The Staphylococcus aureus ArlS Kinase Inhibitor Tilmicosin Has Potent Anti-Biofilm Activity in Both Static and Flow Conditions

Abstract

Staphylococcus aureus can form biofilms on biotic surfaces or implanted materials, leading to biofilm-associated diseases in humans and animals that are refractory to conventional antibiotic treatment. Recent studies indicate that the unique ArlRS regulatory system in S. aureus is a promising target for screening inhibitors that may eradicate formed biofilms, retard virulence and break antimicrobial resistance. In this study, by screening in the library of FDA-approved drugs, tilmicosin was found to inhibit ArlS histidine kinase activity (IC₅₀ = 1.09 μM). By constructing a promoter-fluorescence reporter system, we found that tilmicosin at a concentration of 0.75 μM or 1.5 μM displayed strong inhibition on the expression of the ArlRS regulon genes spx and mgrA in the S. aureus USA300 strain. Microplate assay and confocal laser scanning microscopy showed that tilmicosin at a sub-minimal inhibitory concentration (MIC) had a potent inhibitory effect on biofilms formed by multiple S. aureus strains and a strong biofilm-forming strain of S. epidermidis. In addition, tilmicosin at three-fold of MIC disrupted USA300 mature biofilms and had a strong bactericidal effect on embedded bacteria. Furthermore, in a BioFlux flow biofilm assay, tilmicosin showed potent anti-biofilm activity and synergized with oxacillin against USA300.

Keywords: MRSA; Staphylococcus aureus; biofilm; inhibitor; two-component system.

Figure 1

Impact of arlRS knockout on biofilm production of S. aureus and S. epidermidis strains in vitro. Biofilms produced by the arlRS knockout mutants and complementation strains of the MRSA TCH1516, FPR3757 and USA500 strains; the MSSA Newman strain; and S. epidermidis 1457 strain in TSBG in 96-well tissue-culture-treated polystyrene microplates for 24 h were compared with their parental strains.

Figure 2

Inhibition of ArlS activity by tilmicosin: (A) the purified ArlSHK and the chemical structure of its kinase inhibitor tilmicosin; (B) dose-dependent inhibition of tilmicosin on the ArlSHK kinase activity detected by the Kinase-Glo(R) Luminescent Assay; (C) the effect of tilmicosin on ArlS activity in S. aureus at 4 h and 10 h, indicated by the spx P2 promoter and mgrA P2 promoter-GFP reporter systems (in each panel, from left to right: mock, 0.1% DMSO, 0.75 μM tilmicosin, 1.5 μM tilmicosin, ΔarlRS control).

Figure 3

Inhibition of static staphylococcal biofilm production in vitro by tilmicosin. Effect of tilmicosin at a 1/4 of MIC on biofilms formed by the MRSA TCH1516, FPR3757 and USA500 strains; two clinical MRSA isolates (234 and 15,098); and the S. epidermidis 1457 strain were semi-quantitatively determined as described above.

Figure 4

The impact of tilmicosin on MRSA young biofilms. The 1 μg/mL of tilmicosin or 0.1% DMSO (control) was added into pre-formed 6 h young biofilms of the MRSA strains USA500, 15,098 and 234 in TBSG in fluorodishes. After additional 18 h incubation, the biofilms remaining on the glass bottom were stained with live/dead staining kit. CLSM was performed to acquire fluoresent images, measure the biofilm thickness and analyze cell viability.

Figure 5

Inhibition of biofilm development of the MRSA strain USA300 FPR3757 by tilmicosin under a flowing condition (BioFlux 1000 system). The 16 h biofilm formed in the channels was photographed (Left panel). The amount of biomass was monitored and analyzed with BioFlux Montage software 2.3 in real time (Right panel).

Figure 6

Effect of tilmicosin on MRSA mature biofilms. The 8 μg/mL of tilmicosin, 8 μg/mL of vancomycin or 0.1% DMSO (control) was added into pre-formed 24 h mature biofilm of the MRSA strains USA300 FPR3757 in fluorodishes. After additional 24 h incubation and live/dead staining, the biofilm thickness and fluorescence intensities were measured as described above.