Listeria Monocytogenes ScottA Research Articles

AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

Safety evaluation and identification of key genes from nisin operon in bacteriocinogenic strains isolated from goat milk

Lactococcus lactis bacteriocin producers' strains were differentiated by repPCR. Applied chemicals, pH and temperature showed specific effect on the bacteriocin stability. Addition of CFS to the actively growing cultures of Listeria monocytogenes ScottA, Lactobacillus sakei ATCC 15521 or Enterococcus faecium ATCC 19434 resulted in the complete inhibition of bacterial growth. Different levels of adsorption of bacteriocin produced by studied strains to the test organisms were observed on bacteriocin and test organisms' specific manner, affected by temperature, pH and selected chemicals, important results for the prediction of potential optimal application of studied bacteriocins. Only between 200 AU/mL and 400 AU/mL were levels of adsorption of the bacteriocins to the cells surface on producers’ cells. The five tested strains resulted negative for the presence of different bacteriocins genes, except for nisin. Moreover, the targeted genes, parts of nisin operon, were detected partially in the DNA of DF2Mi and DF60Mi, suggesting that the bacteriocin(s) produced by these two strains differ from nisin. Studied strains were evaluated for presence of different virulence, antibiotics resistance and biogenic amines production associated genes, an important feature for their safety evaluation. Interaction between studied strains and commercial medicaments were evaluated in order to better model their potential application as probiotics.

Bazı gıda kaynaklı patojenler üzerinde geleneksel yoğurt üretiminde kullanılan ticari starter kültürlerin ve probiyotiklerin antimikrobiyal etkisi

Objective: Although many techniques have been developed for food preservation, foodborne diseases are still an important problem. The studies aimed at solving this problem have increased in recent years using lactic acid bacteria with antimicrobial activities in foods. Materials and Methods: In this study, the antimicrobial effects of various lactic acid bacteria species, commercial yoghurt and probiotic yoghurt cultures and their supernatants on pathogen bacteria including Salmonella typmimurium NRLL E4463, Listeria monocytogenes Scott-A, Escherichia coli O157:H7, Staphylococcus aureus 6538P were examined by well diffusion and disc diffusion methods Results: It was determined that there were no statistical differences between the well diffusion and disc diffusion methods in terms of antimicrobial effects. It was also found that all of the lactic acid bacteria in MRS broth had substantial antimicrobial activities against the pathogens in both diffusion methods; however the antimicrobial effects of the supernatants obtained from cultures developed in media prepared from 10% skim milk powder showed diversity in terms of antimicrobial activity. Conclusion: These commercial cultures are thought to make a positive contribution with to help in the control of foodborne diseases.

Antioxidant Properties and Dominant Bacterial Community of Fermented Rohu (Labeo rohita) Sauce Produced by Enzymatic and Fermentation Method

Sauce was produced from Rohu (Labeo rohita) by enzymatic and fermentatively under conditions optimized by response surface method. Salt (20% w/w) and commercial papain (3% w/w) were employed for enzymatic production; while, salt (25%, w/w), sugar (7.5%, w/w) and lactic culture (10%, w/v; Pediococcus pentosaceus FSBP4-40) were used in case of fermentative production. Total antioxidant activity (as ascorbic acid, µg/ml), 2,2`-diphenyl-1-picryl-hydrazyl (DPPH, %) and 2,2`- azinobis-3-ethylbenzothizoline-6 sulphonate (ABTS; %) scavenging activities of fermented sauce samples were higher at 3441±60, 49.57±1.7 and 99.0±0.3, respectively; as compared to enzyme treated samples which showed 803±13, 28.60±1.1 and 78.4±1.0, for the respective parameters, after 180 days storage at 37°C. Superoxide anion scavenging activity and reducing potential were found significantly higher (P<0.05) in both fermented as well as enzymatically prepared sauces in comparison to control. Fermented sauce exhibited excellent antibacterial property against Listeria monocytogenes ScottA. Bacterial counts such as total plate count and halophile count, after 180 days, were significantly lower (P<0.05) in the fermentatively produced sauce than enzymatically prepared sauce. Pediococcus, Micrococcus, Enterococcus and Staphylococcus were the dominant bacterial genera in fermented sauce. The study emphasizes the role of accelerating fermentation process, either enzymatically or fermentatively, to produce biofunctionally and bacteriologically superior rohu sauce.

