PATHOGENIC BACTERIA
20200375931 ยท 2020-12-03
Inventors
Cpc classification
A61K35/742
HUMAN NECESSITIES
A61K38/12
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A23V2002/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to pathogenic bacteria, and is particularly concerned with treating, preventing or ameliorating bacterial infections using novel antibiotic compositions. The invention is especially useful for treating infections of Bacillus and Clostridia species, such as Clostridium difficile, Staphylococcus aureus and Mycobacterium spp. The invention extends to pharmaceutical compositions comprising such formulations. The invention also extends to methods for identifying aerobic Bacillus spp., which exhibit antibacterial activity against other bacteria, such as C. difficile, and to methods for isolating active antibacterial compositions from these aerobic Bacillus spp.
Claims
1. An antibiotic composition comprising: (i) an antibiotic, preferably Chlorotetaine, and (ii) a lipopeptide selected from the group consisting of a member of the Surfactin family, a member of the Iturin family and a member of the Fengycin family or an active derivative of any of these lipopeptides.
2. An antibiotic composition according to claim 1, wherein the antibiotic is selected from the group consisting of Chlorotetaine; a polyketide; Bacilysin; Phoslactomycin; or an active derivative thereof.
3. (canceled)
4. (canceled)
5. An antibiotic composition according to claim 1, wherein the composition forms micelles, and wherein each micelle has an average diameter of between 1 nm and 500 nm, or between 1 nm and 300 nm, or between 1 nm and 160 nm, or between 3 nm and 160 nm.
6. (canceled)
7. (canceled)
8. An antibiotic composition according to claim 1, wherein the composition further comprises a glycolipid, and wherein the glycolipid is a Rhamnolipid or an active derivative thereof, and/or a Sophorolipid or an active derivative thereof.
9. An antibiotic composition according to claim 8, wherein the glycolipid is a Rhamnolipid, and the Rhamnolipid is a Mono or Di Rhamnolipid, optionally wherein the Rhamnolipid is the C.sub.8, C.sub.8:2, C.sub.10, C.sub.12, C.sub.12:2, C.sub.14 or C.sub.14:2 isoform.
10. (canceled)
11. An antibiotic composition according to claim 2, wherein the polyketide is selected from a group consisting of: Amicoumacin C; Difficidin; Oxydifficidin; and Salinipyrone A, or an active derivative thereof, optionally wherein the polyketide is Difficidin or Oxydifficidin.
12. A composition according to claim 1, wherein the composition comprises a member of the Surfactin family; a Rhamnolipid and/or a Sophorolipid; and (i) Chlorotetaine or an active derivative thereof, or (ii) Difficidin or Oxydifficidin.
13. (canceled)
14. An antibiotic composition according to claim 1, wherein the composition further comprises Di-O-acetate lactone, and/or: a further lipopeptide selected from a group consisting of: Mycosubtilin; Mojavensin A; and Kurstakin, or an active derivative of any of these lipopeptides.
15. (canceled)
16. A composition according to claim 1, wherein the lipopeptide is a member of the Iturin family, or active derivative thereof, and a member of the Surfactin family, or active derivative thereof, optionally wherein the member of the Iturin family, or active derivative thereof, and a member of the Surfactin family, or active derivative thereof are used in a ratio of between 1:10 and 10:1, or between 1:5 and 5:1, or between 1:3 and 3:1, or between 1:2 and 2:1.
17. A composition according to claim 1, wherein the member of the Iturin Family is selected from a group consisting of: Iturin A [SEQ ID NO:1], Iturin AL [SEQ ID NO:2], Iturin C [SEQ ID NO:3], Mycosubtilin [SEQ ID NO:4], or Bacillomycin D [SEQ ID NO:5], Bacillomycin F [SEQ ID NO:6], Bacillomycin L [SEQ ID NO:7], Bacillomycin LC [SEQ ID NO:8] and Bacillopeptin A, B or C [SEQ ID NO: 17].
18. A composition according to claim 1, wherein the member of the Iturin family is Iturin A [SEQ ID NO:1], or active derivative thereof, optionally wherein the active derivative is the C14, C15 or C16 isoform.
19. A composition according to claim 1, wherein the member of the Surfactin family is selected from a group consisting of: Esperin [SEQ ID NO: 9], Lichenysin [SEQ ID NO: 10], Pumilacidin [SEQ ID NO: 11] and Surfactin [SEQ ID NO: 12], optionally wherein the active derivative thereof is the C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16, or C.sub.17 isoform.
20. (canceled)
21. A composition according to claim 1, wherein the antibiotic composition comprises the C.sub.15 isoform of Iturin A, and the C.sub.15 isoform of Surfactin.
22. (canceled)
23. A dietary supplement or foodstuff comprising the antibiotic composition according to claim 1.
24. (canceled)
25. A method of treating, preventing or ameliorating a bacterial infection, the method comprising administering or having administered, to a subject in need of such treatment, a therapeutically effective amount of the antibiotic composition according to claim 1, or the dietary supplement or foodstuff according to claim 23.
26. A method according claim 25, wherein the bacterial infection which is treated, prevented or ameliorated is a Gram-positive bacterial infection, and wherein the Gram-positive bacteria includes those in the phylum Firmicutes, which includes Clostridium spp., Bacillus spp., Listeria spp., Mycobacterium spp., Lactobacillus, Staphylococcus spp., Streptococcus spp. and Enterococcus spp.
27. (canceled)
28. A method according to claim 25, wherein the bacterium is Clostridium spp., preferably C. difficile.
29. A method according to claim 25, wherein the bacterium is Staphylococcus spp., preferably S. aureus.
30. A method according to claim 25, wherein the bacterial infection which is treated, prevented or ameliorated is a Gram-negative bacterial infection, and wherein the Gram-negative bacteria includes Enterobaceriaceae, such as Salmonella spp., and Escherichia spp., and Campylobacter spp., Pseudomonas spp. and Vibrio spp.
31. (canceled)
32. A method according to claim 25, where in the bacterium is Vibrio spp., preferably V. harveyi or V. parahaemolyticus.
33. A method according to claim 25, wherein the bacterial infection which is treated, prevented or ameliorated is a Mycobacterium spp., infection, optionally wherein the Mycobacterium spp., is Mycobacterium tuberculosis or Mycobacterium leprae and most preferably Mycobacterium tuberculosis.
34. (canceled)
35. (canceled)
36. A method of treating, preventing or ameliorating a C. difficile infection, the method comprising administering or having administered to a subject in need of such treatment, a therapeutically effective amount of Chlorotetaine or a derivative or analogue thereof.
37. (canceled)
38. (canceled)
Description
[0165] All features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.
[0166] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:
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MATERIALS & METHODS
[0203] General Methods
[0204] C. difficile strains were stored as glycerol stocks and routinely propagated on BHIS agar or medium (Brain heart infusion medium supplemented with 0.1% (w/v) cysteine and 5 mg ml.sup.1 yeast extract (76)). All culturing of C. difficile was made in an anaerobic chamber (80% N.sub.2, 10% H.sub.2, 10% CO.sub.2; Don Whitley, UK).
[0205] Strains
[0206] Clostridium strains C. difficile 630 (erythromycin resistant) was isolated from a patient with pseudomembranous colitis during an outbreak of C. difficile infection (CDI) (77). Other strains of C. difficile including the hypervirulent strain R20291 and the high toxin producing strain VPI 10463 were laboratory stocks.
[0207] Bacillus Strains
[0208] The following strains were deposited at the NCIMB, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB219YA on 15 Feb. 2018.
[0209] Designation number: NCIMB 42971 Referred to herein as: B. amyloliquefaciens SG277
[0210] Designation number: NCIMB 42972Referred to herein as: B. amyloliquefaciens SG297
[0211] Designation number: NCIMB 42973Referred to herein as: B. amyloliquefaciens SG185
[0212] Designation number: NCIMB 42974Referred to herein as: B. subtilis SG140
[0213] Growth of C. difficile and Preparation of Spores
[0214] Spores of C. difficile were prepared by growth on SMC agar plates using an anaerobic incubator (Don Whitley, UK) as described previously (36). After growth for seven days at 37 C. spores were harvested and the spore pellet further purified using centrifugation through a 20% to 50% Histodenz gradient (Sigma) as described elsewhere (85). Spore CFU was determined by heat treatment (60 C., 20 min.) and plating on BHISS agar plates (Brain heart infusion agar containing 0.1% (w/v) L-cysteine, 5 mg/ml yeast extract and the spore germinant sodium taurocholate (0.1% w/v).
[0215] Characterisation of Intestinal Spore Formers in Mice and Hamsters
[0216] C57BL/6 mice (6 weeks, female) housed in groups of 4/cage were dosed with clindamycin (30 mg/kg). Freshly voided faeces was collected 24 h before and after clindamycin treatment and then homogenised in PBS, heat-treated (68 C., 1 h) serially diluted and plated on DSM (Difco sporulation medium; (86)) or BHI agar supplemented with sodium taurocholate (1 g/L) and L-cysteine (i g/L) a medium used for culture of the human gut microbiota (87). Plates were incubated aerobically or anaerobically at 37 C. for 2 days. 500 colonies were randomly picked and restreaked. The presence of spores in colonies was checked microscopically and each colony was grown for 12 h at 37 C. in liquid culture (2 ml) using aerobic or anaerobic conditions as required before sub-culturing ( 1/100) overnight in the same conditions. The cell free supernatant was then obtained using centrifugation and filtering through a 0.45 m syringe filter. Activity against CD630 was determined using a microdilution assay (see below). Biosurfactant activity was determined using an oil displacement assay (88). gyrA sequencing used protocols and primers previously described for Bacillus (40). Antibiotic minimal inhibitory concentrations (MICs) were made using a microdilution method as stipulated by CLIS (Clinical and Laboratory Standards Institute) (89).
[0217] In Vitro Analysis of Anti-C. difficile Activity
[0218] a) Agar Diffusion Assay.
