Proteomic and microscopic analysis of biofilms formed byListeria monocytogenes568

2005 ◽  
Vol 51 (3) ◽  
pp. 197-208 ◽  
Author(s):  
M A Hefford ◽  
S D'Aoust ◽  
T D Cyr ◽  
J W Austin ◽  
G Sanders ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Response ◽  
Physiological State ◽  
Polyacrylamide Gel Electrophoresis ◽  
Microscopic Analysis ◽  
Test Surface ◽  
Food Borne ◽  
Ph Range ◽  
Regulatory Functions ◽  
Response Envelope

Biofilm formation may be important in the colonization of the food-processing environment by the food-borne pathogen Listeria monocytogenes. Listeria monocytogenes 568 formed adherent multicellular layers on a variety of test surfaces following growth at 37 °C with multiple transfers of the test surface into fresh medium. Microscopic examination of these adherent layers suggest that the cells were surrounded by extracellular material. The presence of a carbohydrate containing extracellular polymeric matrix was confirmed by labelling hydrated adherent layers with fluorescein-conjugated concanavalin A, indicating that these adherent layers are biofilms. To gain insight into the physiological state of cells in these biofilms, the proteomes from biofilm- and planktonic-grown cells from the same cultures were compared using 2-dimensional polyacrylamide gel electrophoresis. Nineteen proteins, which exhibited higher levels of expression in biofilm-grown cells, were successfully identified from the 2-D gels using a combination of MALDI-TOF and MS/MS. Proteins that were found to be more highly expressed in biofilm-grown cells were involved in stress response, envelope and protein synthesis, biosynthesis, energy generation, and regulatory functions. In biofilm-grown cells, many proteins in the pH range 4–6 ran as multiple spots arranged horizontally across the 2-D gels.Key words: Listeria monocytogenes, biofilms, proteomics, stress response.

Biofilm Formation and Associated Genes in Listeria Monocytogenes

2017 ◽  
Vol 12 (3) ◽  
Author(s):  
S. R. Warke ◽  
V. C. Ingle ◽  
N. V. Kurkure ◽  
P. A. Tembhurne ◽  
Minakshi Prasad ◽  
...  
Keyword(s):  
Public Health ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Food Processing ◽  
Biofilm Formation ◽  
Milk Samples ◽  
Biofilm Production ◽  
Food Borne ◽  
Serious Infections ◽  
Microtiter Assay

Listeria monocytogenes, an opportunistic food borne pathogen can cause serious infections in immunocompromised individuals. L. monocytogenes is capable of producing biofilm on the surface of food processing lines and instruments.The biofilm transfers contamination to food products and impose risk to public health. In the present study biofilm producing ability of L. monocytogenes isolates were investigated phenotypically and genotypically by microtiter assay and multiplex PCR, respectively. Out of 38 L. monocytogenes isolates 14 were recovered from animal clinical cases, 12 bovine environment and 12 from milk samples. A total of 3 (21.42%) clinical, 2 (16.66%) environment and 3 (25%) milk samples respectively, revealed biofilm production in microtiter assay. Cumulative results showed that 23 (60.52%) out of 38 strains of L. monocytogenes were positive for luxS and flaA gene and 1 (2.63%) was positive only for the flaA gene.

scholarly journals Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1484
Author(s):  
Felice Panebianco ◽  
Selene Rubiola ◽  
Francesco Chiesa ◽  
Tiziana Civera ◽  
Pierluigi Aldo Di Ciccio
Keyword(s):  
Listeria Monocytogenes ◽  
In Vitro Study ◽  
Planktonic Cells ◽  
Free Living ◽  
Biofilm Inhibition ◽  
Additional Tool ◽  
Complete Inactivation ◽  
Gaseous Ozone ◽  
Food Borne

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

scholarly journals Not Just Transporters: Alternative Functions of ABC Transporters in Bacillus subtilis and Listeria monocytogenes

