The secondary metabolite MHL was produced biotechnologically by cultures of the Gram-positive soil bacterium Nonomuraearubra (ATCC 27031, DSM 43768T, IMET13001, 16S rDNA: AF277200; basonyms Nonomuria rubra, Actinomadura rubra, Microtetraspora rubra, Micromonospora rubra [30-33]. MHL was extracted and purified using high-performance liquid chromatography (HPLC) as described [26-28]. The preparation was stored at -20 ºC as a dry powder or dissolved in DMSO until it was used.

The sample was dried and dissolved in methanol (ca. 3 pmol/µl). An Orbitrap/Velos mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nanospray ion source was used. The spectrum was recorded in the positive mode at a resolution of 100 000 and compared with the simulated spectrum generated by the Xcalibur (Thermo Scientific, USA) software (Fig. 1B and 1C).

For biocompatibility testing coatings were prepared as follows. A 10 mg/ml MHL solution in dimethyl sulphoxide (DMSO) was prepared. Ciprofloxacin (Fluka Chemie GmbH, Deisenhofen, Germany) was not soluble in DMSO; therefore a fine suspension was prepared at a concentration of 10 mg/ml ciprofloxacin in DMSO. The wells of a 12-well tissue culture polystyrene (TCPS) plate (BD Falcon, BD Biosciences, Germany) were coated with 1µl each of the MHL solution or with a 1:1 (w/w) mixture of MHL and ciprofloxacin. The coatings were dried overnight at room temperature. Cells were seeded directly on these coatings described below. Zirconium dioxide (zirconia) beads of 1 mm in diameter (BioSpec Products, Inc., Bartlesville, OK, USA) were dip-coated, followed by drying overnight at room temperature. Beads dipped in the solvent DMSO were used as a control. The release of ciprofloxacin from the coatings was determined by submerging the beads in 10µl of 0.1 M phosphate buffered saline (PBS, pH 7.4) in a microcentrifuge tube (Eppendorf, Germany) and kept at 37°C. The liquid was removed at 24h intervals and fresh PBS was added to the beads. The resulting ciprofloxacin concentration in the supernatants was determined by measuring the absorbance at 320nm using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA). MHL released from the coatings was calculated similarly by determination of the OD₄₈₀ of the sample supernatant. Titanium, stainless steel (brushed) and glass discs of 9mm diameter were coated with a single layer of pure MHL dissolved in 70% ethanol at a concentration of 1 mg/ ml. The discs were put on a hot plate that was set to 90ºC and the plate surface was protected by a household aluminum foil. 100 µl MHL solution was added with a pipette and spread out quickly with the pipette tip until the surface was completely covered. After drying the coatings were incubated in phosphate buffered saline (PBS) solution as described in the text and photographed using a STEMI SV11 stereo zoom-microscope (Carl Zeiss, Oberkochen, Germany) equipped with a digital camera DSC-S75 (Sony, Germany).

The Escherichia coli laboratory strains Top 10 (F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara leu) 7697 galU galK rpsL (StrR) endA1 nupG) and DH10B (F– mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara leu) 7697 galU galK rpsL nupG λ–), were purchased from Invitrogen (Germany). Pseudomonas aeruginosa SCV 20265 is a small colony variant with increased antibiotic resistance that was originally isolated from the lung of a cystic fibrosis patient [34, 35]. Pseudomonas aeruginosa strain PA01 CTX::lux is a laboratory strain that has been made luminescent by the insertion of the entire lux operon encoding sequence into the genomic DNA [36, 37]. The strains S. aureus COL and MRSA are both methicillin resistant and their genomes have been completely sequenced [38-44]. Bacteria were grown on LB agar dishes or as suspension cultures in LB broth according to standard procedures [45]. Bacterial growth in suspension cultures was monitored by measuring the optical density at 600 nm (OD₆₀₀). The relative bioluminescence of P. aeruginosa PAO1 CTX::lux in suspension versus the optical density (OD₆₀₀) was determined by serial dilutions of a fresh overnight culture in 100µl LB in a 96-well plate at 37º C using an optical in vivo imaging system (IVIS-200, Xenogen, Alameda, CA).

