Isolation, identification, and characterization of a marine Lactiplantibacillus plantarum strain with antimicrobial activity against Burkholderia contaminans

Abstract

Burkholderia contaminans, an opportunistic pathogen commonly found in the food and cosmetics industries, has serious potential to cause severe human infections and industrial contamination. However, compared to traditional physical or chemical antimicrobial treatment, the novel biological antimicrobial strategies against B. contaminans have not been extensively explored. In this study, a strain with antimicrobial activity against B. contaminans was isolated from a marine grouper aquaculture pond and identified as Lactobacillus plantarum Dys01. The antimicrobial activity of L. plantarum Dys01 mainly originated from its metabolites, with a minimum inhibitory concentration (MIC) of 8 mg/mL. Component analysis indicated that the antibacterial substances of L. plantarum Dys01 primarily included organic acids, proteinaceous substances, and hydrogen peroxide, among which organic acids and proteinaceous substances played the major inhibitory roles. Additionally, the metabolites of L. plantarum Dys01 significantly inhibited the biofilm formation of B. contaminans in a dose-dependent manner. Alkaline phosphatase activity assays and propidium iodide staining revealed that metabolites produced by L. plantarum Dys01 could disrupt the cell wall and cell membrane integrity of B. contaminans. This was further confirmed by scanning electron microscopy, which showed typical morphological damage such as surface indentations and membrane rupture. Therefore, our study provided novel insights into the control of B. contaminans contamination in the food, cosmetic, and pharmaceutical industries, and laid an important theoretical foundation for the development of novel biopreservatives.

Keywords: Burkholderia contaminans; Lactobacillus plantarum; antimicrobial activity; marine microorganisms; metabolites.

Figure 1

Isolation and identification of L. plantarum Dys01 with antimicrobial activity against B. contaminans. (A) Inhibition zone analysis of L. plantarum Dys01 against B. contaminans determined by the Oxford cup assay. (B) Colony morphology of L. plantarum Dys01 on MRS agar. (C) Transmission electron microscopy analysis of L. plantarum Dys01. Scale bar, 10 μm. (D) Neighbor-joining phylogenetic tree of L. plantarum Dys01 and the related bacteria according to 16S rRNA gene sequences. The position of L. plantarum Dys01 in the tree is indicated with red color. The tree was generated by MEGA11 with a bootstrap value of 1,000.

Figure 2

Antimicrobial activity of L. plantarum Dys01 metabolites against B. contaminans. The metabolites of L. plantarum Dys01 were extracted and added to plates containing B. contaminans, the antimicrobial activity of metabolites was evaluated by inhibition zone.

Figure 3

Antimicrobial component analysis of L. plantarum Dys01 metabolites against B. contaminans. The L. plantarum Dys01 metabolites were treated with NaOH, catalase or acidic protease, respectively. After treatment, each sample was co-incubated with B. contaminans for 12 h, followed by detection of OD₆₀₀. BC, B. contaminans suspension without the addition of any metabolites; BC + LP, the control group treated with L. plantarum Dys01 metabolites without any treatment; BC + LP (pH 7.0), the group treated with pH-neutralized metabolites (adjusted to pH 7.0); BC + LP (pH 7.0) + Catalase, the group treated with catalase-treated metabolites (to eliminate hydrogen peroxide); BC + LP + Acidic Protease, the group treated with acidic protease-treated metabolites (to digest proteinaceous substances).

Figure 4

Inhibitory effect of L. plantarum Dys01 metabolites on B. contaminans biofilm formation. The L. plantarum Dys01 metabolites were mixed with B. contaminans suspension at final metabolite concentrations corresponding to 0.5 ×, 1 ×, and 2 × MIC. After incubation for 4 h, the biofilm inhibition rate was ascertained by crystal violet assays.

Figure 5

Effect of L. plantarum Dys01 metabolites on the cell wall integrity of B. contaminans. The L. plantarum Dys01 metabolites were mixed with B. contaminans suspension at final metabolite concentrations of 0.5 ×, 1 ×, and 2 × MIC. The cell wall integrity of B. contaminans was assessed by alkaline phosphatase (AKP) activity assays. The sterile deionized water was employed as a control.

Figure 6

Effect of L. plantarum Dys01 metabolites on the cell membrane integrity of B. contaminans. The B. contaminans treated with L. plantarum Dys01 metabolites at final metabolite concentrations of 0.5 ×, 1 ×, and 2 × MIC were labeled with propidium iodide (red) and then subjected to confocal microscopy analysis. The bacteria with damaged cell membranes were stained red. The sterile deionized water was employed as a control. Scale bar, 20 μm.

Figure 7

Effect of L. plantarum Dys01 metabolites on the morphology of B. contaminans. The B. contaminans were treated with L. plantarum Dys01 metabolites at final metabolite concentrations of 0.5 ×, 1 ×, and 2 × MIC. After 12 h of culture, the bacteria were subjected to field emission scanning electron microscopy analysis. The sterile deionized water was employed as a control. Scale bar, 500 nm.