2021 ◽  
Vol 9 (1) ◽  
pp. 163
Author(s):  
Jeanine Rismondo ◽  
Lisa Maria Schulz
Keyword(s):  
Bacillus Subtilis ◽  
Listeria Monocytogenes ◽  
Abc Transporters ◽  
Organic Molecules ◽  
Atp Hydrolysis ◽  
The Past ◽  
Wide Range ◽  
Regulatory Functions ◽  
Complex Organic ◽  
Detoxification Mechanisms

ATP-binding cassette (ABC) transporters are usually involved in the translocation of their cognate substrates, which is driven by ATP hydrolysis. Typically, these transporters are required for the import or export of a wide range of substrates such as sugars, ions and complex organic molecules. ABC exporters can also be involved in the export of toxic compounds such as antibiotics. However, recent studies revealed alternative detoxification mechanisms of ABC transporters. For instance, the ABC transporter BceAB of Bacillus subtilis seems to confer resistance to bacitracin via target protection. In addition, several transporters with functions other than substrate export or import have been identified in the past. Here, we provide an overview of recent findings on ABC transporters of the Gram-positive organisms B. subtilis and Listeria monocytogenes with transport or regulatory functions affecting antibiotic resistance, cell wall biosynthesis, cell division and sporulation.

scholarly journals Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance

2008 ◽  
Vol 74 (22) ◽  
pp. 6848-6858 ◽  
Author(s):  
F. Abram ◽  
E. Starr ◽  
K. A. G. Karatzas ◽  
K. Matlawska-Wasowska ◽  
A. Boyd ◽  
...  
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Stress Tolerance ◽  
Intact Cell ◽  
Microscopic Analysis ◽  
Phenotypic Characterization ◽  
Carbon Utilization ◽  
Two Dimensional Gel Electrophoresis ◽  
Sigma B ◽  
The Impact

ABSTRACT Sigma B (σB) is an alternative sigma factor that controls the transcriptional response to stress in Listeria monocytogenes and is also known to play a role in the virulence of this human pathogen. In the present study we investigated the impact of a sigB deletion on the proteome of L. monocytogenes grown in a chemically defined medium both in the presence and in the absence of osmotic stress (0.5 M NaCl). Two new phenotypes associated with the sigB deletion were identified using this medium. (i) Unexpectedly, the strain with the ΔsigB deletion was found to grow faster than the parent strain in the growth medium, but only when 0.5 M NaCl was present. This phenomenon was independent of the carbon source provided in the medium. (ii) The ΔsigB mutant was found to have unusual Gram staining properties compared to the parent, suggesting that σB contributes to the maintenance of an intact cell wall. A proteomic analysis was performed by two-dimensional gel electrophoresis, using cells growing in the exponential and stationary phases. Overall, 11 proteins were found to be differentially expressed in the wild type and the ΔsigB mutant; 10 of these proteins were expressed at lower levels in the mutant, and 1 was overexpressed in the mutant. All 11 proteins were identified by tandem mass spectrometry, and putative functions were assigned based on homology to proteins from other bacteria. Five proteins had putative functions related to carbon utilization (Lmo0539, Lmo0783, Lmo0913, Lmo1830, and Lmo2696), while three proteins were similar to proteins whose functions are unknown but that are known to be stress inducible (Lmo0796, Lmo2391, and Lmo2748). To gain further insight into the role of σB in L. monocytogenes, we deleted the genes encoding four of the proteins, lmo0796, lmo0913, lmo2391, and lmo2748. Phenotypic characterization of the mutants revealed that Lmo2748 plays a role in osmotolerance, while Lmo0796, Lmo0913, and Lmo2391 were all implicated in acid stress tolerance to various degrees. Invasion assays performed with Caco-2 cells indicated that none of the four genes was required for mammalian cell invasion. Microscopic analysis suggested that loss of Lmo2748 might contribute to the cell wall defect observed in the ΔsigB mutant. Overall, this study highlighted two new phenotypes associated with the loss of σB. It also demonstrated clear roles for σB in both osmotic and low-pH stress tolerance and identified specific components of the σB regulon that contribute to the responses observed.

