A method for bacteriocin quantification

Cabo, Marta López; Murado, M.A.; González, M.P.; Pastoriza, Laura

doi:10.1046/j.1365-2672.1999.00942.x

Cited by 115 publications

(81 citation statements)

References 25 publications

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“…Recombinant piscicolin 126 produced using this expression system had an antibacterial activity similar to those of bacteriocins purified from their native hosts. The MIC 50 of piscicolin 126 against L. monocytogenes 4A increased over time, as has been reported earlier (6), and was within an order of magnitude of other class IIa bacteriocins tested against the same indicator organism (14).…”

Section: Discussionsupporting

confidence: 57%

“…K ϩ concentrations were measured at 5-s intervals by using a K ϩ ion-selective electrode with an internal reference (Orion Research, Boston, Mass.). Cells of L. monocytogenes 4A from an overnight culture were collected by centrifugation (4,000 ϫ g, 15 min, 4°C), washed twice in a 10% volume of the standard K ϩ buffer (10 mM KCl, 0.2% glucose, 50 mM NaH 2 PO 4 ⅐ Na 2 HPO 4 [pH 6.70]), resuspended to a final OD 600 of 18 (approximately 2.4 ϫ 10 10 cells/ml), and stored on ice for periods no longer than 4 h. Where appropriate, cells were resuspended to 2 ml in buffers of defined pH (pH 5.74, 6.40, 6.70, 7.02, and 7.66) or NaCl concentration (25,50,150, and 300 mM). Cells were equilibrated to 25°C, and K ϩ concentrations in the buffer were determined.…”

Section: Methodsmentioning

confidence: 99%

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Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

Gibbs¹,

Davidson²,

Hillier

2004

Appl Environ Microbiol

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Piscicolin 126 is a class IIa bacteriocin isolated from Carnobacterium piscicola JG126 that exhibits strong activity against Listeria monocytogenes. The gene encoding mature piscicolin 126 (m-pisA) was cloned into an Escherichia coli expression system and expressed as a thioredoxin-piscicolin 126 fusion protein that was purified by affinity chromatography. Purified recombinant piscicolin 126 was obtained after CNBr cleavage of the fusion protein followed by reversed-phase chromatography. Recombinant piscicolin 126 contained a single disulfide bond and had a mass identical to that of native piscicolin 126. This novel bacteriocin expression system generated approximately 26 mg of purified bacteriocin from 1 liter of E. coli culture. The purified recombinant piscicolin 126 acted by disruption of the bacterial cell membrane.Bacteriocins produced by lactic acid bacteria (LAB) have been classified into four classes (23). The class II bacteriocins, which are small (Ͻ10 kDa), heat-stable, non-lanthionine-containing, and membrane-active amphipathic peptides, have been further divided into three groups on the basis of their sequence homology and mechanism of action (23, 29). The class IIa bacteriocins from the LAB are active against Listeria and contain an N-terminal consensus sequence of YGNGV. The antibacterial activity, mechanism of action, and structurefunction relationships of class IIa bacteriocins have been investigated with a view to their development and application in the targeted prevention of growth of human pathogens in food. Class IIa bacteriocins are active at the cell membranes of gram-positive organisms, and their limited spectrum of activity is attributed to the requirement for a specific receptor interaction on the target cell (11,12,16,17,34,38). Class IIa bacteriocins cause the efflux of accumulated compounds and ions (1,5,8,25,39), a dissipation of the membrane potential (1), and depletion of ATP (8), thereby preventing growth of the organism.A requirement for the continued improvement of structurefunction models of class IIa bacteriocins through biophysical studies is the production and purification of large quantities (up to 10 mg) of bacteriocin. Bacteriocin production systems currently utilized include expression in the native strain (19,21), recombinant expression systems in LAB (3, 24), recombinant expression by use of Escherichia coli (27,28), and chemical synthesis (15, 37). The utilization of recombinant expression systems permits the generation of fusion proteins or incorporation of affinity tags that improve purification. Chemical synthesis of class IIa bacteriocins produced large amounts (1 g) of biologically active bacteriocin (15, 37); however, the low purity of the chemically synthesized material resulted in a less-efficient bacteriocin production system. In vitro translation systems for the production of bacteriocins can also be applied. However, a problem with both chemical synthesis and in vitro translation systems is the requirement to form intramolecular disulfide bonds to produce...

