Composition

An antimicrobial composition that includes a rhamnolipid and a cell membrane disruptor, wherein the cell membrane disruptor includes a carboxylic acid selected from the group consisting of caprylic acid, azelaic acid, caproic acid, malic acid, maleic acid, fumaric acid, succinic acid, glutaric acid, adipic acid, tartaric acid, and mixtures thereof, and wherein the cell membrane disruptor is present in the range of 0.01-10 wt % of the composition.

The present invention relates to an improved bactericidal composition comprising a rhamnolipid.

Despite the prior art there remains a need for improved bactericidal rhamnolipid compositions.

Accordingly, and in a first aspect, the present invention provides a bactericidal composition comprising a rhamnolipid and a cell membrane disrupter.

The following data illustrates the bacteriostat efficacy of a composition comprising a rhamnolipid and a cell membrane disruptor, caprylic acid.

Materials and Methods

Microorganisms and Culture Conditions

P. aeruginosaATCC 15442 andStaphylococcus aureusATCC 9144, were maintained in nutrient broth plus 20% glycerol at −20° C. Bacterial growth from a nutrient agar slant incubated for 24 h at 30° C. was used to obtain a bacterial suspension with an optical density at 570 nm adjusted to give 108 cfu/mL.

Cell Membrane Disruptor: Caprylic Acid

Caprylic Acid (CAS RN 0124-07-2) was obtained from Sigma Aldrich (Catalogue number C2875) as >99% purity.

The Mono- and di-rhamnolipid was separated from the sample obtained from Jeneil using the protocol below and the individual R1/R2 fractions obtained were used to produce the 10% rhamnolipid solution mentioned above

A quantified amount of JBR425 was acidified to pH 3 using 12M HCl and placed in a refrigerator overnight. The supernatant was then extracted three times using a 2:1 mixture of Chloroform and Ethanol. The solvent was then removed by rotary evaporation and the isolated rhamnolipid mixture was then re-dissolved in methanol.

The process of separating and characterising the mixture was carried out using an HPLC connected to an Ion Trap Electrospray ionisation Mass Spectrometer. The mode of ionisation was in negative mode with a scanning range of 50-1200 Da. The column used to separate was a Phenomenex luna C18 250×4.6 mm 5 μm column. The mobile phase: water (mobile phase A) and acetonitrile (mobile phase B) were used to separate via a gradient of 60:40 (A:B) changing to 30:70 (A:B) over 30 minutes. The system was then held for 5 minutes before returning to the start conditions all at a flow rate of 0.5 ml/min. The injection volume was 10 μl.

Biofilm Growth on the BioFlux Flowthrough Device.

To analyze biofilm formation under flow conditions, the BioFlux 200 system (Fluxion Biosciences Inc., South San Francisco, Calif.) was used which allows automated image acquisition within specialized multi-well plates. To grow biofilms, the microfluidic channels (depth, 75 μm; width, 350 μm) were primed with TSB (50%) at 10.0 dyn/cm2. Channels were seeded with 107 CFU from an overnight culture ofP. aeruginosaATCC 15442,Staphylococcus aureusATCC 9144 and a mixed culture of both. The plate was then incubated at 30° C. for 48 h to allow cells to adhere. After biofilms had formed, planktonic cells were removed, and PBS 1× (as control) and different treatments were added to the input wells at a flow rate of 279 μL/h for 30 min. The results were recorded with a microscope Evon (10×) (17% Light)

Results

The effect of rhamnolipid together with caprylic acid on pre-formed biofilms byPseudomonas aeruginosaATCC 15442,Staphylococcus aureusATCC 9144 and a mixed culture was determined under Bioflux flowthrough conditions. The disruption produced by the combination of caprylic acid together with rhamnolipids was confirmed. All isolates developed biofilms over 48 h. However, there was considerable variability in all cases in terms of spread around of the microfluidic channel.Pseudomonas aeruginosabiofilms and the mixed culture were well-formed (FIGS. (A), (E) and (G)) under flow conditions, however the biofilms formed byStaphylococcus aureusATCC 9144 (FIG. (C)) were not as thick, but good enough to be considered a multicellular community that represented a fundamentally different physiological state compared to free-living planktonic bacteria.

After 48 h all the plates were rinsed with PBS 1×, and the treatments with the combination of rhamnolipids (0.04% v/v) and caprylic acid (0.01% v/v) were applied for 30 minutes, after which period more than the 90% of the biofilms were disrupted. It is interesting to note that the way that the Gram-positive and Gram-Negative microorganisms respond to the combination between rhamnolipids and caprylic acid varied, suggesting a possible synergy between them compared to the results when the components are applied individually (data not shown). The results are illustrated in the Figure which shows Biofilm formation and disruption in a BioFlux channel. The images are phasecontrast images and show fully formed biofilms after 48 h of incubation at 30° C., and the images were recorded with a microscope Evon (10×) (17% Light) as follows

(F) Mixed Culture (E) after treatment with Rhamnolipid (0.04%) and Caprylic acid (0.01%).