In vitro Anti-Adherent Assessment of Selected Lactic Acid Bacteria Isolates Against S almonella Typhimurium and L isteria monocytogenes to Caco-2 Cells

The present study aimed to evaluate in vitro anti-adherence abilities of the probiotic potential of three selected lactic acid bacteria (LAB) strains isolated from fermented broken rice with two foodborne pathogenic bacteria to Caco-2 cells. Lactobacillus plantarum Ban Pra Doke 110 was the most adherent strain with an adherence of 17.66% at a concentration of added bacterial cells of 2.0 × 108 cfu/mL (multiplicities of infection 100). In the exclusion, competition and displacement tests, Lact. plantarum PD 110 showed the most anti-adherence abilities against Salmonella Typhimurium American Type Culture Collection 13311 and Listeria monocytogenes ScottA to Caco-2 cells. Moreover, the presence of its spent culture supernatant exhibited the highest decrease in the percentage numbers of the adherent pathogenic bacteria used in all tests. The effectiveness of each LAB strain was tested to adhere to Caco-2 cells or to prevent the adherence of the test pathogenic bacteria varied widely depending on the genus and species, source of origin, and assay condition, as well as adherent-promoting factors. Practical Applications Foodborne pathogen infection to the human intestinal epithelial surface cells is a critical step for the survival and cause foodborne illness. The anti-adherent assessment of novel probiotic is of importance for the inhibition of pathogen infections. Lactobacillus plantarum PD 110 isolated from fermented broken rice could be one of these good candidates. The demonstrated in vitro anti-adherent activity of Lact. plantarum PD 110 to Caco-2 cells recommends its possible application as a probiotic candidates in order to inhibit the adherence of Salmonella Typhimurium and Listeria monocytogenes to the human gastrointestinal tract. Therefore, this strain can be considered as a novel potential starter culture to improve the quality and safety of functional foods.

Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources

Probiotics have established their efficacy as dietary adjuncts providing benefits to consumers. However, selection of probiotics before incorporation into diet requires close scrutiny in the form of in vitro as well as in vivo tests. Three bacteriocinogenic Bacillus sp., namely, B. licheniformis Me1, B. flexus Hk1, and B. subtilis Bn1 previously isolated from milk, cheese and fermented beans, respectively, were characterized for typical in vitro probiotic criteria. When compared to probiotic Bacillus coagulans, all three cultures were found to possess better acid and bile tolerance. Cultures Me1 and Bn1, except Hk1, showed bile salt hydrolase activity. A marked difference in adhesion to hydrocarbons and auto-aggregation properties from 10–80 and 60–99%, respectively, were observed for the tested cultures. Highest antioxidant activity was measured for culture Hk1 (66.6%), whereas least activity of 53% was observed for culture Bn1. Cultures Me1 and Bn1 were sensitive to all the antibiotics tested, whereas Hk1 and B. coagulans showed resistance to the penicillin group of β-lactum antibiotics. All the tested cultures showed a broad spectrum of activity against food-borne pathogens. In co-cultivation studies, B. licheniformis Me1 completely inhibited the growth of the indicator pathogen Listeria monocytogenes ScottA. Overall, the test cultures exhibiting potential probiotic characteristics, particularly B. licheniformis Me1, can serve as probiotics of commercial interest.

Evaluation of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 as probiotics by using a gastro-intestinal model with infant milk formulations as substrate

Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 produce bacteriocins with activity against a number of Gram-positive and Gram-negative bacteria. Both strains survived intestinal conditions simulated in a gastro-intestinal model (GIM) with infant milk formulations as substrate and prevented the growth of Listeria monocytogenes ScottA. The strains are inhibited by the antibiotics amoxicillin, cefadroxil, roxithromycin and doxycycline, anti-inflammatory medicaments containing meloxicam, ibuprofen and sodium diklofenak, and analgesics containing paracetamol, codeine phosphate and promethazine. Strain 423 is sensitive to vancomycin and does not contain genes encoding gelatinase, cell aggregation substance (AS), adhesion to collagen (Ace), enterococcus surface protein (Esp), Enterococcus faecalis endocarditis antigen (EfaAfs), cytolysin and non-cytolysin (β-hemolysin III). Genes encoding AS, cytolysin and non-cytolysin (β-hemolysin III) were amplified from the genome of strain ST4SA. Survival of strains ST4SA and 423 improved when used as combined cultures in the GIM and compared well with the survival of commercially available probiotics subjected to the same conditions.