[0219] Aerobic Bacillus strains were grown in LB medium at 37 C. for 16-18 h while anaerobic spore formers were grown in BHI+cysteine+sodium taurocholate overnight in an anaerobic chamber. Samples were centrifuged (microfuge, 8,000 rpm, 10 min.) and supernatants filter-sterilised (0.45 m syringe filter) and stored on ice till use. TGY agar plates were pre-reduced and after spreading with an overnight C. difficile culture (100 l) allowed to dry for 30 min. after which 4-6 wells were cut per plate. TGY medium is, per litre, tryptic soy broth (30 g), glucose (20 g), yeast extract (10 g), L-cysteine (1 g), Resazurin (1 mg) and agar (15 g). Plates were reduced for 4 h in an anaerobic chamber before use. 5 mm diameter wells were cut in the TGY agar plate using a potato borer. 50 l of Bacillus supernatants were applied to labelled wells and the plates incubated at 37 C. for 48 h in an anaerobic chamber and zones of inhibition measured (diameter), typically 9-20 mm.
[0220] b) Microdilution Assay
[0221] Indicator Culture:
[0222] A single colony of the relevant C. difficile strain was inoculated into 10 ml of BHIS and incubated overnight at 37 C. in an anaerobic chamber. The overnight culture was then sub-cultured 1:100 into BHIS (typically 0.1 ml into 10 ml BHIS) and incubated at 37 C. for 6 h after which the culture is ready for use.
[0223] Plate Set Up:
[0224] 180 l of sterile BHIS was pipetted into the first row of a 96-well U-bottom microplate (Sigma CLS3799) and 100 l into each subsequent row. 20 l of the sample (sterile-filtered, 0.45 m) to be tested is pipetted into the first row (1:10 dilution factor) and serially diluted in a 2-fold dilution series until the last row (1:1280 dilution factor) on the microplate. For one serial dilution a media only control is also pipetted into a single well on the first column. 10 l of the 6 h C. difficile indicator culture is pipetted into each well and the plate is incubated overnight at 37 C. in an anaerobic chamber. After overnight growth the microplate contents were agitated on a rotary plate shaker at 200 rpm for 2 min. after which the OD.sub.600 was read using a microplate plate reader. Positive inhibitory activity was defined as an OD.sub.600<50% of the CD630 control.
[0225] c) Co-Culture Assays
[0226] C. difficile strains were grown in BHIS medium overnight at 37 C. under anaerobic conditions. The following day 5 ml BHIS was inoculated with 0.5 ml of overnight culture and incubated at 37 C. until the optical density reached 0.2-0.3 A.sub.600 nm. At this point 1 ml of a freshly prepared (sterile-filtered, 0.45 m) supernatant was aseptically added to the growing CD cultures and growth resumed.
[0227] Preparation of Prophylactic Treatments:
[0228] SG277 (or SG297 or SG378) was grown overnight (18 h, 37 C.) in 25 ml BHIB and after centrifugation the pellet suspended in 2 ml of supernatant. For use of the supernatant an aliquot was filter sterilised (0.45 m). For spores SG277 was grown on DSM (Difco sporulation medium; (85)) agar for 72 h at 37 C. Spore crops were harvested from the plate using a cell scraper, washed three-times in sterile water and then heat-treated to kill residual vegetative cells. Spores were suspended in water to give a concentration of 2.510.sup.10 spores/ml.
[0229] Mouse Colonisation Experiments:
[0230] Animals (C57BL/6, female, aged 6-7 weeks) were dosed i.g. with clindamycin ((clindamycin-2-phosphate, Sigma; 30 mg/kg) and 24 h later challenged with 10.sup.2 spores of CD630. Animal groups were dosed (0.2 ml, i.g.) with prophylactic treatments before and after challenge with CD630 using the schedule shown in
[0231] Hamsters:
[0232] Golden Syrian Hamsters (female) were 16-18 weeks old (Harlan UK Ltd.). For the hamster challenge, animal groups (n=6) were dosed (i.g.) with clindamycin (30 mg/kg body weight) and then challenged 3 days later with 10.sup.2 spores of CD630. Before and after CD630 challenge animals were dosed (i.g.; 2 ml/dose) with the treatments described above. The treatment regimen was six doses before CD630 challenge (48 h, 36 h, 24 h, 12 h, 4 h and 1 h) and then three-times/day post-challenge for 12 days. Animals were monitored for symptoms of disease progression and culled upon reaching the clinical endpoint. The symptoms of CDI were scored as severe/clinical end point (wet tail >2 cm, high lethargy), mild (wet tail <2 cm) or healthy. Caeca were removed and analysed for CD630 CFU and toxins A and B.
[0233] Measurement of Correlates of Colonisation
[0234] The presence of bacterial CFU and toxins in the faeces and/or caecum provides a measurement of colonisation. For determination of CFU faeces was collected 2-days post-challenge, homogenized in 70% ethanol, incubated overnight, serially diluted in sterile water and plated on ChromID plates (BioMerieux). Plates were incubated anaerobically (37 C.) for 2 days before counting. Toxins A and B were recovered from faecal (collected 24 h post challenge) or caecum samples (from dead animals) at a one-fifth (w/v) dilution in extraction buffer (PBS containing 2% (v/v) fetal calf serum, penicillin-streptomycin (Sigma P4333; 10 ml/L) and Pierce protease inhibitor tablets (Thermo 88265). Samples were homogenised in extraction buffer using wooden sticks and incubated for 2 h at 4 C. The supernatant was harvested after centrifugation (14,000, 5 min.), filtered (0.2 m) and was used immediately. Faeces was collected 24 h post challenge. Rabbit anti-toxin A and toxin B (in house reagents) was used to coat ELISA plates ( 1/6,000) and left overnight at RT. After this, plates were blocked for 1 h at 37 C. with 2% BSA. Faecal extraction samples were incubated for 2 h at RT. Replicate samples were used together with a negative control (pre-immune faecal extract). Serial dilutions of toxoid A and B were used as a reference. Detection antibodies were mouse anti-toxin A and anti-toxin B (in house reagents; 1/1000) incubated for 1 h at 30 C. HRP-conjugated anti-mouse IgG (Dako; 1/2000) incubated for 1 h at RT. Reactions were developed using TMB substrate and stopped by 2M H.sub.2SO.sub.4 and OD read at 450 nm.
[0235] Statistical Analysis
[0236] Statistical analysis was calculated and significance determined (p<0.05) using Welch's t-test for unequal variance. All statistical analysis was performed using Graphpad Prism software.
[0237] PEG Precipitation and CsCl Centrifugation
[0238] 1 L of a culture in BHIB was grown from a single colony O/N at 37 C. and supernatant collected after centrifugation at 8,000g for 10 min. The supernatant was then sterile-filtered through a 0.45 m membrane. Saturated polyethylene glycol (PEG) solution (Sigma, 81260) was added to the sterile supernatant to a final concentration of 8% and incubated for 4 h at 4 C. After incubation, the solution was centrifuged (10,000g for 30 min. at 4 C.) and the pellet suspended in 10 ml of SM buffer (per litre; NaCl 5.8 g, MgSO.sub.4.7H.sub.2O 2 g, 50 mL 1M Tris-HCl pH 7.5). 4 ml of the filtrate was then layered on top of a CsCl gradient consisting of: 4 ml of 1.3 g/cm.sup.3 CsCl, 4 ml of 1.4 g/cm.sup.3 CsCl and 4 ml of 1.5 g/cm.sup.3 CsCl in that order. This gradient was centrifuged in an Optima XPN-90 Ultracentrifuge (Beckman Coulter) with a SW32 rotor (150,000g, 18 h, 4 C.). Following centrifugation bands were carefully removed using a pipette.
[0239] Extraction and Purification of the Active Molecule/s
[0240] After cultivating the cells overnight in BHIB broth at 37 C. bacterial cells were removed from the supernatant by centrifugation at 8000g for 10 min and the supernatant was sterile-filtered through a 0.45 m membrane. The sterile supernatant (24 ml) was then precipitated with ammonium sulphate (AmSO4) for 4 h at 4 C. using saturated AmSO4 solution (6 ml) giving a final concentration of 20%. Following centrifugation at 8000g for 15 min the supernatant was removed and the precipitate resuspended in 5 ml PBS. To remove excess AmSO4 the filtrate was dialysed overnight at 4 C. After dialysis, 1 ml of 0.5% (w/v) SDS in PBS was added to the resulting filtrate and 2.5 ml applied to a Superdex 200 column (LI.D. 30 cm10 mm) and fractionated by size-exclusion chromatography under denaturing conditions using PBS/0.1% SDS as the running buffer. Fractions were tested for activity against CD630 using a microdilution assay and positive fractions were dialysed overnight at 4 C. to remove remaining SDS. Fractions showing activity were loaded on a uBondapack Phenyl, 125 m 30 cm3.9 mm (Waters) and separated by RP-HPLC using Waters 600E system controller with a 600 pump and a ABI Kratos 757 absorbance detector. The mobile phase components were (A) 0.5% acetic acid in 60% (v/v) Methanol and (B) 0.5% (v/v) acetic acid in 95% (v/v) Methanol. The fractions were injected in buffer A and the products were eluted at a flow rate of 0.5 ml/min with a linear gradient of solvent B, developed from 0% to 100% (60 min). The elution pattern was monitored by determining absorbance at 220 nm, and resultant fractions were concentrated using a EZ-2 Genevac centrifugal evaporator and then either testing for activity against CD630 using a microdilution assay or identification using mass spectrometry. For MALDI-TOF analysis the RP-HPLC fractions were premixed with -Cyano-4-hydroxycinnamic acid matrix solution (Agilent), which was acidified with TFA (0.01% (w/v) final concentration), and spotted on a 384 polished stainless steel MALDI plate (Bruker). MALDI-TOF analysis was conducted using a Bruker autoflex III smartbeam mass spectrometer. The instrument was calibrated to the mass accuracy of at least 30 ppm.