scholarly journals Gbu Glycine Betaine Porter and Carnitine Uptake in Osmotically Stressed Listeria monocytogenes Cells

2002 ◽  
Vol 68 (11) ◽  
pp. 5647-5655 ◽  
Author(s):  
Mary Lou Mendum ◽  
Linda Tombras Smith
Keyword(s):  
Listeria Monocytogenes ◽  
Transport System ◽  
Glycine Betaine ◽  
Compatible Solutes ◽  
High Salt ◽  
Uptake System ◽  
Food Borne ◽  
Protein Mutants ◽  
Carnitine Uptake ◽  
Do So

ABSTRACT The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a Km of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 μM. This porter has a Km for glycine betaine uptake of about 6 μM. The dedicated carnitine porter, OpuC, has a Km for carnitine uptake of 1 to 3 μM and a V max of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by γ-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.

scholarly journals A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

2021 ◽  
Vol 18 (9) ◽  
pp. 4574
Author(s):  
Kai Chen ◽  
Biao Ma ◽  
Jiali Li ◽  
Erjing Chen ◽  
Ying Xu ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Vibrio Parahaemolyticus ◽  
Pathogenic Bacteria ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Quantitative Detection ◽  
Bacterial Strains ◽  
Food Borne Pathogens ◽  
Food Borne

Food-borne pathogens have become an important public threat to human health. There are many kinds of pathogenic bacteria in food consumed daily. A rapid and sensitive testing method for multiple food-borne pathogens is essential. Europium nanoparticles (EuNPs) are used as fluorescent probes in lateral flow immunoassays (LFIAs) to improve sensitivity. Here, recombinase polymerase amplification (RPA) combined with fluorescent LFIA was established for the simultaneous and quantitative detection of Listeria monocytogenes, Vibrio parahaemolyticus, and Escherichia coliO157:H7. In this work, the entire experimental process could be completed in 20 min at 37 °C. The limits of detection (LODs) of EuNP-based LFIA–RPA were 9.0 colony-forming units (CFU)/mL for Listeria monocytogenes, 7.0 CFU/mL for Vibrio parahaemolyticus, and 4.0 CFU/mL for Escherichia coliO157:H7. No cross-reaction could be observed in 22 bacterial strains. The fluorescent LFIA–RPA assay exhibits high sensitivity and good specificity. Moreover, the average recovery of the three food-borne pathogens spiked in food samples was 90.9–114.2%. The experiments indicate the accuracy and reliability of the multiple fluorescent test strips. Our developed EuNP-based LFIA–RPA assay is a promising analytical tool for the rapid and simultaneous detection of multiple low concentrations of food-borne pathogens.

Studies on diphosphopyridine nucleotidase and platelet damaging factor in an extracellular product of Listeria monocytogenes

1970 ◽  
Vol 16 (10) ◽  
pp. 909-916 ◽  
Author(s):  
I. H. Siddique ◽  
L. C. Ying ◽  
R. A. Chung
Keyword(s):  
Listeria Monocytogenes ◽  
Hemolytic Activity ◽  
Virulent Strain ◽  
Optimal Activity ◽  
Extracellular Product ◽  
Heat Labile ◽  
Ph Range

Hemolysin preparations from a virulent strain of Listeria monocytogenes were chromatographed on Sephadex G-100 and Sephadex DEAE A-50 columns. Three types of activities were identified: DPNase activity, hemolytic activity, and platelet-damaging activity. The separation of the peak with DPN-destroying activity from the peaks with hemolytic and platelet-damaging activities provided evidence that the factor in the solutions responsible for the destruction of DPN was distinct from that causing hemolysis and platelet-damage. The DPNase factor was found to be non-dialyzable, to be heat labile, and to have optimal activity in the pH range of 6.8–7.4.