The antibacterial effects of MHL on solid media were scored by a radial streak assay. 50µg MHL dissolved in DMSO (10mg/ml) were placed in the center of a Petri dish with LB agar. For comparison, ciprofloxacin (50µg) and a combination of 25µg MHL and 25 µg ciprofloxacin and 5µl of the pure solvent DMSO were put on separate Petri dishes. After drying freshly diluted overnight cultures of bacteria were radially streaked from the outer rim towards the center of the dish and the dishes incubated overnight at 37ºC. The antibacterial efficacy was scored by measuring the distance of the growth inhibitory zone from the center (Table 1). The antibacterial activity released from MHL and ciprofloxacin coated zirconia beads was assayed by submerging the beads in 50µl of LB in a microcentrifuge tube at 37°C. The supernatants were collected at 24h intervals and replaced with 50µl of fresh medium. 45µl of each supernatant was inoculated in a 96 well plate with 5µl of a PAO1 CTX::lux culture at an OD₆₀₀ of 0.2. At the times indicated the bacterial luminescence from each sample was determined using an IVIS-200 system. To determine the titer of viable bacteria aliquots of serial dilutions from each of the supernatants were spread on LB agar plates and after overnight incubation at 37°C the colonies were counted. All experiments were performed in triplicates and the data is presented as average value with the standard deviation.

The murine fibroblast cell line NIH 3T3 (ATCC CRL-1658) and human embryonic kidney cell line HEK 293 (ATCC CRL-1573) were stored in liquid nitrogen and cultured according to standard procedures as previously described [46]. NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM; Gibco/BRL), supplemented with 10% (v/v) fetal calf serum, 1% (v/v) glutamine, and 1% (v/v) penicillin and streptomycin (100 U/ml penicillin G and 100 µg/ml streptomycin) at 37°C in a humidified atmosphere with 5% CO₂. A near confluent culture of NIH 3T3 cells was detached using a trypsin solution according to standard procedures and diluted 1:5 in fresh cell culture medium. 2ml of the cell suspension was added to each of the coated wells. After incubating the cells in a humidified atmosphere with 5% CO₂ at 37°C for 24, 48, or 96 h, respectively, the cells were examined using a Zeiss Axio Observer A-1 inverted visible light microscope and images were recorded using the Axiovision Rel. 4.5 software (Carl Zeiss MicroImaging Inc., Germany). The same procedure was used for evaluating the biocompatibility of MHL coatings with HEK 293 cells, except that the culture medium used was minimum essential medium (MEM; Gibco/BRL) supplemented with 10% fetal calf serum, 1% glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. All experiments were conducted in triplicates and the results are given as mean values with the standard deviation.

To confirm the purity and the molecular weight of MHL as calculated by its chemical structure (Fig. 1A), the dark red colored preparation was characterized by electrospray ionization mass spectrometry (ESI-MS). An excess of sodium ions were added to a methanolic solution to obtain a homogeneous sodium salt preparation and to suppress mass peaks of other salts. The most abundant molecular weight species with a mass of 513.080 agrees well with the calculated molecular weight of 513,079 of the sodium adduct of MHL (Fig. 1B and C). Minor peaks at 514.084 and 515,086 correspond to the natural relative abundance and to the calculated molecular weight of the compound with one or two C12 carbon atoms replaced by the C13 isotope, respectively. The peak at m/z 491.098 corresponds to the protonated form of the molecule. The nature of the remaining mass peaks of substances of minor abundance in the preparation was not further investigated. The analysis showed that the preparation used was pure and homogeneous and in agreement with the corrected MHL structure [28].

For coating microscope cover slips MHL was dissolved in DMSO and the solution was dispersed on the glass surface. Upon drying a readily visible orange-red colored MHL film formed. During drying, more material was deposited on the outer rims of the liquid covered area. There were no cracks or holes in the resulting coating. Whereas spray coating turned out to be comparatively wasteful, a more equal distribution was achieved when a MHL solution in ethanol was applied with a pipette onto preheated material samples. With this technique the most widely used metallic implant materials in the clinic, surgical steel and titanium, could be coated with a MHL layer with a more homogeneous appearance (Fig. 2; M). As an example for a ceramic implant material, zirconium dioxide is widely used for tooth repair or as replacement prostheses. ZrO₂ beads were coated by dip coating in a MHL solution in DMSO followed by drying at ambient temperatures. The thickness of the resulting layer was dependent on the MHL concentration in the precursor solution; however, all coatings were firmly attached and had a homogeneous, shiny and smooth appearance (Fig. 3). This indicates that curved ceramic implant surfaces can be coated with a firm layer of MHL.