[0241] For AmSO4 precipitation, AmSO4 (113 g/L) was added to the sterile filtrate to give a 20% w/v solution and incubated O/N at 40 C. The solution was then centrifuged, and the pellet was suspended in PBS at a concentration of 30 (30 ml of initial culture=1 ml AmSO4 ppt). The AmSO4 ppt was dialysed in PBS O/N (at 40 C) to remove excess AmSO4. Activity of AmSO4 ppt= 1/5120. In order to further purify the active species, the AmSO4 precipitate was separated using a Superdex 200 column (10,000 Da-600,000 Da) in PBS+0.1% SDS (w/v). This allowed a crude separation of high MW species. SDS was added to denature/linearize unwanted proteins resulting in a purer separation
[0242] MIC Testing of Compounds Against Mycobacteria
[0243] The minimal inhibitory concentrations (MICs) of the compounds were determined using resazurin microtitre assay method (REMA). The filter-sterilised supernatant of SG277 was precipitated with ammonium sulphate (20%) and subjected to SEC (size exclusion chromotography). SEC fractions 1-20 were examined for activity to M. tuberculosis.
[0244] Briefly, serial dilutions of each compound were made between with MB7H9/ADC (BD Biosciences) media in 96-well U-bottom plates. Mycobacterium tuberculosis H37Rv and multi-drug resistant Mycobacterium tuberculosis (Peru isolate) (MDR-TB) were grown to log phase (OD.sub.600=1.0) and 10.sup.4 cells were added to all wells. The plates were thereafter incubated at 37 C. in 5% CO.sub.2 for 10 days. The plates were internally controlled using standardised serial dilutions of first line TB drugs isoniazid (INH) and rifampicin (RIF) at a concentration range of 4, 2, 1, 0.5, 0.25, 0.125, 0.063 g/ml, in addition to negative (only media) controls. The MICs were determined as the first dilution to show complete growth inhibition. This was determined visually by recording the colour change observed.
[0245] In Vitro Testing Activity Against Staphylococcus aureus
[0246] Sterile filtrates of extracellular material produced by Bacillus strains were made by growing overnight (18 h) cultures of each strain in BHIB (Brain heart infusion broth) at 37 C. (250 ml Bellco flasks). Cultures were centrifuged (9000g, 20 min.) and the supernatant filter-sterilised (0.45 m). Filtrates were kept on ice until use and used within 5 h.
[0247] S. aureus cultures are prepared fresh from single colonies by growth in 25 ml LB medium at 37 C. (in 250 ml flasks). When cultures have reached an approx. OD600 of 1.0 200 l of each culture was plated on dry LB agar plates, (2-4 plates per culture). After the inoculum had dried a sterile potato borer (or similar) was used to excise 5 mm circular wells in plates (4-5 holes per plate). 100 l of sterile extracellular filtrate was added to wells, plates were then incubated (plates up, not inverted) at 37 C. for 2 days and zones of inhibition read after 1 or 2 days (diameter of zone of inhibition, or radius). Each plate would carry a control (sterile PBS or water) and duplicates used for each test strain.
[0248] In Vivo Testing Activity Against Staphylococcus aureus
[0249] On the same day the S. aureus strain was used to inoculate LB medium. Cultures were grown at 37 C. until mid-log phase of growth (0.2-0.8 OD600). The culture was then split and to one flask sterile extracellular filtrate ( 1/10 diln.) were added. Growth was maintained at 37 C. and OD600 readings taken hourly (
[0250] Testing Activity Against Vibrio harveyi and Vibrio parahemolyticus
[0251] Strains
[0252] SG527 V. harveyi 16.6
[0253] SG528 V. harveyi 27.4
[0254] SG529 V. parahemolyticus
[0255] SG530 V. parahemolyticus
[0256] V. harveyi SG527 was grown in LB+2% NaCl overnight at 28 C. The following day 1/500 dilution was subcultured into fresh LB+2% NaCl and incubated at 28 C. in a shaking water bath at 200 rpm. The Optical Density (600 nm) and viable counts were measured every hour. Once the OD reached 0.6 the culture was split into two parts and the filter-sterile culture supernatant of SG277 was added to give a final concentration of 1/10 and OD measurements taken thereafter.
[0257] V. parahemolyticus SG529 was grown in LB+2% NaCl overnight at 28 C. The following day 1/500 dilution was subcultured into fresh LB+2% NaCl and incubated at 28 C. in a shaking water bath at 200 rpm. The Optical Density (600 nm) and viable counts were measured every hour. Once the OD reached 0.481 the culture was split into two parts and filter sterile culture supernatant of SG277 was added to the final concentration of 1/10 and OD measurements taken thereafter.
[0258] Testing the stability of AmyCide in Lypohilised Form
[0259] SG277 was prepared (o/n growth at 37 C. in BHIB) and the bacteria centrifuged and one portion lyophilised. Portions of wet material and lyophilised material were stored at RT, 4 C. and frozen and aliquots taken for analysis of anti-C. difficile activity using a microplate assay.
[0260] Testing the Stability of AmyCidem in Lypholised Form
[0261] Groups of mice (n=8/gp) were dosed (oral, intra-gastric (i.g.), i.g., 0.2 ml/dose) with different forms of test material. The regimen used is shown below with the 1st oral administration of material 4 h before challenge with CD630 (C. difficile strain 630; 100 spores). Animals were housed individually in IVCs (independently ventilated cages). The basic animal model of CDI was the colonisation model in which symptoms of CDI do not develop but colonisation is indicated by the presence of C. difficile toxins and bacterial cfu in the cecum or faeces [3-5].
[0262] Animal Groups
[0263] Gp.1. SG277 SP+VC Cells of SG277 were grown for 16 h at 37 C. in 25 ml BHIB (brain heart infusion broth). The cells were harvested from 100 ml culture (4 flasks) and the pellet resuspended in 2 ml of PBS. 1 dose=0.2 ml and 510.sup.9 CFU consisting of spores (70-100%).
[0264] Gp.2. Freeze-Dried SG277 SP+VC (SG277 SP+VC.sup.LYO)
[0265] Cells of SG277 were grown for 16 h at 37 C. in 25 ml BHIB. The cell pellet was then frozen and lyophilized o/n. On the day of use, material was resuspended in 2 ml of PBS. 1 dose=0.2 ml and 510.sup.9 CFU consisting of spores (70-100%).
[0266] Gp.3. SG277 SUP
[0267] SG277 was grown overnight at 37 C. in BHIB. After 18 h the cells removed by centrifugation and supernatant (SUP) sterilised by filtration through a 0.45 m filter. Stored at 20 C. till use. 1 dose=0.2 ml.
[0268] Gp.4. SG277 SEC Fraction (SG277 SUPSEC)
[0269] The SG277 sterile supernatant was precipitated with 20% ammonium sulphate and purified by size-exclusion chromatography (SEC) and fractions carrying anti-CD630 activity pooled (determined using an in vitro microdilution assay). Sample aliquots were stored at 20 C. and thawed on the day of use. 1 dose=0.2 ml. Gp.5. Naive 1 dose=0.2 ml of PBS.
[0270] Results
Example 1: Intestinal Aerobic Spore-Forming Bacteria, Rather than Anaerobic Spore Formers, Inhibit C. difficile
[0271] 10 days before the start of the experiment, C57BL/6 mice were kept in independently ventilated cages (IVCs) with autoclaved water, UV treated food and sterile bedding. Cages were changed every day. Faecal samples were taken from mice before and after treatment with clindamycin. Clindamycin, administered by intra-gastric (i.g.) gavage, was used at a concentration (30 mg/kg) sufficient to induce CDI but here the animals were not challenged with C. difficile. Homogenised faeces were heat-treated to kill vegetative cells and serial dilutions made on agar plates, which were incubated aerobically or anaerobically. Resultant colonies would arise from heat-resistant spores that had germinated and a total of 500 colonies from the aerobic or anaerobic plates were colony purified for further analysis. First, the inventors determined the number of colonies that inhibited growth of C. difficile strain 630 (CD63 using an agar-diffusion assay that measured activity from a cell-free supernatant of the cultured bacterium, and the results are given in Table 1.
TABLE-US-00009 TABLE 1 Intestinal spore formers pre- and post-clindamycin treatment from mouse faeces Pre-clindamycin Post-clindamycin No. anti- No. anti- No. CD No. CD Spores.sup.a isolated activity.sup.b % isolated activity.sup.b % aerobic 500 40 8 500 4 0.8 Spore formers anaerobic 500 0 0 500 0 0 Spore formers .sup.aspores isolated by aerobic or anaerobic incubation of heat-treated faeces. .sup.bactivity against CD630 using a well-diffusion assay of sterile cell free supernatants from colony cultures.
[0272] Surprisingly, the inventors found no anaerobic isolates either before or after clindamycin treatment able to inhibit CD63. On the other hand, 8% of the aerobic, spore-forming, isolates (n=40) carried anti-C. difficile activity in the extracellular material but, after clindamycin treatment, the percentage of isolates carrying anti-C. difficile activity had reduced to 0.8% (n=4). Interestingly, the inventors could not detect Bacillus species in mouse faecal samples using 16S metagenomic sequencing. This probably indicates that faecal samples may mostly contain only Bacillus spores but not vegetative cells in agreement with a recent murine study (39)
[0273] Next, the inventors characterized the aerobic isolates that carried anti-C. difficile activity, and the results are shown in Table 2.