Adhesion of the probiotic strains Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 cells under conditions simulating the intestinal tract, and in the presence of antibiotics and anti-inflammatory medicaments

Adhesion of Enterococcus mundtii ST4SA and Lactobacillus plantarum 423 to Caco-2 (human carcinoma epithelial) cells was visualized by fluorescent staining. Both strains showed good adhesion compared to L. casei MB1, L. casei Shirota, L. johnsonii La1 and L. rhamnosus GG. No correlation was found between hydrophobicity, aggregation and adhesion to Caco-2 cells. Presence of antibiotics and anti-inflammatory medicaments reduced adhesion of bacterial strains to Caco-2 cells. Proteins sensitive to pepsin, trypsin and pronase are involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. Adhesion of Listeria monocytogenes ScottA to Caco-2 cells was not prevented by E. mundtii ST4SA and L. plantarum 423. Cell-free culture supernatants of strains ST4SA and 423, containing the antimicrobial peptides plantaricin 423 and peptide ST4SA, prevented the invasion of L. monocytogenes ScottA into Caco-2 cells.

Oxygen restriction increases the infective potential of Listeria monocytogenes in vitro in Caco-2 cells and in vivo in guinea pigs

BackgroundListeria monocytogenes has been implicated in several food borne outbreaks as well as sporadic cases of disease. Increased understanding of the biology of this organism is important in the prevention of food borne listeriosis.The infectivity of Listeria monocytogenes ScottA, cultivated with and without oxygen restriction, was compared in vitro and in vivo. Fluorescent protein labels were applied to allow certain identification of Listeria cells from untagged bacteria in in vivo samples, and to distinguish between cells grown under different conditions in mixed infection experiments.ResultsInfection of Caco-2 cells revealed that Listeria cultivated under oxygen-restricted conditions were approximately 100 fold more invasive than similar cultures grown without oxygen restriction. This was observed for exponentially growing bacteria, as well as for stationary-phase cultures.Oral dosage of guinea pigs with Listeria resulted in a significantly higher prevalence (p < 0.05) of these bacteria in jejunum, liver and spleen four and seven days after challenge, when the bacterial cultures had been grown under oxygen-restricted conditions prior to dosage. Additionally, a 10–100 fold higher concentration of Listeria in fecal samples was observed after dosage with oxygen-restricted bacteria. These differences were seen after challenge with single Listeria cultures, as well as with a mixture of two cultures grown with and without oxygen restriction.ConclusionOur results show for the first time that the environmental conditions to which L. monocytogenes is exposed prior to ingestion are decisive for its in vivo infective potential in the gastrointestinal tract after passage of the gastric barrier. This is highly relevant for safety assessment of this organism in food.

Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions

To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco-2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219. Bacteriocin HV219 activity against Gram-positive and Gram-negative bacteria was confirmed by leakage of DNA and beta-galactosidase, and atomic force microscopy. Adsorption of bacteriocin HV219 to bacteria is influenced by pH, temperature, surfactants and salts. Initially, only 3% of HV219 cells adhered to Caco-2 cells. However, after 2 h, adherence increased to 7%. Strain HV219 and Listeria monocytogenes ScottA did not compete for colonization. Strain HV219 is sensitive to most antibiotics tested, but resistant to amikacin, ceftazidime, nalidixic acid, metronidazole, neomycin, oxacillin, streptomycin, sulphafurazole, sulphamethoxazole, sulphonamides, tetracycline and tobramycin. Ibuprofen, ciprofloxacin, diklofenak and nonoxylol-9 inhibited the growth of strain HV219. Strain HV219 is resistant to hostile conditions in the intestinal tract, including therapeutic levels of specific antibiotics and binds to Caco-2 cells, but not in competition with L. monocytogenes. Strain HV219 will only be effective as probiotic if taken with specific antibiotics and not with anti-inflammatory drugs and spermicides.

Essential Oils of Satureja, Origanum, and Thymus Species: Chemical Composition and Antibacterial Activities Against Foodborne Pathogens

