either C. perfringens or S. aureus is not likely to occur.

The effect of intermittent shaking, headspace and temperature on the growth of Alicyclobacillus acidoterrestris in stored apple juice

SummaryThe presence of Alicyclobacillus acidoterrestris in stored juices can be difficult to detect. In this study the effects of storage temperature, headspace and agitation of juice containers was investigated. The results indicate that the amount of headspace has a significant effect on growth of vegetative cells and spores of A. acidoterrestris at 35 °C. Intermittent shaking before sampling increased growth and therefore probable detection rates at 30 °C, Agitating containers and sampling from several areas within containers is therefore recommended for determining whether A. acidoterrestris is present or absent from stored juice, especially in large containers.

A family of autocrine growth factors in Mycobacterium tuberculosis

Mycobacterium tuberculosis and its close relative, Mycobacterium bovis (BCG) contain five genes whose predicted products resemble Rpf from Micrococcus luteus. Rpf is a secreted growth factor, active at picomolar concentrations, which is required for the growth of vegetative cells in minimal media at very low inoculum densities, as well as the resuscitation of dormant cells. We show here that the five cognate proteins from M. tuberculosis have very similar characteristics and properties to those of Rpf. They too stimulate bacterial growth at picomolar (and in some cases, subpicomolar) concentrations. Several lines of evidence indicate that they exert their activity from an extra-cytoplasmic location, suggesting that they are also involved in intercellular signalling. The five M. tuberculosis proteins show cross-species activity against M. luteus, Mycobacterium smegmatis and M. bovis (BCG). Actively growing cells of M. bovis (BCG) do not respond to these proteins, whereas bacteria exposed to a prolonged stationary phase do. Affinity-purified antibodies inhibit bacterial growth in vitro, suggesting that sequestration of these proteins at the cell surface might provide a means to limit or even prevent bacterial multiplication in vivo. The Rpf family of bacterial growth factors may therefore provide novel opportunities for preventing and controlling mycobacterial infections.

Expression of a P-type Ca 2+-transport ATPase in Bacillus subtilis during sporulation

The open reading frame designated yloB in the genomic sequence of Bacillus subtilis encodes a putative protein that is most similar to the typically eukaryotic type IIA family of P-type ion-motive ATPases, including the endo(sarco)plasmic reticulum (SERCA) and PMR1 Ca 2+-transporters, located respectively in the SERCA and the Golgi apparatus. The overall amino acid sequence is more similar to that of the Pmr1s than to the SERCAs, whereas the inverse is seen for the 10 amino acids that form the two Ca 2+-binding sites in SERCA. Sporulating but not vegetative B. subtilis cells express the predicted protein, as shown by Western blotting and by the formation of a Ca 2+-dependent phosphorylated intermediate. Half-maximal activation of phosphointermediate formation occurred at 2.5 μM Ca 2+. Insertion mutation of the yloB gene did not affect the growth of vegetative cells, did not prevent the formation of viable spores, and did not significantly affect 45 Ca accumulation during sporulation. However, spores from knockouts were less resistant to heat and showed a slower rate of germination. It is concluded that the P-type Ca 2+-transport ATPase from B. subtilis is not essential for survival, but assists in the formation of resistant spores. The evolutionary relationship of the transporter to the eukaryotic P-type Ca 2+-transport ATPases is discussed.

Optimized, One-Step, Recovery-Enrichment Broth for Enhanced Detection of Listeria monocytogenes in Pasteurized Milk and Hot Dogs

A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM D-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of D-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.