TABLE-US-00010 TABLE 2 Phenotype of aerobic spore formers with activity against C. difficile isolated from mouse faeces.sup.a # Pre- # Post- Bacillus species Phenotype.sup.b Clindamycin Clindamycin B. Clin.sup.R BS.sup.+ 0 0 amyloliquefaciens Clin.sup.R BS.sup. 0 0 Clin.sup.S BS.sup.+ 19 3 Clin.sup.S BS.sup. 0 0 B. subtilis Clin.sup.R BS.sup.+ 0 0 Clin.sup.R BS.sup. 0 0 Clin.sup.S BS.sup.+ 10 0 Clin.sup.S BS.sup. 0 0 B. licheniformis Clin.sup.R BS.sup.+ 0 0 Clin.sup.R BS.sup. 11 1 Clin.sup.S BS.sup.+ 0 0 Clin.sup.S BS.sup. 0 0 .sup.aspores isolated by aerobic or anaerobic incubation of heat-treated faeces .sup.bClin.sup.R = clindamycin resistant, Clin.sup.S, clindamycin sensitive, BS.sup.+, biosurfactant activity, BS.sup., no biosurfactant activity. Resistance to clindamycin was determined using a microdilution assay with resistance defined as an MIC of >4 mg/L (83).
[0274] Using gyrA sequencing, which is more informative than 16S rRNA sequencing (40), the inventors demonstrated that only three Bacillus species were present, B. amyloliquefaciens (n=22), B. subtilis (n=10) and B. licheniformis (n=12). Using an assay for biosurfactant activity the inventors demonstrated that the B. amyloliquefaciens (n=22) and B. subtilis (n=10) isolates all carried biosurfactant activity in their cell-free supernatants, were mucoid and sensitive to clindamycin, see Table 2. However, the B. licheniformis isolates (n=12) were all resistant to clindamycin and did not carry biosurfactant activity.
[0275] The inventors also conducted a similar study using hamsters (Golden Syrian), as shown in Table 3, and observed a similar phenomenon with 26% of aerobic spore formers carrying activity against C. difficile and this being reduced to 1% post-clindamycin treatment.
TABLE-US-00011 TABLE 3 Intestinal spore formers pre- and post-clindamycin treatment from hamster faeces Pre-clindamycin Post-clindamycin No. anti- No. anti- No. CD No. CD Spores.sup.a isolated activity.sup.b % isolated activity.sup.b % aerobic 100 26 26 100 11 11 Spore formers anaerobic l00 0 0 0 0 0 Spore formers .sup.aspores isolated by aerobic or anaerobic incubation of heat-treated faeces. .sup.bactivity against CD630 using a well-diffusion assay of sterile cell free supernatants from colony cultures.
Example 2: Bacillus Spore-Formers Isolated from the Murine GI-Tract with In Vitro Activity Against C. difficile Inhibit CDI In Vivo
[0276] To determine whether in vitro activity to C. difficile could translate to inhibition in vivo, the inventors dosed (intra-gastric, i.g.) two groups of mice (n=4) with suspensions of bacteria isolated from the pre-clindamycin faecal samples shown in Table 1. Group 1 were dosed with a mixture of three Bacillus species (B. amyloliquefaciens, B. subtilis and B. licheniformis) that showed activity against C. difficile while Group 2 were dosed with three Bacillus isolates (one isolate each of B. amyloliquefaciens, B. subtilis and B. licheniformis) that showed no in vitro activity against C. difficile. For each group the oral dose comprised a total of 310.sup.9 bacteria consisting of 110.sup.9 bacteria from each isolate tested. A third group consisted of nave animals dosed only with PBS. As shown in
[0277] All animals in Group 1 showed no evidence of CDI as shown from the absence of toxin A or C. difficile CFU in caecum samples, see
Example 3: The Intestinal Cohort of Aerobic Spore Formers Represents an Allochthonous Population
[0278] The inventors housed groups of mice in either conventional cages (CCs) or independently ventilated cages (IVCs) using three animals per cage. IVCs carry HEPA-filtration and prevent exposure of animals to airborne bacteria. Animals received sterile food and water together with regular changes of sterile bedding. Every ten days faeces was examined for the presence of aerobic bacteria including total and heat-resistant (representing bacterial spores) CFU following aerobic incubation.
[0279] As shown in
Example 4: Human Isolates of B. amyloliquefaciens and B. subtilis that have Anti-C. difficile Activity
[0280] The inventors have previously characterised Bacillus species isolated from human faeces (19). From their collection of human isolates, they screened for strains that carried extracellular activity against C. difficile (CD630) using an agar-diffusion assay. They were able to identify a number of B. subtilis and B. amyloliquefaciens strains that carried potent activity and, using a more robust microdilution assay, quantified the level of extracellular inhibitory activity, see
[0281] B. amyloliquefaciens strains SG277 and SG297 that demonstrated the highest levels of inhibitory activity were studied further. Using a co-culture assay the inventors added sterile supernatants of SG277 or SG297 to logarithmic cultures of different C. difficile strains which revealed a clear bactericidal effect resulting in rapid and complete lysis of the C. difficile cultures, see
[0282] C. difficile cultures were grown in BHIS for 10 h at 37 C. 180p of the C. difficile culture was added to a microplate well followed by 20 l of a sterile SG277 filtrate. Plates were incubated anaerobically 18 h at 37 C. after which the OD.sub.600 was read. A SG277 sterile filtrate was also incubated overnight as a control and showed no growth. As shown in Table 4, the inventors showed that the SG277 filtrate had activity against a large number of different C. difficile ribotypes.
TABLE-US-00012 TABLE 4 Activity against different C. difficile ribotypes.sup.a C. difficile OD.sub.600 % strain Ribotype Untreated +SG277 inhibition CD630 RT012 0.756 0.059 92 SH1 RT078 0.845 0.086 90 SH101 RT115 0.772 0.081 89.5 SH102 RT176 0.981 0.072 93 R20291 RT027 0.857 0.091 89 CD196 RT027 0.798 0.056 93 SH104 RT023 0.534 0.056 89.5 VPI 10463 RT087 0.687 0.054 92 CD10 non-tox 0.824 0.102 88 SH242 RT111 0.672 0.089 87 SH200 RT056 0.914 0.076 92 SH203 RT038 0.882 0.064 93 SH218 RT001 0.732 0.081 89 SH210 RT002 0.655 0.055 92 SH213 RT014 0.758 0.064 92 SH215 RT54 1.025 0.056 95 SH220 RT336 0.783 0.049 94 SH222 RT401 0.952 0.082 91 SH231 RT56 0.791 0.073 91 SH236 RT005 0.883 0.091 90 SH239 RT103 0.966 0.086 91 SH103 RT075 0.761 0.106 86 SH3 RT017 0.692 0.055 92 SH1 RT005 0.883 0.091 90
[0283] The extracellular activity of SG277 and SG297 supernatants was characterised using a microdilution assay to measure inhibitory activity against CD strain 630. Bacillus supernatants were filter-sterilised (0.45 m) exposed to various treatment conditions. The highest dilution factor that showed inhibitory activity for the treated sample is shown in Table 5. All assays were conducted on the same day and with the same filtrate.
[0284] The various treatment conditions were: [0285] Heatfiltrates (0.5 ml) were incubated in an oven at the selected temperature for 30 min. and allowed to cool to RT before assay. [0286] Autoclavingfiltrates were autoclaved at 121 C. and 20 psi for 20 min. [0287] Simulated gastric fluid (SGF)three solutions at pH, 2, 3 and 4 were made using HCl to adjust pH. Supernatants (0.5 ml) were incubated with an equal volume of SGF (0.2% w/v NaCl, 3.5 mg/ml pepsin) and incubated for 1 h at 37 C. before assay. [0288] Enzymessupernatants (0.5 ml) were incubated with the following enzymes (all from Sigma) at 1 g/ml final concentration for 1 h at 37 C. before assay, lysozyme (L7651), lipase (L3126), amylase (A3176). For the proteases, pronase (P5147), trypsin (T8003) and proteinase K (Thermo Scientific E00491) the final concentration of enzyme was 1 mg/ml. [0289] Solventsfiltrate (0.5 ml) was vortexed for 1 min. with 0.5 ml of solvent. [0290] 0.1M NaOH or 0.1% SDSfiltrates (0.5 ml) were combined with 0.5 ml solutions of 0.2M NaOH or 0.2% (w/v) SDS, to give samples comprising 0.1M NaOH or 0.1% (w/v) SDS and incubated overnight at 37 C. [0291] 0.1%-1.0% glutaraldehydefiltrates (0.5 ml) were combined with 0.5 ml solutions of solutions (v/v) of 0.2%, 0.5%, 1.0% or 2.0% glutaraldehyde neutralised with glycine at a molar ration of 1:10 to give samples comprising of 0.1%, 0.25%, 0.5% or 1.0% (v/v) glutaraldehyde and incubated for 2 h at 37 C.
TABLE-US-00013 TABLE 5 Characterization of Extracellular Activity Treatment SG277 filtrate SG297 filtrate No treatment 1/80 1/80 Heat 60 C. 1/80 1/80 70 C. 1/80 1/80 80 C. 1/80 1/80 90 C. 1/40 1/80 100 C. 1/40 1/40 Autoclaving 0 0 SGF pH2 1/40 1/40 pH3 1/40 1/40 pH4 1/40 1/40 Enzymes Lysozyme 1/80 1/160 Lipase 1/80 1/80 Amylase 1/80 1/160 Pronase 1/80 1/80 Trypsin 1/80 1/80 Proteinase K 1/80 1/160 Solvents toluene 1/80 1/80 chloroform 1/80 1/80 acetone 1/40 1/80 0.1 M NaOH 1/40 1/80 0.1% SDS 1/40 1/80 0.1% glutaraldehyde 1/40 1/40 0.25% glutaraldehyde 1/40 1/40 0.5% glutaraldehyde 1/40 1/40 1% glutaraldehyde 1/40 1/40
[0292] As shown in Table 5, the cell free activity of SG277 and SG297 supernatants was shown to be resistant to a number of treatments including organic solvents, proteases, simulated gastric fluid (SGF) and notably heat with partial resistance to 100 C.
