Patent ID: 12214070

EXAMPLES

In the present examples, the inventors developed engineered postbiotics suitable to tackle causes, symptoms and recurrence of unaesthetic manifestations of skin dysbiosis due to the growth ofS. aureus, and demonstrated:aS. aureusspecific killing activity based on a lysate containing Lysostaphin,a synergistic increase in Lysostaphin killing activity in presence of bacterial lysate,a beneficial effect on host commensal microbiota, anda soothing effect on irritated skin after skin tape-stripping

As a proof of principle, probiotic strainL. rhamnosusGG (LrOs11721) was engineered to express, in the cytoplasm, lysostaphin, a bacteriocin with high specificity forS. aureus. Lysostaphin was cloned on a plasmid (FIG.1) under the control of the sakacin inducible promoter PorfX (Sørvig et al. (2005) Microbiology (Reading, England) 151:2439-2449) and transformed intoL. rhamnosus. Transformants were grown, lysostaphin expression was induced at mid-log phase and cells were harvested at high density (OD˜1). Bacterial cells were concentrated 40× in 20 mM acetate buffer pH 5 by centrifugation before being lysed mechanically and filtered sterilized leading to a lysate (herein called lysolysate) containing bothL. rhamnosuscell components and Lysostaphin.

EXAMPLE 1

S. aureusSpecific Killing Using aL. rhamnosusLysolysate

In order to check the staphylolytic activity of the lysolysate, a turbidity reduction experiment was performed usingS. aureusNewman strain mixed with lysolysate (FIG.2). As shown inFIG.2, a rapid decrease of theS. aureuspopulation, as measured by absorbance at 600 nm, can be observed in presence of the lysolysate. No decrease in absorbance was observed whenS. aureuscells were put in presence of theL. rhamnosuslysate indicating that the expressed Lysostaphin is responsible for the turbidity reduction and so the staphylolytic activity. Inventors decided to test the lysolysate killing specificity towardsS. aureus.

Specificity of endolysin is generally at the genus level meaning that for example staphylococcal endolysins are able to kill both non-commensal species such asS. aureusbut also commensal species such asS. epidermidis. Unlike endolysins, lysostaphin has been shown to be specifically targetingS. aureusand show a much lower activity against otherStaphylococcalspecies such asS. epidermidis. To test if the Lysostaphin-containingL. rhamnosuslysate has also a highS. aureusspecificity, a killing assay was performed (FIG.3) using lysolysate in presence of both Coagulase positive strains (CoPS) among which 75S. aureusstrains and Coagulase negative strains (CoNS) among which 35S. epidermidisstrains. An average of 4.58 log reduction was obtained forS. aureusstrains against a 1.54 log reduction obtained against the 35S. epidermidisstrains. Thus lysolysate shows a high specificity towardsS. aureusspecies.

To quantify the effect of the bacterial lysate on lysostaphin activity, a turbidity reduction experiment and a CFU experiment were performed (FIG.4-5). The IC50, time to decrease initial OD by half, was measured for theL. rhamnosuslysate alone, 20 μg/ml of purified Lysostaphin (Sigma reference L9043) resuspended in 20 mM acetate buffer pH 5 (NaOAc) buffer and 20 μg/ml of purified Lysostaphin resuspended inL. rhamnosuslysate. Surprisingly, the inventors observed a higher turbidity reduction (lower IC50) and a faster decrease in CFU for the Lysostaphin in lysate compared to Lysostaphin in acetate buffer. No difference in absorbance or CFU counts were measured betweenL. rhamnosuslysate and acetate buffer indicating that there is no activity of theL. rhamnosuslysate alone and the improvement of Lysostaphin activity in lysate is not the result of an additive effect of Lysostaphin activity andL. rhamnosuslysate activity but rather a synergistic effect of the lysate on the Lysostaphin activity. Such synergistic effect of Lysostaphin and bacterial lysate has not been documented and offer an advantageous and non-obvious effect for the killing ofS. aureuswith lysolysate compare to Lysostaphin alone.

This synergistic effect between Lysostaphin and theL. rhamnosuslysate was observed for different concentrations of lysate (FIG.6) and for different concentrations of Lysostaphin (FIG.7).

To test if this effect was specific ofL. rhamnosus lysate, S. aureuskilling activity of Lysostaphin mixed with lysates from different bacteria (Lactobacillus plantarumandEscherichia coli) was measured. Synergistic effect was observed for bothLactobacillus plantarumandE. colieven if at high Lysostaphin concentration (60 μg/ml)E. colilysate killing activity was similar to the activity in buffer (FIG.7).

The inventors also tested if the pH of the lysate could explain this synergy as it is known that acidic pH is not optimum for Lysostaphin activity. Lysate fromL. rhamnosus, L. plantarumandE. coliwere respectively pH 6.8, 6.2, 6.2.S. aureusbactericidal activity was measured for different concentrations of Lysostaphin in acetate buffer with equivalent pH (6.2 and 6.8). A higher killing was still observed for the lysate compared to buffer at same pH (FIG.7) indicating that the pH cannot explain the synergy observed.

