Effects of bacteriocins on methicillin-resistant Staphylococcus aureus biofilm

Abstract

Control of biofilms formed by microbial pathogens is an important subject for medical researchers, since the development of biofilms on foreign-body surfaces often causes biofilm-associated infections in patients with indwelling medical devices. The present study examined the effects of different kinds of bacteriocins, which are ribosomally synthesized antimicrobial peptides produced by certain bacteria, on biofilms formed by a clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA). The activities and modes of action of three bacteriocins with different structures (nisin A, lacticin Q, and nukacin ISK-1) were evaluated. Vancomycin, a glycopeptide antibiotic used in the treatment of MRSA infections, showed bactericidal activity against planktonic cells but not against biofilm cells. Among the tested bacteriocins, nisin A showed the highest bactericidal activity against both planktonic cells and biofilm cells. Lacticin Q also showed bactericidal activity against both planktonic cells and biofilm cells, but its activity against biofilm cells was significantly lower than that of nisin A. Nukacin ISK-1 showed bacteriostatic activity against planktonic cells and did not show bactericidal activity against biofilm cells. Mode-of-action studies indicated that pore formation leading to ATP efflux is important for the bactericidal activity against biofilm cells. Our results suggest that bacteriocins that form stable pores on biofilm cells are highly potent for the treatment of MRSA biofilm infections.

Fig 1

Structures of bacteriocins. The shaded residues indicate modified amino acids. Ala-S-Ala, lanthionine; Abu-S-Ala, 3-methyllanthionine; Dha, dehydroalanine; Dhb, dehydrobutyrine; fMet, formylmethionine.

Fig 2

Bactericidal activities of bacteriocins and vancomycin against planktonic cells. Peptides at final concentrations of 4× MIC were incubated with S. aureus MR23 in BHI medium and further incubated at 37°C with shaking. The CFU were enumerated following different incubation periods (0, 1, 4, and 24 h). Data points represent the means and standard deviations of triplicate determinations.

Fig 3

Bactericidal activities of bacteriocins and vancomycin against biofilm cells. Peptide solutions at a concentration of 4× MIC were incubated with preformed biofilms of S. aureus MR23 at room temperature. The numbers of CFU of biofilm cells were measured after 1-h (white bars) and 24-h (black bars) incubations. The means and standard deviations of triplicate determinations are presented.

Fig 4

Three-dimensional images of S. aureus MR23 biofilms after treatment with bacteriocins and vancomycin. Biofilms formed on 35-mm glass-bottomed dishes were incubated with peptide solutions at a concentration of 4× MIC for 1 h at room temperature. After washing, the living and dead cells in the biofilm were stained by SYTO9 (green) and PI (red), respectively. (A) PBS (60 min); (B) nisin A (5 min); (C) nisin A (60 min); (D) lacticin Q (60 min); (E) nukacin ISK-1 (60 min); (F) vancomycin (60 min); (A′ to D′) enlarged views of x-z sections of panels A to D, respectively. Bars, 5 μm.

Fig 5

Dose-dependent effects of bacteriocins and vancomycin against S. aureus MR23 biofilm. Peptide solutions at a concentration of 4× MIC were incubated with preformed S. aureus MR23 biofilms for 1 h at room temperature. Subsequently, biofilms were stained using a FilmTracer LIVE/DEAD biofilm viability kit. Fluorescence derived from PI was measured at excitation and emission wavelengths of 485 and 635 nm, respectively. The means and standard deviations of triplicate determinations are presented. A.U., arbitrary units.

Fig 6

ATP efflux from S. aureus MR23 biofilm cells. After incubation of S. aureus MR23 biofilm cells with bacteriocins and vancomycin at a concentration of 4× MIC for 1 h at room temperature, the amount of ATP in the supernatants was measured by luciferase assay. ATP concentrations were determined using standard ATP solutions. The means and standard deviations of triplicate determinations are presented.

Fig 7

Membrane potential of S. aureus MR23 biofilm after incubation with bacteriocins and vancomycin. Fluorescence derived from DiBAC₄(3), a membrane-potential-sensitive probe, was measured at excitation and emission wavelengths of 485 and 535 nm, respectively. The means and standard deviations of triplicate determinations are presented. A.U., arbitrary units.

Fig 8

Localization analysis of BODIPY-vancomycin. (A) S. aureus MR23 biofilm was incubated with BODIPY-vancomycin at a concentration of 4× MIC for 0, 5, 15, 30, and 60 min at room temperature. Fluorescence derived from BODIPY-vancomycin was measured at excitation and emission wavelengths of 485 and 535 nm, respectively. The means and standard deviations of triplicate determinations are presented. van, vancomycin; A.U., arbitrary units. (B) After a 60-min incubation with BODIPY-vancomycin, the biofilm resuspended in PBS was observed by fluorescence microscopy. Arrows, division septa of the cells. (Inset) Enlargement of boxed area. Bar, 1 μm.

Fig 9

Live/dead images of staphylococcal biofilms after treatment with bacteriocins and vancomycin. Biofilms formed on 35-mm glass-bottomed dishes were incubated with peptide solutions at a concentration of 4× MIC for 1 h at room temperature. By way of exception, biofilms of SE4 and SE21 were treated with 20 μM lacticin Q. After washing, the living and dead cells in the biofilm were stained by SYTO9 (green) and PI (red), respectively. x-z sections of three-dimensional images are shown. MR10, S. aureus MR10; MR11, S. aureus MR11; SH1000, S. aureus SH1000; SE4, S. epidermidis SE4; SE21, S. epidermidis SE21. Bar, 5 μm.