[0293] Furthermore, the stability of the SG277 sterile supernatant was measured over 50 days and, as shown in
[0294] SG277 cells when repeatedly washed, then mixed (1:1) with CD630 and applied to a semi-soft agar and, when incubated anaerobically overnight, revealed clear lysis of the bacterial lawn, see
TABLE-US-00014 TABLE 6 Results of CD630 applied to agar with additional treatments CD630 Treatment Plate Lysis Untreated +SG277 supernatant + +SG277 supernatant (80 C.) + +SG277 washed cells + +SG277 washed cells (80 C.) + +, lysis of CD630 lawn, , no lysis of CD630 lawn, i.e., profuse growth (ref to FIG. 5)
[0295] The activity of SG277 and SG297 sterile filtrates were assessed for their spectrum of activity against a range of Gram-positive and Gram-negative bacteria, as shown in Table 7. Activity was determined using an agar-diffusion method. SG247 is a strain of B. amyloliquefaciens shown not to have activity against C. difficile and was used as a control.
TABLE-US-00015 TABLE 7 Antimicrobial Spectrum of B. amyloliquefaciens extracellular activity Species Strains SG247 SG277 SG297 Gram-positives Bacillus anthracis Sterne DL1090.sup.b + + Bacillus pumilus SF216 + + Bacillus subtilis PY79 +/ + Bacillus clausii SF150 + + Bacillus cereus GN105 + + Bacillus megaterium QMB1551 +++ +++ Bacillus firmus SF203 +++ +++ Bacillus aquimaris SF222 ++ ++ Listeria monocytogenes ATCC 7644 ++ ++ Staphylococcus aureus ATCC 6538 +/ + Staphylococcus epidermidis ATCC 12228 +++ +++ Enterococcus fecalis ATCC 29212 ++ ++ Lactobacillus rhamnosus GG ++ +++ Lactobacillus fermentum DRL38 ++ +++ Lactobacillus mucosae SF1146 ++ Mycobacterium smegmatis mc2 155 +/ + Gram-negatives Pseudomonas aeruginosa NCTC 12903 ++ ++ Vibrio harveyi SG528 +(1/32).sup.b +(1/32).sup.b Vibrio parahemolyticus SG530.sup.c +(1/8).sup.b +(1/8).sup.b .sup.a, Activity was determined using an agar-diffusion method. + = 1-3 mm; ++ = 4-5 mm; +++ > 5 mm .sup.bactivity against these strains was bacteriostatic and inhibition was demonstrated using a microdilution assay and the highest dilution factor required to inhibit growth is shown in brackets.
[0296] Activity is represented by +=1-3 mm; ++=4-5 mm; and +++>5 mm. Activity of Pseudomonas aeruginosa, V. harveyi and V. parahemolyticus was bacteriostatic and inhibition was demonstrated using a microdilution assay and the highest dilution factor required to inhibit growth is shown in brackets.
[0297] Activity was found against Gram-positives, including a number of important pathogens, Bacillus anthracis, Listeria monocytogenes and Staphylococcus aureus. With a number of exceptions the inventors did not observe much activity against Gram-negatives. Those exceptions all exhibited bacteriostatic rather than bacteriocidal inhibition. It is of course possible that both SG277 and SG297 produce other antimicrobials that can inhibit growth but it is worthwhile noting that the strains that showed inhibition included a number of important pathogens (V. harveyi, V. parahemolyticus and P. aeruginosa).
[0298] In conclusion, the inventors show that isolates of B. subtilis and B. amyloliquefaciens have the potential to produce a potent biosurfactant that is associated with the cell surface. The inventors refer to this biosurfactant as AmyCidem.
Example 5: Inhibition of CDI In Vivo
[0299] The inventors used mouse and hamsters to determine whether the biosurfactant, AmyCide, could prevent CDI using SG277 as an exemplar. The inventors considered a number of different administrations. First, use of the sterile, cell-free supernatant (SUP), second, a suspension of spores (SPORES), and finally a suspension of an SG277 culture either (i) washed and suspended in PBS (277-PBS), or (ii) suspended in the supernatant (277-SUP). For the latter approach, the inventors used overnight cultures of SG277 that were found to contain a mixture of vegetative cells and spores. Controls were provided by an overnight culture of SG378 suspended in their sterile cell-free supernatant (378) and by using a PBS buffer (nave).
[0300] For evaluation in mice, the inventors used a model of CDI where two attributes are used to define colonisation, the presence of toxins A and B, and C. difficile CFU in the caecum (42). Animals were dosed with the four different SG277 preparations using the regimen shown in
[0301] Hamsters are considered a gold-standard for evaluation of CDI (43). The inventors used them to evaluate the ability of the same SG277 treatments described above for mice to prevent CDI. As explained in the Method section, the inventors' dosing strategy involved multiple doses of each treatment using groups of six hamsters per treatment. Animals showing symptoms of CDI were sacrificed, and the survival curves of the groups is shown in
[0302]
[0303] The inventors have conducted three other hamster studies with similar results and, combined with the mouse data, this has clearly demonstrated that a suspension of 277-SUP or 277-PBS prevents C. difficile colonisation.
[0304] Two further points can be made. First, SPORES (i.e., a suspension of SG277 spores only) have limited efficacy, for which the inventors must assume that insufficient numbers of spores can germinate in the GI-tract to secrete the biosurfactant, AmyCide. Second, despite the in vitro data, the cell-free supernatant was not as effective as when combined with cells. As shown above, the inventors predict that AmyCidem remains partially attached to the cell envelope and possibly is more efficacious when associated with the cell wall, and so this contribution to efficacy is likely important. In addition, the inventors predict that cells transiently proliferate in the GI-tract, further boosting the levels of AmyCidem. In data not shown, the inventors have found that SG277 administered to mice as a single dose persists (as determined by faecal shedding) for up to 10 days post-dosing.
Example 6: Identification of the Active Compounds
[0305] Using centrifugal concentrators of different molecular weight (mwt.) cut-offs the inventors determined the approximate molecular weight of AmyCide contained within the filter-sterilised (0.45 m) supernatant from SG277, as shown in Table 8.
TABLE-US-00016 TABLE 8 Activity against CD630 measuring for filter-sterilised supernatant from SG277 with different molecular weight cut-offs using a microdilution assay Cut-off (kDa) Activity titre % Untreated 1/80 100 <10 0 0 10-30 0 0 30-100 1/40 50 >100 1/40 50
[0306] Approximately 50% of total activity was present in the 30-100 kDa fraction with approximately another 50% being present in the >100 kDa fraction, suggesting that AmyCide might exist as a complex, be physically labile and could dissociate while retaining some activity.
[0307] Using 20% ammonium sulphate (AmSO.sub.4), the inventors found that the precipitate carried activity against C. difficile as well as biosurfactant activity. Surprisingly, SDS-PAGE analysis of the functionally active AmyCidem preparation yielded no Coomassie stained protein bands in the mwt. range 5-200 kDa but a single, white-coloured feature (labelled GB), resembling a band, with an estimated mwt. of the order of 1 kDa.
[0308] Proteins were only apparent following a combined 20%-70% precipitation. When SDS was eluted from the gel functional activity against live CD630 bacteria could be observed corresponding to the same band (labelled GB), see
[0309] Gel filtration of the 20% AmSO.sub.4 precipitate also confirmed that the active, AmyCide, component did not correspond to the protein fractions, see
[0310] AmSO4 precipitation was also performed on the sterile filtrate. This method enabled precipitation of the large molecular weight species responsible for the functional activity (as evidenced in MWCO experiments), and to reduce the amount of protein which would be co-purified alongside them. The active species were further purified by crude separation of high MW species.
[0311] Electron microscopy revealed distinct aggregates present in the active fractions following both CsCl gradient centrifugation and size exclusion chromatography, see
[0312] The inventors also confirmed the presence of gamma-polyglutamic acid (-PGA) in the AmSO.sub.4 precipitate as well as the presence of -PGA biosynthetic genes on the SG277 and SG297 genomes. Taken together AmyCide must constitute a high mwt. and primarily, non-proteinaceous complex, carrying a combination of exopolysaccharides and -PGA derived from the cell surface mucilage.
[0313] To characterise AmyCidem further, the inventors used size-exclusion chromatography (SEC) using the microdilution assay to identify active fractions that were then analysed further by RP-HPLC. Fifteen distinct fractions were obtained by RP-HPLC, see
TABLE-US-00017 TABLE 9 Activity of RP-HPLC fractions of AmyCide against CD630 determined using a microdilution assay Fraction.sup.a Activity.sup.b M wt..sup.c Identity.sup.c 1 1/5 1065.5 [M + Na].sup.+ C.sub.14 Iturin A.sup.d 1081.5 [M + K].sup.+ C.sub.14 Iturin A.sup.d 1043.5 [M + H].sup.+ C.sub.14 Iturin A.sup.d 2 1065.5 [M + Na].sup.+ C.sub.14 Iturin A.sup.e 3 1/5 1079.5 [M + Na].sup.+ C.sub.15 Iturin A 1095.5 [M + K].sup.+ C.sub.15 Iturin A 1057.5 [M + H].sup.+ C.sub.15 Iturin A 4 1093.5 [M + Na].sup.+ C.sub.16 Iturin A 1109.5 [M + K].sup.+ C.sub.16 Iturin A 1071.5 [M + H].sup.+ C.sub.16 Iturin A 5 1093.5 [M + Na].sup.+ C.sub.16 Iturin A 1109.5 [M + K].sup.+ C.sub.16 Iturin A 6 1107.5 [M + Na].sup.+ Mycosubtilin 1123.5 [M + N].sup.+ Mycosubtilin 7 1471.8 [M + Na].sup.+ C.sub.15 Fengycin A and/or C.sub.13 Fengycin B.sup.f 1449.8 [M + H].sup.+ C.sub.15 Fengycin A and/or C.sub.13 Fengycin B.sup.f 1487.8 [M + K].sup.+ C.sub.15 Fengycin A and/or C.sub.13 Fengycin B.sup.f 8 1463.8 [M + H].sup.+ C.sub.16 Fengycin and/or C.sub.14 Fengycin B 1503.8 [M + H].sup.+ C.sub.16 Fengycin A.sup.g and/or C.sub.14 Fengycin B.sup.g 9 1/20 1477.7 [M + H].sup.+ C.sub.17 Fengycin and/or C.sub.15 Fengycin B 1517.8 [M + H].sup.+ C.sub.17 Fengycin and/or C.sub.15 Fengycin B.sup.h 1463.8 [M + H].sup.+ C.sub.16 Fengycin A and/or C.sub.14 Fengycin B 10 1/40 1016.6 [M + Na].sup.+ C.sub.12 Surfactin 1032.6 [M + K].sup.+ C.sub.12 Surfactin 1054.6 [M + Na].sup.+ C.sub.12 Surfactin.sup.i 1070.6 [M + K].sup.+ C.sub.12 Surfactin.sup.i 1491.8 [M + H].sup.+ C.sub.18 Fengycin A and/or C.sub.16 Fengycin B 1477.8 [M + H].sup.+ C.sub.17 Fengycin A and/or C.sub.15 Fengycin B 1499.8 [M + Na].sup.+ C.sub.17 Fengycin A and/or C.sub.15 Fengycin B 1515.8 [M + K].sup.+ C.sub.17 Fengycin A and/or C.sub.15 Fengycin B 11 1/80 1046.6 [M + K].sup.+ C.sub.13 Surfactin 1030.6 [M + Na].sup.+ C.sub.13 Surfactin 1068.6 [M + Na].sup.+ C.sub.13 Surfactin.sup.j 1084.6 [M + K].sup.+ C.sub.13 Surfactin.sup.j 1491.8 [M + H].sup.+ C.sub.18 Fengycin A and/or C.sub.16 Fengycin B 1513.8 [M + Na].sup.+ C.sub.18 Fengycin A and/or C.sub.16 Fengycin B 1529.8 [M + K].sup.+ C.sub.18 Fengycin A and/or C.sub.16 Fengycin B 12 1/80 1060.6 [M + K].sup.+ C.sub.14 Surfactin 1044.6 [M + Na].sup.+ C.sub.14 Surfactin 1082.6 [M + Na].sup.+ C.sub.14 Surfactin.sup.k 1098.6 [M + K].sup.+ C.sub.14 Surfactin.sup.k 13 1/80 1058.6 [M + Na].sup.+ C.sub.15 Surfactin 1074.6 [M + K].sup.+ C.sub.15 Surfactin 1096.5 [M + Na].sup.+ C.sub.15 Surfactin.sup.l 1112.5 [M + K].sup.+ C.sub.15 Surfactin.sup.l 1080.6 [M + H].sup.+ C.sub.15 Surfactin.sup.l 14 1/40 1072.6 [M + Na].sup.+ C.sub.16 Surfactin 1088.6 [M + K].sup.+ C.sub.16 Surfactin 1058.6 [M + Na].sup.+ C.sub.15 Surfactin.sup.m 1074.6 [M + K].sup.+ C.sub.15 Surfactin.sup.m 1110.6[M + Na].sup.+ C.sub.16 Surfactin.sup.n 1126.6 [M + K].sup.+ C.sub.16 Surfactin.sup.n 15 1086.6 [M + Na].sup.+ C.sub.17 Surfactin 1102.6 [M + K].sup.+ C.sub.17 Surfactin 1072.6 [M + Na].sup.+ C.sub.16 Surfactin.sup.o 1058.6 [M + Na].sup.+ C.sub.15 Surfactin.sup.o 1124.6 [M + Na].sup.+ C.sub.17 Surfactin.sup.p .sup.aRP-HPLC fraction from FIG. 10A. .sup.bactivity of fraction against CD630 determined using a microdilution assay. .sup.cMonoisotopic mass, identity determined using MALDI-TOF. .sup.dtrace levels of C.sub.13 Iturin A .sup.epotential evidence of Mojavensin A .sup.ftrace levels of C.sub.13 Kurstakin .sup.gacetylated .sup.hminor components, acetylated C.sub.17 Fengycin and/or C.sub.15 Fengycin B .sup.iC.sub.12 & C.sub.13 surfactins with amino acid modifications .sup.jsay C.sub.13 & C.sub.14 surfactins with amino acid modifications .sup.ksay C14 & C.sub.15 surfactins with amino acid modifications .sup.lminor components, C.sub.15 & C.sub.16 surfactins with amino acid modifications, C.sub.17 Fengycin B and C.sub.16 Fengycin B .sup.mminor components .sup.nay C.sub.16 & C.sub.17 surfactins with amino acid modifications .sup.ominor components .sup.pC.sub.17 & C.sub.18 surfactins with amino acid modifications
[0314] The identity of the iturins, fengycins and surfactins was also confirmed using NMR (not shown). In addition, the inventors also observed evidence of two additional lipopeptides, mojavensin A (44) and kurstakin (45). As shown in
TABLE-US-00018 TABLE 11 Activity of different molecular weight cut-offs using different solvents Concentrator m wt. cut-off Solvent <5 kDa. <10 kDa. <30 kDa. <50 kDa. <100 kDa. water + + PBS + + 50% + + + + + methanol
[0315] Interestingly, the inventors found that if the 7 positive RP-HPLC fractions were combined the activity against C. difficile was increased almost 3-times compared to the cumulative activity of the individual fractions. If RP-HPLC fractions 1-15 were reconstituted, the anti-C. difficile activity was at least four-times greater than the cumulative activity of individual RP-HPLC fractions, see
[0316] Accordingly, the inventors have shown that AmyCidem produced by B. amyloliquefaciens and B. subtilis is a water-soluble complex that associates with the cell envelope and comprises different isoforms of iturin A, surfactin, mycosubtilin, fengycin A and B. The inventors have shown that when combined, the apparent mwt. of the active fraction is higher than that of the individual monomers indicating that micelles are formed, a characteristic of many biosurfactants (49). Advantageously, these micelles have been shown to aggregate into nanostructures, are more stable and resistant to degradation, have enhanced solubility and carry a higher antimicrobial activity than the monomeric form (50-52). Without wishing to be bound to any particular theory, the inventors suspect that AmyCide is mostly likely a mixture of different factors, including lipopeptides that in some B. amyloliquefaciens strains are produced in sufficient concentrations to form micelles.
[0317] It is possible that mixed micelles are being formed. Micelles might be considered a laboratory-based phenomenon, but the SEM images of aggregates of spherical-like granules obtained following SEC resemble synthetically produced micelles (53). The inventors believe that AmyCide micelles might, in some way, be stabilised or entrapped in the copious exopolysaccharides that encase the vegetative cell mucilage. The active strains examined here produced profuse biofilms and produced mucoid colonies, both attributes requiring the production of large amounts of extracellular polysaccharide (54).
[0318] Surfactant molecules form micelles at concentrations higher than the critical micelle concentration (CMC) (49). If surfactants were produced at sufficiently high concentrations by the inventor's B. amyloliquefaciens strains then this might explain how activity was associated with the high mwt fractions. Ultrafiltration has been used to purify and concentrate biosurfactants where at concentrations greater than the CMC surfactants can be purified (58).
[0319] As shown in
[0320] Accordingly, the inventors have shown that methanol (50%) was able to disrupt this activity enabling activity to correlate with a mwt. of less than 5 kDa. If the filtrate was then dissolved in water, then the mwt of activity reverted to >5 KDa (and >30 kDa). This suggests that the apparent high mwt of anti-C. difficile activity corresponds to the formation of surfactant micelles. Surfactants are produced at sufficiently high levels by these bacteria enabling them to form micelles either comprised of individual surfactants or mixed populations.
[0321] Using dynamic light scattering (DIS), the inventors examined the SEC fraction of an SG277 AmSO.sub.4 precipitate in dH.sub.2O revealing one large population, most likely micelles, with an average size of 3 nm (*1 nm; see
[0322] C.sub.15 iturin A is an example of a lipopeptide able to form micelles and is water-soluble. The inventors suspect that C.sub.15 iturin A may enable other lipopeptides to be solubilised in water and potentially form mixed micelles enabling them to target the bacterial cell. To test this, the inventors mixed water solubilised C.sub.15 iturin A with a commercial surfactin (Sigma S3523; derived from B. subtilis and a mixture of C.sub.13-C.sub.15 surfactins). The commercial surfactin was not soluble in water and showed no activity against C. difficile while C.sub.15 iturin A had a functional activity of 1/40 using the microplate assay. Confirming their hypothesis, the combined C.sub.15 iturin A+C.sub.15 surfactin exhibited a higher activity against C. difficile ( 1/160). This then reveals a possible mechanism for activity against C. difficile hypothesised by the inventors. The presence of C.sub.15 iturin A enables the solubilisation of Cis surfactin in water and probably all or many of the other lipopeptides present in or on the cell wall of SG277 and other Bacillus species. The important requirements for activity are the concentration of lipopeptides that are produced (since micelles can only be formed at levels above a critical threshold). The micelles formed are most likely to be mixed micelles (i.e., carrying different lipopeptide species but all carrying C.sub.15 iturin A)(at least for B. amyloliquefaciens).
[0323] The 3 nm size of water-soluble micelles in the SEC fraction is believed to be important because it may also explain how the micelles are able to make contact with the cell envelope of C. difficile. As biosurfactants, the molecule must interact with the phospholipid bilayer of C. difficile (or other sensitive bacteria).
[0324] Comparison of RP-HPLC fractions from B. amyloliquefaciens SG277 and SG297 (see
[0325] Comparison of SG277 and two B. subtilis positive strains (SG83 and SG140) showed that the B. subtilis strains did not produce iturins (
[0326] Lastly, a positive B. licheniformis strain (SG130) was shown to produce no biosurfactants (see
[0327] Returning to the original mouse experiments reported in Example 1, the inventors verified that first, surfactins and iturins were detectable in mouse faeces as well as in intestinal homogenates (jejunum, ileum, caecum), and second, that the three mouse-Bacillus subtilis strains (SG17, SG83 and SG140) produced surfactins.
Example 7: Bacteriolytic Vs Bacteriostatic Activity
[0328] To further examine the constituent species of the crude SEC active fraction, separation was performed using a SEC-HPLC column (
[0329] Fractions 1-3 were analysed for their ability to inhibit growth of cultures of CD630 by addition of test material at the log phase of cell growth (OD.sub.6000.3). Test materials were diluted in dH.sub.2O to normalise so that each sample to be tested carried the same activity/volume. 120 ml of diluted test material was added to 1 ml of CD630 culture and 0.2 ml removed hourly for OD600 readings. For this study the test materials were as shown in Table 12.
TABLE-US-00019 TABLE 12 Test materials Test Original activity Dilution material Description (1/n).sup.a required.sup.b 1 sterile extracellular 160 10 filtrate 2 AmSO.sub.4 ppt 5120 320 3 SEC crude fraction.sup.c 2560 160 4 fraction 1.sup.d 320 20 5 fraction 2.sup.d 0 20 6 fraction 3.sup.d 320 20 7 fraction 1 + 3 320 20 .sup.aoriginal activity determined using a microplate assay .sup.bthe test material is dissolved in dH2o to normalise activity to 16. The dilution factor is indicated. .sup.cthe active fraction determined by SEC of the AmSO4 ppt .sup.dactive fractions following SEC-HPLC analysis of the crude SEC active fraction
[0330] Optical density (OD.sub.600) was measured before and after addition of test material. A decline in OD.sub.600 indicates bacteriolytic activity while stalling of the increase in OD.sub.600 indicates either bacteriostatic or bacteriocidal growth. On the other hand a decline in CFU/ml indicates both bacteriolytic and/or bacteriocidal activity. (
TABLE-US-00020 TABLE 13 Summary of anti-CD activity Bacteriolytic.sup.1 Bacteriocidal2 Bacteriostatic.sup.3 Extracellular + + Filtrate.sup.4 AMSO.sub.4 + + SEC.sup.5 + fraction 1 + + fraction 2 fraction 3 + fraction 4 not tested not tested not tested .sup.1decline in OD600 of the CD630 culture .sup.2cessation of cell growth (CD630 cfu/ml) .sup.3inhibition of cell growth (CD630 cfu/ml) .sup.4sterile extracellular filtrate .sup.5SEC fraction prior to HPLC analysis. This produced partial lysis.
[0331] The basic identity of each fraction is shown in Table 14 below.
TABLE-US-00021 TABLE 14 SEC-HPLC Fractions Fraction Composition anti-CD activity other.sup.1 1 surfactin + cloudy solution (1/320) 2 Iturins & fengycin clear solution 3 Chlorotetaine + clear solution (1/320) 4 n/a (tail from 3) .sup.1clear solution can indicate good solubility
[0332] Fraction 3 was bacteriocidal while fraction 1 (surfactins) was bacteriolytic. The SEC crude fraction showed partial bacteriolytic activity. Without wishing to be bound to any particular theory, the inventors believe this is because of the SDS used in the preparation of the SEC fraction from the AmSO4 precipitate may have disrupted micelles and thus activity. Taken together, this suggests that functionality of AmyCide complex is dependent on molecular composition and variations in the concentration of individual components influences activity. Interestingly, the inventors have observed that fraction 3 is significantly reduced and sometimes absent in B. subtilis strains possibly explaining their lowered activity to C. difficile.
[0333] Further analysis was performed on factions 1-3, whereby individual fractions from SEC-HPLC analysis were now run on an RP-HPLC column (
[0334] SEC-HPLC fraction 3 was applied to an RP-HPLC column and 36 fractions analysed for anti-CD630 activity. In parallel the SG277 total crude SEC fraction was run on RP-HPLC and 36 fractions examined (
[0335] Fractions 1-5 of SEC-HPLC fraction 3 showed clear activity to CD630 and the MS analysis of these fractions is shown in Table 15.
[0336] For SG277 run by RP-HPLC in parallel Fraction 4 and fractions 30-32 of the SG277 crude SEC showed activity. Fractions 30-32 are Surfactins while the identity of fraction 4 using mass spec is shown in Table 16 below. Note that low activity was sometimes found with Iturin fractions but not found in the analysis shown here. The species clearly identifiable in fraction 4 was the antibiotic Chlorotetaine. This was the only compound is important for understanding how how AmyCidem kills C. difficile.
[0337] Fractions 1-9 of SEC-HPLC fraction 3 do not absorb at 220 nm and therefore the active fractions (1-9) are unlikely to be proteinaceous or lipopeptides. Surfactin however, is also a component in inhibiting CD suggesting a complex.
TABLE-US-00022 TABLE 15 Mass-Spc ID of Fractions 1-5 of the RP-HPLC fractionation of SG277 SEC ID m/z other Chlorotetaine 311 Chlorotetaine (.sup.35Cl) [M + Na] 313 Chlorotetaine (.sup.37Cl) [M + Na] 327 Chlorotetaine (.sup.35Cl) [M + K] or Hydroxychlorotetaine (.sup.35Cl) [M + Na] 329 Chlorotetaine (.sup.37Cl) [M + K] or Hydroxychlorotetaine (.sup.37Cl) [M + Na] 333 Chlorotetaine (.sup.35Cl) [M H + 2 Na] 335 Chlorotetaine (.sup.37Cl) [M H + 2 Na] 343 Hydroxychlorotetaine (.sup.35Cl) [M + K] 345 Hydroxychlorotetaine (.sup.37Cl) [M + K]
TABLE-US-00023 TABLE 16 Mass-Spec ID of Fraction 4 of the SEC-HPLC Fraction 3 by RP-HPLC (as shown in Fig. 36) monois- monoisotopic otopic experimental theoretical ERROR ERROR measurement m/z Interpretation m/z D.sup.a PPM 311.094 [M + Na] 288.104 Chlorotetaine (.sup.35Cl) 288.088 0.016 57 313.092 [M + Na] 290.102 Chlorotetaine (.sup.37Cl) 290.085 0.017 60 327.079 [M + K] 288.116 Chlorotetaine (.sup.35Cl) 288.088 0.028 96 327.079 [M + Na] 304.090 Hydroxychlorotetaine 304.083 0.007 22 (.sup.35Cl) 329.083 [M + K] 290.120 Chlorotetaine (.sup.37Cl) 290.085 0.035 119 Hydroxychlorotetaine 329.083 [M + Na] 306.094 (.sup.37Cl) 306.080 0.014 44 333.072 [M H + 2 Na] 288.101 Chlorotetaine (.sup.35Cl) 288.088 0.013 43 335.084 [M H + 2 Na] 290.112 Chlorotetaine (.sup.37Cl) 290.085 0.027 93 343.067 [M + K] 304.103 Hydroxychlorotetaine 304.083 0.020 66 (.sup.35Cl) 345.067 [M + K] 306.103 Hydroxychlorotetaine 306.080 0.023 76 (.sup.37Cl) .sup.aabsolute error
[0338] Cryo-EM analysis was performed on the SEC fraction and showed small disc-like objects tightly packed and <10 nm in size. Also, occasional large size discs were apparent with a diameter of 60 nm. These objects resembled micelles of <10 nm and 160 nm in size (
[0339] DIS Analysis (Dynamic Light Scattering) showed that the SEC fraction exhibited 3 nm particles agreeing with cyro-EM analysis (
[0340] The inventors also assessed the synergistic effect of the identified factors using DLS (
[0341] In a similar study it has been shown that lipopeptides of B. subtilis undergo a change in micelle size when combined (Jauregi, Coutte et al. 2013). For example, combining surfactin (5-105 nm) with mycosubtilin (8-18 nm) creates mixed micelles of 8 nm. This is similar to what the inventors observed.
[0342] The inventors also assessed activity against CD630 using a microdilution assay (Table 17).
TABLE-US-00024 TABLE 17 Activity against CD630 measured using a microdilution assay. Cut-off (kDa).sup.a Activity titre Untreated 1/80 <10 0 10-30 0 30-100 1/40 >100 1/40
[0343] Molecular weight cut off shows the size of molecules (table above) to be above 3 kDa and yet all lipopeptides demonstrated a range of sizes that are universally small in size (400-1.5 kDa). When separated on a SEC column the elution of the molecule would suggest a size of >20 kDa, comparing to protein standards (
[0344] Without wishing to be bound to any particular theory, these two observations would imply that these components are monomers in solution but interacting with each other leading to a single complex which elutes in the same fraction during SEC separation. Interestingly if methanol is added to the SEC solution all interactions between components break down and molecular weight reduces to below 5 kDathe size of lipopeptides as monomers. Addition of methanol to a solution increases the hydrophobicity of the solution resulting in the dissolution of the micelle as the intermolecular force between the more hydrophobic solvent and hydrophobic tail of the surfactant molecules increases. Cheng et al., 2013 provide an example of a similar blend of lipopeptides and glycolipids in B. subtilis. (Cheng, Tang et al. 2013). Note that discrepancy in size is also observed when considering CsCl2 ultracentrifugation which implies large molecular weight in comparison to the band of activity seen in SDS page gels which runs alongside the dye front. The SDS-page seems to separate the complex into individual, smaller components.
[0345] An assessment of the combination of different factors was performed. Fractions 1-3 from SEC-HPLC (
[0346] fraction 1=surfactins
[0347] fraction 2=iturins and fengycins
[0348] fraction 3=Chlorotetaine
[0349] Addition of fractions 1+2+3 showed activity greater ( 1/640) than either 1, 2 or 3 alone or 1+2.1+3 or 2+3. The original SEC fraction was 1/1280 in this case (TOTAL).
[0350] Fractions from RP-HPLC fractionation (see
[0351] Commercial (SIGMA) samples of iturin (I), fengycin (F) and surfactin (S) were used in this analysis and none showed activity to CD630 alone or combined.