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Section: Discussionsupporting

confidence: 57%

Section: Methodsmentioning

confidence: 99%

Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

Gibbs¹,

Davidson²,

Hillier

2004

Appl Environ Microbiol

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“…The ECP extraction was performed according to the method of Cabo et al (3). Briefly, all the LAB strains were grown overnight at 30°C in MRS broth.…”

Section: Antibacterial Activity Of Ecps Of Labmentioning

confidence: 99%

Evaluation of Immunomodulatory Effects of Lactic Acid Bacteria in Turbot (Scophthalmus maximus)

Villamil

Tafalla

Figueras

et al. 2002

Clin Vaccine Immunol

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In the present work, the effects of several lactic acid bacteria on the immune response of turbot (Scophthalmus maximus) macrophages have been studied both in vitro and in vivo. Out of six lactic acid bacterial strains tested, only heat-killed Lactococcus lactis significantly increased the turbot head kidney macrophage chemiluminescent (CL) response after 24 h of incubation. Nitric oxide (NO) was also significantly enhanced by this bacterium after 72 h of incubation with either viable (10 3 and 10 6 cells/ml) or heat-killed (10 6 cells/ml) bacteria. Viable Leuconostoc mesenteroides (10 6 cells/ml) was also capable of significantly increasing NO production. Since L. lactis proved to be the strain with more effects on the host immune function, further in vivo and in vitro experiments were conducted with this bacterium. The in vitro capacity of L. lactis to adhere to turbot intestinal mucus was positively confirmed. When orally administered, L. lactis significantly increased the macrophage CL response and the serum NO concentration after 7 days of daily administration. The antibacterial effect of the extracellular products from the six LAB strains against the fish-pathogenic bacterium Vibrio anguillarum was also demonstrated in vitro.The increased intensification of aquaculture has led to a high number of disease outbreaks with an increasing range of pathogens. Consequently, the extensive use of broad-spectrum antibiotics in aquaculture has led, as in other fields, to drug resistance problems (32). In order to improve health and welfare in the rearing of these animals, several alternatives such as improved husbandry, nutrition, and water quality; lower stocking densities; and use of vaccines, nonspecific immunostimulants (7), and bacterial probiotics such as lactic acid bacteria (LAB) (9, 26) have been proposed.Probiotics are defined as microbial dietary adjuvants that beneficially affect the host physiology by modulating mucosal and systemic immunity, as well as improving nutritional and microbial balance in the intestinal tract (23).The role of LAB within the digestive tract of endothermic animals and humans has been extensively studied (14,18,28), while only a few studies have demonstrated that LAB are part of the normal intestinal microflora in larvae and juvenile fish (25). Most studies with probiotics conducted to date with fish have been undertaken with strains isolated and selected from aquatic environments and cultured animals. The main bacteria tested belonged to the genera Bacillus, Vibrio, and Pseudomonas (9, 22). Few authors have tested in vivo the protection conferred by probiotics in fish experimentally infected with pathogens. Gastesoupe (8) found that the Lactobacillus or Carnobacterium strain isolated from rotifers increased the resistance of turbot larvae against a pathogenic Vibrio sp. Gildberg et al. (10) demonstrated that Carnobacterium divergens decreased the mortality rate of Atlantic cod fry challenged with Vibrio anguillarum but not the mortality of salmon fry challenged with Aeromonas hy...

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“…The 50% inhibitory concentration (IC 50 ) was determined using a broth inhibition assay described by Cabo et al (4). Briefly, 996 l of nutrient broth (inoculated to 0.5% with E. faecalis) was taken in sterile Eppendorf tubes.…”

Section: Methodsmentioning

confidence: 99%

“…Resistant variants were selected using the method described by Rekhiff et al (22). In brief, culture containing 1 ϫ 10 8 to 5 ϫ 10 8 CFU was inoculated in nutrient broth containing alamethicin (4,6,8, and 10 times the IC 50 of the wild-type strain) and incubated at 37°C. After visible growth, the broth culture was serially diluted and plated onto nutrient hard agar without alamethicin.…”

Section: Methodsmentioning

confidence: 99%

Substantiation in Enterococcus faecalis of Dose-Dependent Resistance and Cross-Resistance to Pore-Forming Antimicrobial Peptides by Use of a Polydiacetylene-Based Colorimetric Assay

Mehla

Sood

2011

Appl Environ Microbiol

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A better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and for in vitro assessment, variants of Enterococcus faecalis that are resistant to different doses of the fungal AMP alamethicin (Alm r ) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Alm r variants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Alm r variants showed crossresistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Alm r variants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using an in vitro colorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.

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A method for bacteriocin quantification

Cited by 115 publications

References 25 publications

Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

Novel Expression System for Large-Scale Production and Purification of Recombinant Class IIa Bacteriocins and Its Application to Piscicolin 126

Evaluation of Immunomodulatory Effects of Lactic Acid Bacteria in Turbot (Scophthalmus maximus)

Substantiation in Enterococcus faecalis of Dose-Dependent Resistance and Cross-Resistance to Pore-Forming Antimicrobial Peptides by Use of a Polydiacetylene-Based Colorimetric Assay

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