Osmotic Stress Leads to Decreased Intracellular pH of Listeria monocytogenes as Determined by Fluorescence Ratio-Imaging Microscopy

Intracellular pH (pH_i) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH_i, and recovery on solid medium was impaired compared to that in liquid medium. N,N′-dicyclohexylcarbodiimide abolished pH_i recovery, and lowering a_w with glycerol showed no effect on pH_i.     

Sensitization of Listeria monocytogenes to Low pH, Organic Acids, and Osmotic Stress by Ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

Specificity of the Anamnestic Response Produced by Listeria monocytogenes or Mycobacterium tuberculosis to Challenge With Listeria monocytogenes

When mice immunized with Listeria monocytogenes were given a second injection of listeria, they showed an anamnestic immune response to intravenous challenge with listeria, as measured by enumeration of the viable infecting organisms in the spleens of the infected animals. This response was independent of the effects of the challenge dose. When mice immunized with living or heat-killed attenuated mycobacterial cells were boosted with living H37Ra, there was also an accelerated response to listeria challenge. The response was greater in the mice given the primary immunization with living cells than in those immunized with heat-killed cells. The response to listeria challenge in mice immunized and boosted with mycobacteria was of less magnitude than that in the mice immunized and boosted with listeria. Growth of listeria in the mice immunized and boosted with mycobacteria was retarded only during the first 2 days of the infection, whereas the infecting listeria in mice immunized and boosted with listeria were permanently inactivated. Mice immunized with mycobacterial ribosomal fraction and restimulated with living mycobacterial cells showed no accelerated response to listeria challenge. It is evident from these results that resistance to these organisms is specifically evoked, but that once evoked it is not completely nonspecific in action. Also, the resistance produced by the mycobacterial ribosomal fraction to challenge with mycobacteria is completely specific in action. Therefore, it has been shown that there are two mechanisms involved in acquired immunity to facultative, intracellular parasites. One is nonspecific and mediated by activated macrophages. The other is specific and mediated by a mechanism as yet unknown.     

Influence of Stress on Single-Cell Lag Time and Growth Probability for Listeria monocytogenes in Half Fraser Broth

The impacts of 12 common food industry stresses on the single-cell growth probability and single-cell lag time distribution of Listeria monocytogenes were determined in half Fraser broth, the primary enrichment broth of the International Organization for Standardization detection method. First, it was determined that the ability of a cell to multiply in half Fraser broth is conditioned by its history (the probability for a cell to multiply can be decreased to 0.05), meaning that, depending on the stress in question, the risk of false-negative samples can be very high. Second, it was established that when cells are injured, the single-cell lag times increase in mean and in variability and that this increase represents a true risk of not reaching the detection threshold of the method in the enrichment broth. No relationship was observed between the impact on single-cell lag times and that on growth probabilities. These results emphasize the importance of taking into account the physiological state of the cells when evaluating the performance of methods to detect pathogens in food.Listeria monocytogenes has been involved in severe food-borne outbreaks with high mortality rates. This pathogen is widespread in many environments (16) and can be isolated from a large variety of foods which are the major routes of infection in humans. Ready-to-eat foods that can support the growth of L. monocytogenes may pose a major risk for public health, and the European Union legislation generally requires absence in 25 g at the production stage as a food safety criterion for this type of food (4).In food, L. monocytogenes is often affected by one or more stresses caused by a variety of processing treatments, including heating, freezing, and exposure to acids and to high osmotic pressures (15, 25, 29, 39). Recovering stressed L. monocytogenes from food is of great importance in food safety since sublethally injured bacteria may repair themselves under suitable conditions and regain or even increase their pathogenicity (19, 30).The injury of microbial cells has two major consequences for pathogen behavior in enrichment broths. First, injured cells become sensitive to selective components present in enrichment broths to which they normally show resistance (9, 10, 11, 42). Therefore, some cells of the stressed bacterial population do not initiate growth in enrichment broth, eventually resulting in an inefficient detection of pathogenic bacteria in food samples (50). This phenomenon can explain results obtained in several studies showing the effect of inoculum size on the growth limits of bacterial populations (26, 27, 37). Second, due to repair time, stressed cells show a longer lag phase than do healthy cells (5, 7, 37). This situation results in a true risk of not reaching the bacterial concentration necessary for the detection of the pathogen (in the range of 10² to 10⁴ CFU ml⁻¹) within the enrichment duration.The recent development of gene-based or immunologically based procedures, such as PCR, gene probes, and enzyme-linked immunosorbent assay, has facilitated the development of more-rapid methods which can identify positive samples in considerably shorter time periods. Nevertheless, these relatively rapid tests also require efficient enrichment steps to increase target organism numbers to detectable levels.At the moment of pathogen detection, low numbers of sublethally injured cells, as often encountered in naturally contaminated foods, show a wide distribution of lag-phase durations (45) and may not be able to multiply in broth containing selective components (11, 42). The challenge of the enrichment stage is to obtain appropriate enrichment conditions (2) which will favor pathogen resuscitation and limit the food microflora growth.In our study, we have focused on the primary enrichment phase of the International Organization for Standardization 11290-1 L. monocytogenes detection method (3), i.e., the half Fraser broth (1/2FB). The objectives were to investigate the impact of 12 different stresses on the single-cell growth probability and single-cell lag time of L. monocytogenes in 1/2FB. The intraspecific variability and the impact of food components and background microflora on single-cell growth probability were also studied.     

Three Transporters Mediate Uptake of Glycine Betaine and Carnitine by Listeria monocytogenes in Response to Hyperosmotic Stress

The uptake and accumulation of the potent osmolytes glycine betaine and carnitine enable the food-borne pathogen Listeria monocytogenes to proliferate in environments of elevated osmotic stress, often rendering salt-based food preservation inadequate. To date, three osmolyte transport systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and a carnitine transporter OpuC. We investigated the specificity of each transporter towards each osmolyte by creating mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state osmolyte accumulation data together with growth rate experiments demonstrated that osmotically activated glycine betaine transport is readily and effectively mediated by Gbu and BetL and to a lesser extent by OpuC. Osmotically stimulated carnitine transport was demonstrated for OpuC and Gbu regardless of the nature of stressing salt. BetL can mediate weak carnitine uptake in response to NaCl stress but not KCl stress. No other transporter in L. monocytogenes 10403S appears to be involved in osmotically stimulated transport of either osmolyte, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown under elevated osmotic stress.     

Osmotic stress leads to decreased intracellular pH of Listeria monocytogenes as determined by fluorescence ratio-imaging microscopy

Intracellular pH (pH(i)) of Listeria monocytogenes was determined after exposure to NaCl or sorbitol in liquid and solid media (agar). Both compounds decreased pH(i), and recovery on solid medium was impaired compared to that in liquid medium. N,N'-dicyclohexylcarbodiimide abolished pH(i) recovery, and lowering a(w) with glycerol showed no effect on pH(i).     

Identification of Listeria monocytogenes Genes Expressed in Response to Growth at Low Temperature

Listeria monocytogenes is a food-borne bacterial pathogen that is able to grow at refrigeration temperatures. To investigate microbial gene expression associated with cold acclimation, we used a differential cDNA cloning procedure known as selective capture of transcribed sequences (SCOTS) to identify bacterial RNAs that were expressed at elevated levels in bacteria grown at 10°C compared to those grown at 37°C. A total of 24 different cDNA clones corresponding to open reading frames in the L. monocytogenes strain EGD-e genome were obtained by SCOTS. These included cDNAs for L. monocytogenes genes involved in previously described cold-adaptive responses (flaA and flp), regulatory adaptive responses (rpoN, lhkA, yycJ, bglG, adaB, and psr), general microbial stress responses (groEL, clpP, clpB, flp, and trxB), amino acid metabolism (hisJ, trpG, cysS, and aroA), cell surface alterations (fbp, psr, and flaA), and degradative metabolism (eutB, celD, and mleA). Four additional cDNAs were obtained corresponding to genes potentially unique to L. monocytogenes and showing no significant similarity to any other previously described genes. Northern blot analyses confirmed increased steady-state levels of RNA for all members of a subset of genes examined during growth at a low temperature. These results indicated that L. monocytogenes acclimation to growth at 10°C likely involves amino acid starvation, oxidative stress, aberrant protein synthesis, cell surface remodeling, alterations in degradative metabolism, and induction of global regulatory responses.     

