Inhibition of Staphylococcus aureus biofilm by Lactobacillus isolated from fine cocoa

Abstract

Background: Biofilm production represents an important virulence and pathogenesis factor for Staphylococcus aureus. The formation of biofilms on medical devices is a major concern in hospital environments, as they can become a constant source of infection. Probiotic bacteria, such as Lactobacillus fermentum and L. plantarum, have been found to inhibit biofilm formation; however little is known about the underlying mechanism. In this study, we tested the activity of supernatants produced by L. fermentum TCUESC01 and L. plantarum TCUESC02, isolated during the fermentation of fine cocoa, against S. aureus CCMB262 biofilm production. We measured inhibition of biofilm formation in vitro and analyzed biofilm structure by confocal and electronic microscopy. Additionally, we quantified the expression of S. aureus genes icaA and icaR involved in the synthesis of the biofilm matrix by real-time PCR.

Results: Both Lactobacillus supernatants inhibited S. aureus growth. However, only L. fermentum TCUESC01 significantly reduced the thickness of the biofilm, from 14 μm to 2.83 μm (at 18 mg∙mL^-1, 90 % of the minimum inhibitory concentration, MIC), 3.12 μm (at 14 mg∙mL^-1, 70 % of the MIC), and 5.21 μm (at 10 mg∙mL^-1, 50 % of the MIC). Additionally, L. fermentum TCUESC01 supernatant modulated the expression of icaA and icaR.

Conclusions: L. fermentum TCUESC01 reduces the formation of S. aureus biofilm under subinhibitory conditions. Inhibition of biofilm production probably depends on modulation of the ica operon.

Keywords: Antistaphylococcal; Biofilm; Lactobacillus fermentum; Probiotic properties; ica.

Fig. 1

a Effect of minimum inhibitory concentration (MIC) of Lactobacillus fermentum TCUESC01 and L. plantarum TCUESC02 supernatants on Staphylococcus aureus CCMB262 growth. MRS Control, MRS medium without lyophilized Lactobacillus cells; PC, positive control for S. aureus; NC, negative control, S. aureus with 12.5 μg∙mL⁻¹ chloramphenicol; MC, sterility control of MH medium. b Bactericidal/bacteriostatic activity of L. fermentum TCUESC01 and L. plantarum TCUESC02 supernatants (at MIC doses) against S. aureus CCMB262 grown on MHA

Fig. 2

Biofilm formation index for S. aureus CCMB262 treated or not with TCUESC01 and TCUESC02 supernatants. ■, L. fermentum TCUESC01; □, L. plantarum TCUESC02; N, untreated control; 90, 70, and 50 %, MIC doses used for the treatment; *, statistical significance compared to the control (p < 0.001)

Fig. 3

S. aureus CCMB262 growth in the presence or not of L. fermentum TCUESC01 supernatant at a subinhibitory dose (50 % of MIC).● and continuous line, standard growth curve with S. aureus CCMB262 (control); □ and dashed line, growth curve of S. aureus CCMB262 with addition of L. fermentum TCUESC01 supernatant at 50 % of the MIC (10 mg∙mL⁻¹). The result represents the average of three experiments

Fig. 4

Confocal microscopy of S. aureus CCMB262 treated with L. fermentum TCUESC01 and L. plantarum TCUESC02 supernatants at subinhibitory doses. Treatments were as follows: a untreated S. aureus CCMB262; b TCUESC02 at 90 % of the MIC (2.25 mg∙mL⁻¹); c TCUESC02 at 70 % of the MIC (1.75 mg∙mL⁻¹); d TCUESC01 at 90 % of the MIC (18 mg∙mL⁻¹); e TCUESC01 at 70 % of the MIC (14 mg∙mL⁻¹); f TCUESC01 at 50 % of the MIC (10 mg∙mL⁻¹). The biofilm was stained with DAPI (blue, panel 2) and PI (red, panel 3); panel 1 shows the overlapping of panels 2 and 3

Fig. 5

Thickness of S. aureus CCMB262 biofilm treated with L. plantarum TCUESC02 supernatant at subinhibitory doses. Treatments were as follows: C, untreated control; 70 %, 70 % of the MIC (1.75 mg∙mL⁻¹); 90 %, 90 % of the MIC (2.25 mg∙mL⁻¹). No significant difference could be observed

Fig. 6

Thickness of S. aureus CCMB262 biofilm treated with L. fermentum TCUESC01 supernatant at subinhibitory doses. Treatments were as follows: C, untreated control; 50 %, 50 % of the MIC (10 mg∙mL⁻¹); 70 %, 70 % of the MIC (14 mg∙mL⁻¹); 90 %, 90 % of the MIC (18 mg∙mL⁻¹).*, statistical significance at p < 0.05, analysis of variance

Fig. 7

Scanning electron microscopy of S. aureus CCMB262 biofilm treated or not with L. fermentum TCUESC01 supernatant. Treatments were as follows: a untreated control; b 50 % of the MIC

Fig. 8

Real-time quantification of icaA and icaR expression in S. aureus CCMB262 treated or not with L. fermentum TCUESC01 supernatant. a Relative quantification (RQ) of icaA. b RQ of icaR. Treatments were as follows: 1, untreated control; 2, 50 % of the MIC. Values correspond to RQs (2^-∆∆CT) normalized to16S rRNA.*, statistical significance at p < 0.05, t-test