Antimicrobial Effects of d-3-Phenyllactic Acid on Listeria monocytogenes in TSB-YE Medium, Milk, and Cheese

1998 ◽  
Vol 61 (10) ◽  
pp. 1281-1285 ◽  
Author(s):  
VIRGINIE DIEULEVEUX ◽  
MICHELINE GUÉGUEN
Keyword(s):  
Listeria Monocytogenes ◽  
Growth Phase ◽  
Physiological State ◽  
Geotrichum Candidum ◽  
Bactericidal Effect ◽  
Exponential Growth Phase ◽  
Bacteriostatic Effect ◽  
Experimental Conditions ◽  
Phenyllactic Acid ◽  
Whole Milk

d-3-Phenyllactic acid is a compound with anti-Listeria activity which is produced and secreted by the yeastlike fungus, Geotrichum candidum. This compound has a bactericidal effect independent of the physiological State of Listeria monocytogenes when added at a concentration of 7 mg/ml to tryptic soy broth supplemented with yeast extract (TSB-YE). An initial L. monocytogenes population of 105 CFU/ml was reduced 100-fold (2 log) after 4 days of culture at 25 °C in TSB-YE containing d-3-phenyllactic acid. The Listeria population was reduced 1,000-fold (3 log) when the compound was added during the exponential growth phase, and was reduced to less than 10 CFU/ml when it was added during the stationary phase. d-3-Phenyllactic acid had a bacteriostatic effect in UHT whole milk, reducing the population by 4.5 log, to give fewer cells than in the control after 5 days of culture. The results obtained with L. monocytogenes at concentrations of 105 and 103 CFU/ml in cheese curds were less conclusive. d-3-Phenyllactic acid was 10 times less active than nisin in our experimental conditions (TSB-YE at 25°C).

scholarly journals Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

1978 ◽  
Vol 175 (2) ◽  
pp. 743-750 ◽  
Author(s):  
P Calvo ◽  
A Reglero ◽  
J A Cabezas
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mixed Substrates ◽  
Ph Optimum ◽  
Terrestrial Mollusc ◽  
Buffer Solutions ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Ph Range

1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8–4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site.

Growth, Inhibition, and Survival of Listeria monocytogenes as Affected by Acidic Conditions

1990 ◽  
Vol 53 (8) ◽  
pp. 652-655 ◽  
Author(s):  
DONALD E. CONNER ◽  
VIRGINIA N. SCOTT ◽  
DANE T. BERNARD
Keyword(s):  
Lactic Acid ◽  
Listeria Monocytogenes ◽  
Growth Inhibition ◽  
Propionic Acid ◽  
Cell Populations ◽  
Growth And Survival ◽  
Viable Cells ◽  
Acidic Conditions ◽  
Ph Range ◽  
Lactic Acids

Growth and survival of four strains of Listeria monocytogenes under acidic conditions were investigated. Tryptic soy broth with yeast extract (TSBYE) was acidified with acetic, citric, hydrochloric, lactic, or propionic acid to pH 4.0–6.0, inoculated with L. monocytogenes and incubated at 30 or 4°C. The minimum test pH at which L. monocytogenes did not grow (inhibitory pH) was determined for each acid. In the pH range tested, this inhibitory pH was 5.0 for propionic acid, 4.5 for acetic and lactic acids, and 4.0 for citric and hydrochloric acids. All four strains gave similar results. Subsequent studies were conducted at 10 and 30°C to determine changes in cell populations in TSBYE adjusted to each inhibitory pH. Initial populations of viable cells (104 CFU/ml) were reduced to <10 CFU/ml within 1–3 weeks at 30°C, whereas at 10°C, L. monocytogenes survived for 11–12 weeks in acetic, citric, or propionic acid-adjusted media and for 6 weeks in media adjusted with hydrochloric or lactic acid. The concentration of undissociated lactic acid was 0.002 M at pH 4.5.

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