One difference between Lysostaphin in acetate buffer and Lysostaphin in lysate is the presence of a large amount of proteins and other cellular molecules that might provide a high molecular crowding environment for Lysostaphin to act. In order to test if molecular crowding could explain the increased activity of Lysostaphin in bacterial lysate the inventors performed a turbidity reduction experiment in presence of increasing concentrations of Bovine Serum Albumin (BSA) (FIG.8). All concentrations of BSA led to a slower decrease in turbidity indicating a lower Lysostaphin activity. Thus molecular crowding does not seem to explain the synergy effect observed between lysate and Lysostaphin.

The inventors have shown that a lysolysate, produced from the lysis ofL. rhamnosusbacterial cells heterologously expressing cytoplasmic Lysostaphin, allows highly efficient and specific killing ofS. aureusstrains. Surprisingly inventors demonstrated a synergistic effect between Lysostaphin andL. rhamnosuslysate increasing Lysotaphin killing activity. This synergistic effect is not specific toL. rhamnosuslysate and depends on the lysate concentration.

Materials and Methods:

Bacterial Strains:

L. plantarums15998 was isolated from fermented cabbage. Lysolysate was produced from strain s18195 (L. rhamnosus+p1016).

Production of Bacterial Lysates:

Overnight cultures ofL. plantarums15998 was inoculated from cryostock in 50 mL of MRS (NutriSelect Merck) and incubated in anaerobic conditions at 37° C. Overnight culture ofL. rhamnosuswas inoculated from cryostock in 50 mL of SPY2 (Heenan, C. N., et al.(2002). Lwt-Food Sci Technology 35, 171-176) and incubated in anaerobic conditions at 37° C. Overnight culture ofE. coliK-12 MG1655 liquid culture was grown in LB (Difco) and incubated overnight in aerobic conditions at 37° C.

Overnight cultures were diluted 1/10 in 500 mL of the appropriate media pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions except forE. colithat was incubated at 37° C. in aerobic conditions. At OD600 nm≈[1-2], bacterial cultures were put on ice, and following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes, placing the sample on ice for 2 minutes in between cycles. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU was performed before and after bead beating treatment to measure lysis efficiency.

Production of Lysolysate:

Overnight culture ofL. rhamnosus+p1016 was inoculated from cryostock in 50 mL of SPY2 medium (Heenan, C. N., et al. (2002). Lwt-Food Sci Technology 35, 171-176) with erythromycin at final concentration 5 μg/mL and incubated in anaerobic conditions at 37° C. Overnight culture was diluted 1/10 in 500 mL of SPY2 medium pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions. At OD600nm=0.3 the culture was induced with 200 ng/mL of inducing peptide IP-673 (Novopro Cat. #: 300935) and incubated at 37° C. until OD600nm=1.0. Bacterial culture was put on ice, and every following step was performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes, placing the sample on ice for 2 minutes in between cycles. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. Measure of CFU was performed before and after bead beating treatment to measure lysis efficiency.

Turbidity Reduction Experiment:

Overnight culture ofS. aureusstrainNewmanwas inoculated from an isolated colony in 15 mL of TSB (Tryptic Soy Broth, Difco) and incubated at 37° C., aerobically. Overnight culture was diluted 1/100 in a final volume of 1.5 L of TSB and incubated aerobically at 37° C. At OD600 nm=1 culture was washed twice with deionised water at 4° C., centrifuged at 4° C., 4000 g for 10 minutes, resuspended in 7.5 mL of 1× PBS (Phosphate Buffered Saline, Fisher BioReagents, pH 7.4) and finally frozen as 500 μL aliquots at −20° C.

Bacterial suspension for the turbidity reduction assay was prepared from an aliquot of the frozen stock ofS. aureusstrainNewman. Lysostaphin solution and bacterial suspension were mixed in a ratio 1:10 in duplicates in a 96-well plate (Microlon 200, transparent, flat bottom) final volume 200 μL and absorbance at 600 nm (Tecan Infinite 200 pro) was measured every 1.3 minutes, at 37° C. without agitation for 1 hour.

EXAMPLE 2

Beneficial Effect of Lysate on Skin Microbiota

The approach of the inventors aims at killing specificallyS. aureus, as shown in example 1, but also helping the skin commensal bacteria to restore homeostasis by helping them grow and occupe the niche left empty fromS. aureusdecolonization.

To test such an effect, the inventors investigated the effect of the lysate on the growth ofS. epidermidis(FIG.9).S. epidermidis(ATCC® 12228TM) was grown in poor nutrient conditions supplemented or not withL. rhamnosuslysate and cell density was followed by absorbance using OD600 nm.S. epidermidisshows a higher growth rate and final density in presence ofL. rhamnosuslysate compared to buffer indicating a beneficial effect of the lysate onS. epidermidis.

