Use of PCR-Restriction Fragment Length Polymorphism of inlA for Rapid Screening of Listeria monocytogenes Strains Deficient in the Ability To Invade Caco-2 Cells

A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA. inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA. Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened. Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles. Thirteen L. monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA. Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells. However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA. Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L. monocytogenes strains which were isolated from food. This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin. Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L. monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied.     

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.     

Exposure to salt and organic acids increases the ability of Listeria monocytogenes to invade Caco-2 cells but decreases its ability to survive gastric stress

The effects of environmental stress exposure on Listeria monocytogenes growth and virulence-associated characteristics were investigated. Specifically, we measured the effects of temperature (7 or 37 degrees C), pH (5.5 or 7.4), the presence of salt and organic acids (375 mM NaCl, 8.45 mM sodium diacetate [SD], 275 mM sodium lactate [SL], or a combination of NaCl, SD, and SL), and deletion of sigB, which encodes a key stress response regulator, on the ability of L. monocytogenes to grow, invade Caco-2 cells, and survive exposure to synthetic gastric fluid (pH 2.5 or 4.5). Our results indicate that (i) L. monocytogenes log-phase generation times and maximum cell numbers are not dependent on the alternative sigma factor sigmaB in the presence of NaCl and organic acids at concentrations typically found in foods; (ii) growth inhibition of L. monocytogenes through the addition of organic acids is pH dependent; (iii) the ability of L. monocytogenes to invade Caco-2 cells is affected by growth phase, temperature, and the presence of salt and organic acids, with the highest relative invasion capabilities observed for cells grown with SL or NaCl at 37 degrees C and pH 7.4; (iv) growth of L. monocytogenes in the presence of NaCl, SD, or SL reduces its ability to survive exposure to gastric fluid; and (v) exposure of L. monocytogenes to gastric fluid reduces the enhanced invasiveness caused by growth in the presence of NaCl or SL. These findings suggest that virulence-associated characteristics that determine the L. monocytogenes infectious dose are likely to be affected by food-specific properties (e.g., pH or the presence of salt or organic acid).     

Invasion assay of Listeria monocytogenes using Vero and Caco-2 cells

The invasion ability of Listeria monocytogenes into cultured cells has been used to evaluate its pathogenicity. In this study, invasive ability was investigated using Vero and Caco-2 cell lines. The form of invasion showed no morphological differences between both cell lines inoculated with L. monocytogenes L89-H2 or L96-23C1 strains when double fluorescence stained with rhodamine and FITC or with Giemsa staining. Recovery count and recovery rate of L. monocytogenes from Vero cells was related to the number of inoculated bacteria (2 x 10(5) to 2 x 10(7)/ml) in a bell-shape pattern, though the relationship was unclear in Caco-2 cells. Recovery rate of L. monocytogenes was higher in Vero cells than Caco-2 cells at a multiplicity of infection (MOI) 10, though the rates in both cells showed different stable stages over a considerably wide range of MOI. The recovery rate of all five L. monocytogenes strains from listeriosis patients was 15% at MOI 10 from infected Vero cells, while meat-derived strains showed variable rates regardless of the serovar. These results suggest that the Vero cell line is suitable for an invasion assay and that a recovery rate of 15% may be the critical limit for the expression of pathogenicity in the host.     

Lactobacillus casei DN-114 001 Inhibits the Ability of Adherent-Invasive Escherichia coli Isolated from Crohn's Disease Patients To Adhere to and To Invade Intestinal Epithelial Cells

Ileal lesions in 36.4% of patients with Crohn's disease are colonized by pathogenic adherent-invasive Escherichia coli. The aim of this study was to determine the in vitro inhibitory effects of the probiotic strain, Lactobacillus casei DN-114 001, on adhesion to and invasion of human intestinal epithelial cells by adherent-invasive E. coli isolated from Crohn's disease patients. The experiments were performed with undifferentiated Intestine-407 cells and with undifferentiated or differentiated Caco-2 intestinal epithelial cells. Bacterial adhesion to and invasion of intestinal epithelial cells were assessed by counting CFU. The inhibitory effects of L. casei were determined after coincubation with adherent-invasive E. coli or after preincubation of intestinal cells with L. casei prior to infection with adherent-invasive E. coli. Inhibitory effects of L. casei on adherent-invasive E. coli adhesion to differentiated and undifferentiated intestinal epithelial cells reached 75% to 84% in coincubation and 43% to 62% in preincubation experiments, according to the cell lines used. Addition of L. casei culture supernatant to the incubation medium increased L. casei adhesion to intestinal epithelial cells and enhanced the inhibitory effects of L. casei. The inhibitory effects on E. coli invasion paralleled those on adhesion. This effect was not due to a bactericidal effect on adherent-invasive E. coli or to a cytotoxic effect on epithelial intestinal cells. As Lactobacillus casei DN-114 001 strongly inhibits interaction of adherent-invasive E. coli with intestinal epithelial cells, this finding suggests that the probiotic strain could be of therapeutic value in Crohn's disease.     

Factors Influencing the Ability of Listeria monocytogenes To Pass through a Membrane Filter by Active Infiltration

Listeria monocytogenes infiltrated the reticulate structure of a membrane filter and passed through a filter with pore sizes of 0.45 μm and 0.2 μm in 6 to 24 h and 5 to 6 days, respectively. Flagellar motility and expansive pressure generated by the growing bacterial population were indicated as the driving forces of infiltration.     

Survival and Heat Resistance of Listeria monocytogenes after Exposure to Alkali and Chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of L. monocytogenes incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log₁₀ CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log₁₀ CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log₁₀-unit reductions is not compromised in these cells.     

