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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 (R1) treated with culture supernatants from: None (R1 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-f005: Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R1) treated with culture supernatants from: None (R1 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: Microplate inhibition assays were also performed with the L. monocytogenes 39-2 R1 BacR variant (Figure 3B) and growth inhibition assays were again evaluated with non-neutralized and neutralized cell-free culture supernatants (Figure 5). As observed previously, the R1 strain with non-neutralized supernatants from the Bac− control strain showed intermediate inhibition (Figure 5A) whereby the neutralized supernatants from the Bac− control showed no inhibition (Figure 5B). This time, three of six neutralized Bac+ preps showed no inhibition of R1 due to the bacteriocin resistance incurred by strain R1 (Figure 5B). All of the bacteriocins that are no longer inhibitory to L. monocytogenes 39-2 (R1) are presumed to have the same or highly similar MOA while those bacteriocins that were still inhibitory to R1 were considered to have a different MOA. The data obtained in microplate growth assays with L. monocytogenes 39-2 (R1) suggests that curvaticin FS47, curvaticin Beef3, and pediocin Bac3 belong to the same mode of action, MOA1.


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 (R1) treated with culture supernatants from: None (R1 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-f005: Microplate growth inhibition assays showing the activity of Listeria monocytogenes 39-2 (R1) treated with culture supernatants from: None (R1 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: Microplate inhibition assays were also performed with the L. monocytogenes 39-2 R1 BacR variant (Figure 3B) and growth inhibition assays were again evaluated with non-neutralized and neutralized cell-free culture supernatants (Figure 5). As observed previously, the R1 strain with non-neutralized supernatants from the Bac− control strain showed intermediate inhibition (Figure 5A) whereby the neutralized supernatants from the Bac− control showed no inhibition (Figure 5B). This time, three of six neutralized Bac+ preps showed no inhibition of R1 due to the bacteriocin resistance incurred by strain R1 (Figure 5B). All of the bacteriocins that are no longer inhibitory to L. monocytogenes 39-2 (R1) are presumed to have the same or highly similar MOA while those bacteriocins that were still inhibitory to R1 were considered to have a different MOA. The data obtained in microplate growth assays with L. monocytogenes 39-2 (R1) suggests that curvaticin FS47, curvaticin Beef3, and pediocin Bac3 belong to the same mode of action, MOA1.

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