Materials and Methods:

Production ofL. rhamnosusLysate:

Overnight culture ofL. rhamnosuswas inoculated from cryostock in 50 mL of SPY2 (Heenan, C. N., et al. (2002). Lwt-Food Sci Technology 35, 171-176) and incubated in anaerobic conditions at 37° C. Overnight culture was diluted 1/10 in 500 mL of the appropriate media pre-reduced in anaerobic conditions and incubated at 37° C. in anaerobic conditions. At OD600 nm≈1, bacterial culture was put on ice, and following steps were performed at 4° C. First cells were washed twice in deionized water using centrifugation and finally resuspended in 12.5 mL of 20 mM acetate buffer pH 5 (40× concentration of the initial cell culture). The concentrated culture was then lysed using bead beating at 30 Hz for 2 cycles of 20 minutes, placing the sample on ice for 2 minutes in between cycles. Bacterial lysate was centrifuged for 10 min at 10 000 g and supernatant was then filtered (0.4 μm) and stored at 4° C. CFU was performed before and after bead beating treatment to measure lysis efficiency.

Growth Curve Experiment:

A preculture ofS. epidermidis(ATCC® 12228TM) was inoculated from cryostock into 5 mL TSB and incubated at 37° C. overnight. Overnight culture was resuspended, normalised to OD600nm=1 and diluted 1/10 in 12.5% (v/v) TSB. In a 96 well plate, 180 μL of normalized bacterial culture was supplemented with 20 μL ofL. rhamnosuslysate or 20 μL of 20 mM acetate buffer pH 5. Absorbance at 600 nm (Tecan Infinite 200 pro) was measured every 10 minutes, at 37° C. with agitation for a total of 6.8 hours.

EXAMPLE 3

L. rhamnosusLysate is Non-Irritating and has Soothing Effect

Soothing effect ofL. rhamnosuslysate on the skin of healthy volunteers submitted to mechanical stress via skin stripping was tested.

First a test was performed to test the irritating potential of the bacterial lysate. Briefly, three differentL. rhamnosuslysate concentrations were put in contact with the skin of healthy volunteers during 48 hours. After 48 hours,L. rhamnosuslysates were removed from skin and skin reaction (erythema and oedema) was evaluated by a dermatologist 15 minutes, 1 hour and 24 hours after removal. These evaluations showed that the three different concentrations ofL. rhamnosuslysate were non-irritating according to the amended Draize classification.

To measure the soothing effect of theL. rhamnosuslysate, a mechanical stress was applied on the skin forearm of 20 volunteers by removing superficial layers of the skin (FIG.10). Following skin stripping, the bacterial lysate and a placebo (20 mM acetate buffer pH 5) were applied in different treated zones. Redness of skin prior skin stripping (T-1), just after skin stripping (T0) and 30 min, 60 min, and 120 min after lysate and placebo applications was followed. A significant decrease in redness at all time after application (paired t-test p-value<0.0001 at T=30 min, T=60 min, T=120 min) was measured forL. rhamnosuslysate compared to the placebo control indicating a soothing effect of theL. rhamnosuslysate (FIG.11).

Materials and Methods:

Production ofL. rhamnosuslysate: 2L ofL. rhamnosusculture was grown in MRS until OD600 nm=5,8. Centrifugation was performed at 10 000 g for 15 min and cells were resuspended in 200 mL of acetate buffer pH 5.

Cohort Recruitment for Skin Irritation Potential Test:

10 healthy female volunteers between 18 and 70 years old were informed about test purposes and were recruited under the supervision of a dermatologist.

Skin Irritation Potential Test:

The lysate was applied as it is using a Finn Chamber fixed to the skin with a tape already been tested for its safety to ensure the occlusive application of the product. The lysate was left in contact with the skin surface for 48 hours. The cutaneous reactions were analysed 15 minutes, one hour and 24 hours after Finn Chamber removal. A Finn Chamber containing a blotting paper disk soaked with demineralized water was applied and used as a negative control. For each experimental time, erythema reaction and oedema reaction were evaluated and their mean value combined to calculate a Mean Irritation Index (IIM) according to the amended Draize classification.

Cohort Recruitment for Skin Stripping Test:

20 healthy female volunteers between 18 and 70 years old were informed about test purposes and were recruited under the supervision of a dermatologist.

Skin Stripping Test:

4 skin areas on the volar surface of the forearms of each subject were stripped in order to induce transient and not harmful increase of skin redness. The skin stripping procedure consists in removing serial layers of stratum corneum by standardized repeated applications of adhesive tapes to the skin's surface.L. rhamnosuslysate was applied in one of the 4 surfaces and as a placebo the 20 mM acetate buffer pH 5 was also applied in another of the 4 surfaces. Skin redness was measured, using a colorimeter, at baseline (T-1, before stripping procedure), after stripping procedure (T0) and 30, 60 and 120 minutes after the single product applications.
Percentage variation=(skin redness T−skin redness T0)/skin redness T0.

In conclusion the engineered postbiotics of the present invention show multiple activities that once combined on human skin should help resolve dysbiosis-induced unaesthetic manifestations and reach homeostasis faster by:killing specifically the most frequent aetiological agent that isS. aureus, without affecting the commensal skin population,stimulating growth of commensal skin population such asS. epidermidis, andhaving a soothing effect on the skin.

Some of these activities should act synergistically to address dysbiosis-induced unaesthetic manifestations.