Antimicrobial activities of therapeutic herbal plants against Listeria monocytogenes and the herbal plant cytotoxicity on Caco-2 cell

Aims: This study investigated the antimicrobial effect of various therapeutic herbal plants on Listeria monocytogenes, and their cytotoxicity effect on mammalian cells. Methods and Results: The extracts from 69 therapeutic herbal plants were used to investigate the effect on the growth inhibition of L. monocytogenes, and their minimal inhibition concentrations and minimal bactericidal concentrations were determined. Among the plants, Psoraleae semen L. (Bogolji) and Sophorae radix L. (Gosam) extracts, which showed obvious antilisterial activity, were examined for the stability to heat, NaCl and acidic condition. Moreover, cytotoxicities of Bogolji and Gosam were tested, using Caco‐2 cells. L. monocytogenes growth was completely inhibited by Bogolji and Gosam extracts at 3·2–6·3 and 50–100 AU ml^?1, respectively, and heat, NaCl and acidic condition did not affect the antilisterial activity of Bogolji and Gosam. Cytotoxic activities were observed only at high concentration (50 AU ml^?1) of Bogolji extract. Conclusion: Bogolji and Gosam could be considered as potential phytochemicals to control L. monocytogenes. Significance and Impact of the Study: Use of therapeutic herbal plants should be useful in controlling L. monocytogenes, because most consumers have better acceptance for phytochemicals than synthetic chemicals.     

N-Terminal Polypeptide of Annexin A2 Decreases Infection of Mycoplasma hyorhinis to Gastric Cancer Cells

Mycoplasma infection in human and its contamination in cell cultures are worldwide problems. The drugs currently available for preventing or treating mycoplasma infection suffer from low sensitivity, strong resistance and high toxicity. Our previous work showed that Mycoplasma hyorhinis (M. hyorhinis) infection was mediated by the interaction between p37 of M. hyorhinis and Annexin A2 (ANXA2) of host cells, however the translational value of this mechanism was unknown. Herein, we synthesized the N-terminal of ANXA2 polypeptide (A2PP) and found that A2PP could decrease the infection of M. hyorhinis to gastric cancer cells and block M. hyorhinis infection-induced cell migration. Furthermore, we found that A2PP could reduce M. hyorhinis contamination of passage cells. Moreover, compared with the commercial antibiotics commonly used in cell culture to prevent M. hyorhinis infection, A2PP demonstrated a more effectiveness but a low toxicity on cell growth. Thus, our study for the first time revealed A2PP’s potential for the treatment and prevention of M. hyorhinis infection.     

Lactobacillus casei NY1301 Increases the Adhesion of Lactobacillus gasseri NY0509 to Human Intestinal Caco-2 Cells

In the presence of Lactobacillus casei NY1301, the adhesion of Lactobacillus gasseri NY0509 to cultured human intestinal Caco-2 cells was significantly increased (P<0.01). In contrast, L. gasseri NY0509 did not affect the adhesion of L. casei NY1301. A heat-stable cell component of L. casei NY1301 was involved in this increase of adhesion. These results suggest that a combination of these strains may have synergistic effects of adhesion to human intestinal mucosa.     

Characterization of Glycine Betaine Porter I from Listeria monocytogenes and Its Roles in Salt and Chill Tolerance

Listeria monocytogenes is a pathogenic bacterium that can grow at low temperatures and elevated osmolarity. The organism survives these stresses by the intracellular accumulation of osmolytes: low-molecular-weight organic compounds which exert a counterbalancing force. The primary osmolyte in L. monocytogenes is glycine betaine, which is accumulated from the environment via two transport systems: glycine betaine porter I, an Na⁺-glycine betaine symporter; and glycine betaine porter II, an ATP-dependent transporter. The biochemical characteristics of glycine betaine porter I were investigated in a mutant strain (LTG59) lacking the ATP-dependent transporter. At 4% NaCl, glycine betaine uptake in LTG59 was about fivefold lower than in strain DP-L1044, which has both transporters, indicating that the ATP-dependent transporter is the primary means by which glycine betaine enters the cell. In the absence of osmotic stress, cold-activated uptake by both transporters was most rapid between 7 and 12°C, but a larger fraction of the total uptake was via the ATP-dependent transporter than was observed under salt-stressed conditions. Twelve glycine betaine analogs were tested for their ability to inhibit glycine betaine uptake and growth of stressed cultures. Carnitine, dimethylglycine, and γ-butyrobetaine appear to inhibit the ATP-dependent transporter, while trigonelline and triethylglycine primarily inhibit glycine betaine porter I. Triethylglycine was also able to retard the growth of osmotically stressed L. monocytogenes grown in the presence of glycine betaine.     

Adhesion,invasion, and translocation characteristics of Listeria monocytogenes serotypes in Caco-2 cell and mouse models

Adhesion is a crucial first step in Listeria monocytogenes pathogenesis. In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model. Adhesion characteristics of all 13 serotypes of L. monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles. The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R(2)) was only 0.1236. In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles. However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R(2) = 0.3371). Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.     

Adhesion, Invasion, and Translocation Characteristics of Listeria monocytogenes Serotypes in Caco-2 Cell and Mouse Models

Adhesion is a crucial first step in Listeria monocytogenes pathogenesis. In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model. Adhesion characteristics of all 13 serotypes of L. monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles. The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R²) was only 0.1236. In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles. However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R² = 0.3371). Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Microplate Fluorescence Assay for Measurement of the Ability of Strains of Listeria monocytogenes from Meat and Meat-Processing Plants To Adhere to Abiotic Surfaces

Listeria monocytogenes is a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for the ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30°C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25°C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40°C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments.