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A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

Vijayakumar PP, Muriana PM - Biomolecules (2015)

Bottom Line: Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients.In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives.In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Oklahoma State University, Monroe Street, Stillwater, OK 74078, USA. paul.v@uky.edu.

ABSTRACT
Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

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Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R0) treated with culture supernatants from: None (R0 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Microplate assay using non-neutralized culture supernatants; (B) Microplate assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in order to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
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biomolecules-05-01178-f004: Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R0) treated with culture supernatants from: None (R0 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Microplate assay using non-neutralized culture supernatants; (B) Microplate assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in order to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.

Mentions: Differences were observed with Bac− preps in microplate growth inhibition assays in which L. monocytogenes 39-2 (R0) was treated with neutralized or non-neutralized cell-free culture supernatants (Figure 4) that were not observed during agar spot activity titer assays (Figure 1C). When non-neutralized cell-free supernatants of the Bac− control strain (Lb. delbrueckii 4797-2) were added to L. monocytogenes R0, some inhibition was observed whereby R0 did not grow as well as in the Bac− control assay while all the Bac+ preparations showed baseline-level inhibition (Figure 4A). However, when neutralized cell-free supernatants of the Bac− 4797-2 strain were added to R0, it grew equally well and without any significant difference from the R0 control (Figure 4B). Trials with the neutralized preps also showed late recovery of L. monocytogenes R0 treated with the Beef3 Bac+ prep (Figure 4B) suggesting possible outgrowth of spontaneous resistant variants which is consistent with observations in the agar spot assay for FS47 (Figure 2B) and other bacteriocins (Figure 3A). The baseline-level inhibition of L. monocytogenes by bacteriocin Beef3 observed with non-neutralized preparations (Figure 4A) could have been due to acid-assisted inhibition that was removed by neutralization; such differences are likely only noticeable with microplate growth inhibition data, offering real-time growth curve comparisons, that would otherwise appear as similar fully-grown endpoints on agar surface spot assays.


A Microplate Growth Inhibition Assay for Screening Bacteriocins against Listeria monocytogenes to Differentiate Their Mode-of-Action.

Vijayakumar PP, Muriana PM - Biomolecules (2015)

Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R0) treated with culture supernatants from: None (R0 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Microplate assay using non-neutralized culture supernatants; (B) Microplate assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in order to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496717&req=5

biomolecules-05-01178-f004: Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R0) treated with culture supernatants from: None (R0 Cont.), Lb. delbrueckii 4797-2 (Bac−), Lb. curvatus FS47, P. acidilactici Bac3, Lb. curvatus Beef3, En. faecium FS56-1, En. thailandicus FS92, and Lc. lactis FLS1. (A) Microplate assay using non-neutralized culture supernatants; (B) Microplate assay using neutralized culture supernatants. Data points represent the means of triplicate replications and error bars represent the standard deviations from the means (error bars were not used for all curves in order to prevent clutter). Treatments with different lowercase letters are significantly different (p < 0.05); letters in brackets are for entire group of graph lines.
Mentions: Differences were observed with Bac− preps in microplate growth inhibition assays in which L. monocytogenes 39-2 (R0) was treated with neutralized or non-neutralized cell-free culture supernatants (Figure 4) that were not observed during agar spot activity titer assays (Figure 1C). When non-neutralized cell-free supernatants of the Bac− control strain (Lb. delbrueckii 4797-2) were added to L. monocytogenes R0, some inhibition was observed whereby R0 did not grow as well as in the Bac− control assay while all the Bac+ preparations showed baseline-level inhibition (Figure 4A). However, when neutralized cell-free supernatants of the Bac− 4797-2 strain were added to R0, it grew equally well and without any significant difference from the R0 control (Figure 4B). Trials with the neutralized preps also showed late recovery of L. monocytogenes R0 treated with the Beef3 Bac+ prep (Figure 4B) suggesting possible outgrowth of spontaneous resistant variants which is consistent with observations in the agar spot assay for FS47 (Figure 2B) and other bacteriocins (Figure 3A). The baseline-level inhibition of L. monocytogenes by bacteriocin Beef3 observed with non-neutralized preparations (Figure 4A) could have been due to acid-assisted inhibition that was removed by neutralization; such differences are likely only noticeable with microplate growth inhibition data, offering real-time growth curve comparisons, that would otherwise appear as similar fully-grown endpoints on agar surface spot assays.

Bottom Line: Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients.In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives.In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, Oklahoma State University, Monroe Street, Stillwater, OK 74078, USA. paul.v@uky.edu.

ABSTRACT
Lactic acid bacteria (LAB) have historically been used in food fermentations to preserve foods and are generally-recognized-as-safe (GRAS) by the FDA for use as food ingredients. In addition to lactic acid; some strains also produce bacteriocins that have been proposed for use as food preservatives. In this study we examined the inhibition of Listeria monocytogenes 39-2 by neutralized and non-neutralized bacteriocin preparations (Bac+ preps) produced by Lactobacillus curvatus FS47; Lb. curvatus Beef3; Pediococcus acidilactici Bac3; Lactococcus lactis FLS1; Enterococcus faecium FS56-1; and Enterococcus thailandicus FS92. Activity differences between non-neutralized and neutralized Bac+ preps in agar spot assays could not readily be attributed to acid because a bacteriocin-negative control strain was not inhibitory to Listeria in these assays. When neutralized and non-neutralized Bac+ preps were used in microplate growth inhibition assays against L. monocytogenes 39-2 we observed some differences attributed to acid inhibition. A microplate growth inhibition assay was used to compare inhibitory reactions of wild-type and bacteriocin-resistant variants of L. monocytogenes to differentiate bacteriocins with different modes-of-action (MOA) whereby curvaticins FS47 and Beef3, and pediocin Bac3 were categorized to be in MOA1; enterocins FS92 and FS56-1 in MOA2; and lacticin FLS1 in MOA3. The microplate bacteriocin MOA assay establishes a platform to evaluate the best combination of bacteriocin preparations for use in food applications as biopreservatives against L. monocytogenes.

Show MeSH
Related in: MedlinePlus