The biocompatibility of MHL coatings was investigated by monitoring cultures of human and murine cells seeded directly on MHL coated glass slides. Cell attachment, cell spreading and cell proliferation were evaluated microscopically at various time points. Initially cells did not attach to MHL coatings, the cells remained rounded and in suspension. A few individual rounded cells showed an intense red staining (Fig. 4, arrows). 4 days after seeding both cell types investigated eventually adhered to the coating and showed a spread out physiology (Fig. 4A and 4B). Similar to the control, the cells then proliferated on the MHL layer until confluence was reached (Fig. 4E). This shows that MHL coatings are biocompatible in cell culture assays and after some delay they can even serve as a substrate for mammalian cell adhesion.

MHL has been reported to have antimicrobial effects against Gram-positive staining bacteria [29]. The antibacterial efficacy of MHL was examined with a plate assay using Gram-positive S. aureus as well as Gram- negative E. coli laboratory strains and P. aeruginosa, a drug-resistant biofilm forming pathogen. All bacterial strains tested were highly resistant to MHL in plate assays (Table 1). Since MHL showed promising properties as a slow release coating but only limited effects against bacteria, combinations with a well established antibiotic with proven activity were used to increase the antibacterial efficacy of the coatings. Among various MHL to ciprofloxacin ratios tested, the 1:1 (w/w) mixture formed a well adhering coating and was highly effective against all bacterial strains tested (Table 1). Therefore, it was selected for further investigations.

The coating remained attached to these materials even after immersion in a phosphate buffered solution (Fig. 2). To determine the stability of the coatings under physiological conditions the MHL coated implant material discs were incubated in PBS at 37°C and 5% CO₂ atmosphere in a cell culture incubator with a daily exchange of the supernatant. The single layer coatings gradually dissolved in the course of one to two weeks while remaining attached to the metal surface (Fig. 2). In contrast, under these conditions flakes of the coating peeled off from the glass surfaces that were used here as controls, indicating a less firm attachment to this material. These results show that MHL fulfills some basic criteria for applications as a slow release coating for metallic implants with a time course that limits its suitability to some specific short-term applications.

To determine the characteristics of the coating on zirconium dioxide beads these were incubated in a physiological phosphate buffer, whereby the buffer was exchanged at daily intervals. The ciprofloxacin and MHL concentrations in the supernatant could be determined by measuring the absorption at 320 nm and 480 nm, respectively, with a nearly linear correlation of the optical density and the concentration within the range investigated (Fig. 5A and 5B). The release of pure MHL from coatings was even slower whereas ciprofloxacin from coatings without MHL was largely dissolved in one initial burst on the first day (Fig. 5C). The concentration of ciprofloxacin released from the mixed coating with MHL was highest in the first day of the experiment and decreased gradually until day 3 when the absorption reached background levels and was no longer detectable. Therefore, for measuring the small amounts released after longer incubation periods the absorption measurements were not sensitive enough.

To establish a more sensitive antibiotic release scoring method genetically engineered luminescent Pseudomonas aeruginosa bacteria were used. This allowed the contact-free quantification of the antibacterial effects in small assay volumes. The luminescence was linear proportional to the viable bacterial cell density over a wide range of several orders of magnitude (Fig. 6). Therefore the luminescence could be directly used as a measure of cell growth and viability. Using this assay the supernatant from the beads coated with a 1:1 (w/w) mixture of MHL and ciprofloxacin significantly inhibited bacterial proliferation until day 7 (Fig. 7). The highest efficacy was observed during the first day, with a gradual decrease over the time of the experiment and resulting in a marginal antibacterial effect at day 7. In contrast, the bactericidal effect of a pure ciprofloxacin coating was reduced over 90% after one day incubation. This is in good agreement with the absorption measurements of the supernatant of the incubation buffer, showing that this test was suitable and sufficiently sensitive. The results indicated that the combined coating of ciprofloxacin with MHL was gradually released when exposed to an aqueous environment.