Solubility and antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli as influenced by pH

The antimicrobial efficacy of protamine on Listeria monocytogenes and Escherichia coli was evaluated at concentrations from 50 to 10 000 microgram ml-1 and pH levels from 5.5 to 8.0. The minimum inhibitory concentrations decreased with increasing pH. Protamine inhibited E. coli at all pH values while L. monocytogenes was inhibited at pH 6.5 and above. The antimicrobial efficacy of protamine decreased in the presence of negatively charged gelatine B but remained almost unchanged with addition of the positively charged gelatine A. Binding studies showed that the amount of protamine adsorbed to culture media components in tryptic soy broth and bacterial cells increased with increasing pH values. The increased efficacy of protamine at alkaline pH may be explained on the basis of an increase in electrostatic affinity for the cell surface of target cells. E. coli produced a protamine-degrading enzyme, however, was still susceptible to protamine.     

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.     

Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis

Listeria monocytogenes is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700 L. monocytogenes mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm² relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (fliP, flhB, flgD, flgL) and motor control (motB, fliM, fliY), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (P < 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.     

Sensitization of Listeria monocytogenes to low pH, organic acids, and osmotic stress by ethanol

The killing of Listeria monocytogenes following exposure to low pH, organic acids, and osmotic stress was enhanced by the addition of 5% (vol/vol) ethanol. At pH 3, for example, the presence of this agent stimulated killing by more than 3 log units in 40 min of exposure. The rate of cell death at pH 3.0 was dependent on the concentration of ethanol. Thus, while the presence 10% (vol/vol) ethanol at pH 3.0 stimulated killing by more than 3 log units in just 5 min, addition of 1.25% (vol/vol) ethanol resulted in less than 1 log unit of killing in 10 min. The ability of 5% (vol/vol) ethanol to stimulate killing at low pH and at elevated osmolarity was also dependent on the amplitude of the imposed stress, and an increase in the pH from 3.0 to 4.0 or a decrease in the sodium chloride concentration from 25 to 2.5% led to a marked reduction in the effectiveness of 5% (vol/vol) ethanol as an augmentative agent. Combinations of organic acids, low pH, and ethanol proved to be particularly effective bactericidal treatments; the most potent combination was pH 3.0, 50 mM formate, and 5 % (vol/vol) ethanol, which resulted in 5 log units of killing in just 4 min. Ethanol-enhanced killing correlated with damage to the bacterial cytoplasmic membrane.     

The effect of culture medium and aeration on growth of Listeria monocytogenes at pH 4.5

Listeria monocytogenes multiplied at 20°C in medium adjusted to pH 4.5 with HCl, and the lag before growth was eliminated when the inoculum was grown to log phase in the same medium. In a tryptone soya medium with yeast extract and added glucose, growth at pH 4.5 was more rapid than in a tryptose phosphate medium, and this difference was greater in air than under nitrogen. The results show that the bacterium was capable of more rapid growth in air than under nitrogen at this pH and suggest that the tryptose phosphate medium was nutritionally limiting for growth.     

Proteolytic Pathways of Activation and Degradation of a Bacterial Phospholipase C during Intracellular Infection by Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment. Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC). PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L. monocytogenes metalloprotease (Mpl). Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway. Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity. Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner. Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1–positive vacuoles. Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases. Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A₁, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment. These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.