The chemical composition of the essential oils obtained from the species restricted to Greece and the eastern Mediterranean region, Satureja spinosa L. and Thymus longicaulis L.; species endemic to central and south Greece, Satureja parnassica ssp. parnassica Heldr. and Sart ex Boiss.; species endemic to the island of Crete, Origanum dictamnus L.; and species widely distributed in the Mediterranean region, Satureja thymbra L. and Origanum vulgare L. subsp. hirtum, were determined by gas chromatography (GC) and GC/mass spectrometry (MS) analysis. The in vitro antibacterial activities of the essential oils were evaluated against a panel of five foodborne bacteria (Escherichia coli 0157:H7 NCTC 12900, Salmonella enteritidis PT4, Staphylococcus aureus ATCC 6538, Listeria monocytogenes ScottA, and Bacillus cereus FSS 134). The analytical data indicated that various monoterpene hydrocarbons and phenolic monoterpenes constitute the major components of the oils, but their concentrations varied greatly among the oils examined. The antibacterial assay results showed that 5 muL doses of the essential oils extracted from the endemic Satureja species in Greece possess remarkable bactericidal properties, which are clearly superior as compared to those of Origanum and Thymus species essential oils. Therefore, they represent an inexpensive source of natural mixtures of antibacterial compounds that exhibit potentials for use in food systems to prevent the growth of foodborne bacteria and extend the shelf life of the processed food.

Effects of divercin V41 combined to NaCl content, phenol (liquid smoke) concentration and pH on Listeria monocytogenes ScottA growth in BHI broth by an experimental design approach.

To investigate the main effects and interactions of different factors : divercin V41 (0-4 ng ml(-1)), NaCl content (0.5-5.5% w v(-1)), phenol (liquid smoke) concentration (0-8 ppm), and pH (5.5-7.5) on Listeria monocytogenes ScottA growth. Experiments were carried out in BHI broth using a central composite design. Divercin V41 (div41), NaCl content and pH were found to be the most influential factors whereas phenol concentration in liquid smoke had no effect on L. monocytogenes ScottA growth in our experimental domain. The combined effects of div41, NaCl content and pH decreased L. monocytogenes ScottA maximum specific growth rate (mu(max)) from 0.34 to 0.01 h(-1) and led to a significant increase in lag time (t(lag)) from 5.5 to 25 h. In this study, NaCl, pH and phenol conditions were similar to those currently observed in smoked salmon production. This shows that L. monocytogenes ScottA growth could be efficiently delayed by the use of div41 in addition to the usual technological hurdles. In conclusion, the technological hurdles of cold smoked salmon production could be further optimized and combined with the use of div41 or the div41 producer strain to improve the food safety of the product.

Synthesis and characterization of poly(1,3‐thiazol‐2‐yl‐carbomoyl) methyl methacrylate: Its metal complexes and antimicrobial activity studies

AbstractPoly(1,3‐thiazol‐2‐yl‐carbomoyl) methyl methacrylate [poly(TCMMA)] is prepared in dimethyl sulfoxide using 2,2′‐azobisisobutyronitrile as an initiator at 60°C. Poly(TCMMA) is characterized by IR and 1H‐NMR spectroscopic techniques. Cadmium(II), copper(II), and nickel(II) chelates of poly(TCMMA) were synthesized. An elemental analysis of the polychelates suggests a metal/ligand ratio of 1:2. The polychelates are further characterized by IR and magnetic susceptibility measurements. The thermal properties of the polymer and metal chelates are also discussed. The molecular weights of the poly(TCMMA) are determined by the gel permeation chromatography technique. The antimicrobial activities of the polymer and metal chelates are tested against Staphylococcus aureus COWAN I (bacteria), Escherichia coli ATCC 25922 (bacteria), Listeria monocytogenes SCOTTA (bacteria), Bacillus subtilis LMG (bacteria), Enterobacter aeroginosa CCM 2531 (bacteria), Klebsiela pneumania FMCS (bacteria), Candida albicans CCM 314 (Mayo yeast), and Saccharamyces cerevisiae UGA 102 (Mayo yeast). © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 3244–3251, 2003

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence.

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Antimicrobial activity studies of the metal complexes derived from substituted cyclobutane substituted thiazole Schiff base ligands

Two novel bidentate Schiff base ligands derived from 4-(1-methyl-1-mesitylcyclobutane-3-yl)-2-aminothiazole (LH) have been prepared and characterized by elemental analyses, i.r., 13C- and 1H-n.m.r. spectra, u.v.–vis. and magnetic susceptibility measurements. The complexes are mononuclear. Antimicrobial activities of the ligands and their complexes have been tested: Bacillus megaterium DSM 32, Candida albicans FMC 17, Enterobacter aeroginosa CCM 2531, Escherichia coli ATCC 25922, Listeria monocytogenes SCOTTA, Micrococcus luteus LA 2971, Proteus vulgaris FMC 11, Pseudomonas aeruginosa DSM 50071, Saccharomyces cerevisiae FMC 16 and Staphylococcus aureus COWAN I. The thermal properties of the ligands and their complexes have been studied by t.g.a.