[0352] Fractions 1-3=iturin A
[0353] Fractions 7-9=fengycins
[0354] Fractions 10-14=surfactins
[0355] For RP-HPLC fractions iturins and fengycins showed low activity and less than surfactins (fr 10-14). The sum of individual peaks was 1/320 and when iturins (fr 1-3) were combined with fengycins (fr 7-9) and surfactins (fr 10-14) the activity increased to 1/640 but still less than the entire SEC fraction ( 1/1280).
[0356] Taken together this data for SEC-HPLC and RP-HPLC fractions shows a synergistic effect when individual active fractions are combined that is greater than the mathematical sum.
Example 8: Activity Against Mycobacterium tuberculosis
[0357] AmSO4 material from SG277 was subjected to SEC chromotography and 20 fractions analysed for activity to M. tuberculosis. The minimal inhibitory concentrations (MICs) of SEC fractions described above were determined for inhibition of drug-sensitive and drug-resistant M. tuberculosis growth and compared with the antibiotics. High inhibitory activity was observed for fractions 1, 2, 3 15 and 16 for both mycobacterial cultures (Table 18 and
TABLE-US-00025 TABLE 18 MIC of compounds determined for drug sensitive M. tuberculosis H37Rv and multi-drug resistant M. tuberculosis (Peru isolate) by the REMA method. MIC (g/ml) Crude SEC M. tuberculosis MDR-TB Peru Fractions H37Rv isolate 1 1/64 1/64 2 1/64 1/32 3 1/64 1/32 4 1/8 1/4 5 >1/2 >1/2 11 >1/2 >1/2 12 >1/2 >1/2 13 >1/2 >1/2 14 1/8 1/8 15 1/64 1/32 16 1/64 1/64 17 1/32 1/8 18 1/4 1/4 19 >1/2 >1/2 20 >1/2 >1/2 Isoniazid 0.25 >4.0 Rifampicin 0.5 >4.0
[0358] Rifamycin is considered a standard for treating Tuberculosis. The data here shows that RP-HPLC fractions carried activity to M. tuberculosis, notably, fractions 1-3 and 15 and 16.
Example 9: Activity Against Staphylococcus aureus
[0359] In Vitro Activity
[0360] An in vitro assay was used to assess activity against S. aureus (DL1065). B. amyloliquefaciens strains were all strains that showed extracellular activity against C. difficile and carried an AmyCide complex. SG378 was a B. amyloliquefaciens strain that showed no anti-CD activity. The results are shown in Table 19.
TABLE-US-00026 TABLE 19 In vitro activity of B. amyloliquefaciens strains to S. aureus B. amyloliquefaciens Zone of inhibition strain (diameter, mm) Activity SG57 16 + SG137 26 ++ SG277 15 + SG297 18 + SG185 22 ++ SG378 0
[0361] In Vivo Activity
[0362] The inventors also performed an in vivo assay as described under the methods section above, to assess activity against S. aureus.
[0363] Sterile filtrates of a variety of B. amyloliquefaciens strains were added to exponentially growing cultures of S. aureus. Cell growth was monitored by optical density readings (
[0364] Extracellular activity to S. aureus was clearly present in three B. amyloliquefaciens strains, SG185, SG277 and SG297.
Example 10: Activity Against V. harueyi and V. parahemolyticus
[0365] The culture supernatant from SG277 and SG297 was added to cultures of V. harveyi and V. parahemolyticus at a final concentration of 1/10.
[0366] Both SG277 and SG297 supernatants exhibited activity against both Vibrio strains (V. harveyi and V. parahemolyticus) (
Example 11: Testing the Stability and Activity of AmyCide in Lyphophilised Form
[0367] The stability of lyphophilised SG277 was tested at varying temperatures and compared with fresh SG2776.
[0368] Lyophilised bacteria (a mixture of spores and vegetative cells) does not impair the efficacy of bacteria to prevent CDI. 100% protection is achieved (
[0369] Administration of supernatant material shows 66% protection while SEC purified material 33% protection. Even for animals still colonised with CD the levels of CD CFU is reduced. This suggests that the failure to obtain 100% protection most likely results from dosage and/or formulation. That is, using higher doses should confer 100% protection.
DISCUSSION
[0370] Targeted restoration of the gut microbiota has proven efficacious for the treatment of CDI but the mechanism of action has remained elusive, and includes rebalancing the gut microbiota, competitive exclusion and the contribution of predatory bacteriophages (10-12). The inventors show that these methods appear to have overlooked the contribution of aerobic spore-formers for control of CDI. There are several reasons for this. First, methods have employed sequence or metagenomic-based methods to identify candidates for bacteriotherapy. Second, previous methods all focus on anaerobic bacteria, and assume that minority bacterial populations are unlikely to play a predominant role in the control of intestinal infections.
[0371] By contrast, the inventors' approach has focused on traditional microbiological methods for identification of bacteria with functional activity coupled with a focus on aerobic spore-forming bacteria rather than anaerobic bacteria. Using CDI as a model gastrointestinal pathogen, the cohort of aerobic spore-formers from the inventor's work is mostly Bacillus species and, of course, these would ordinarily be mostly undetectable using standard metagenomic- or sequence-based approaches. These bacteria constitute an allochthonous population that implies the inventors' exposure to aerobic spore formers plays an important role in controlling CDI.
[0372] Rates of CDI are highest in industrialised countries, most notably the USA followed by the UK (62, 63) and it could be argued then that the increase in processed food and decreased exposure to environmental bacteria might be an important factor in why rates are so high in these countries. The hygiene hypothesis attempts to link changes in lifestyle in industrialised countries that have produced a decrease in infectious disease with a concurrent rise in allergic and autoimmune disease (64-67). The inventors speculate that, broadly, the same phenomenon may be a contributing factor underlying the high rates of CDI observed in the USA and UK (68, 69). Decreased exposure to environmental microbes through for example, diet and increased antibiotic usage, might be expected to lower our exposure to Bacilli and may, in part, account for the increased rates of CDI. As noted by others, the root cause is likely to be multifactorial (70) but the inventors believe that the contribution of exposure to environmental microbes has, hitherto, been unnoticed. Although the lack of thorough or proper diagnosis may partly explain the low incidence rates of CDI in developing countries it is intriguing that these are the same countries that have a record of abuse and overuse of antibiotics (71). It is also interesting that CDI is now beginning to emerge in many developing countries in parallel with improved living standards, hygiene and nutrition (72,73). The inventors suspect then that our exposure to environmental bacteria plays an important role in controlling CDI and also in other gastrointestinal diseases.
[0373] The ability of Bacillus species, including B. amyloliquefaciens and B. subtilis, to produce antimicrobials is well known and the, non-ribosomally produced, cyclic lipopeptides (iturins and surfactins and fengycins) are considered particularly powerful biosurfactants (47, 74-78). A number of these are used commercially as anti-fungal agents for the control of plant disease (79-81). The inventors show here that the functional biosurfacant or antibiotic, referred to herein as AmyCidem, produced by B. amyloliquefaciens and B. subtilis is a large complex that associates with the cell envelope and comprises lipopeptide biosurfactants together with Chlorotetaine. The combination of biosurfactants is synergistic and at high concentrations creates micelles, most probably mixed micelles. The primary composition of these AmyCide micelles is surfactin but other lipopeptides (iturin A, surfactin, mycosubtilin and fengycin A and B) or glycolipids could substitute at high concentrations to produce similar micelles. These micelles appear to form complexes that also have the unique ability to combine or concentrate antimicrobials produced by Bacillus species, notably Chlorotetaine. Although Chlorotetaine is clearly bacteriocidal to C. difficile while the lipopeptides are by themselves bacteriolytic, without wishing to be bound to any particular theory, the inventors assume that the complex of lipopeptides and Chlorotetaine facilitates greater activity, possibly by enhancing the stability of one or both components (lipopeptides and/or Chlorotetaine) or increasing avidity, that is, the rate of killing. For example, but without wishing to be bound to any particular theory, the inventors believe that in the presence of biosurfactant micelles or as a complex the antibiotic, namely Chlorotetaine, has improved stability or a higher density, and thus more targeted activity. Chlorotetaine is a non-ribosomally synthesized dipeptide antibiotic similar to Bascilysin, an antibiotic commonly produced by B. subtilis (Phister et al, Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp. strain CS93 isolated from pozol, a Mexican fermented maize dough, Appl Environ Microbiol 70(1): 631-634. The B. amyloliquefaciens strains described here all produce Bacilysin and it is likely that they use the same or a modified biosynthetic pathway. Bacilysin is a dipeptide composed of L-alanine and L-anticapsin and is known as a Trojan Horse antibiotic. Susceptible cells use dipeptide permeases to import Bacilysin after which peptidases release the anticapsin inside the cell. Anticapsin, as an analogue of glutamine, can inhibit glucosamine synthase. The irreversible inhibition of glucosamine synthase results in the lysis of bacterial or fungal cells. Chlorotetaine is a dipeptide carrying an unusual chlorine-containing amino acid (3-chloro-4-oxo-2-cyclo-hexenyl) alaninc fused to L-alaninc but most probably has a similar mode of action as Bacilysin.
[0374] AmyCide is particularly unusual because it comprises a mixture of lipopeptides and antibiotic that form a complex and are associated with the cell wall. The inventors suspect that aggregation occurs by virtue of the copious exopolysaccharides that encase the vegetative cell in mucilage. This polysaccharide component also contains -PGA which is typically present in the mucilage of B. amyloliquefaciens (82).
[0375] Mucoid colonies and exopolysaccharide production are characteristic of many Bacillus strains (54) and probably assist biofilm formation. The inventors speculate that an additional role is in the entrapment of biosurfactants. The aggregation of biosurfactants must also improve the performance of these antibiotics since as shown here, as individual moieties, their activity is greatly reduced. The inventors observe that activity of these biosurfactants is greatly increased when combined. An intriguing question is how AmyCide lyses C. difficile since C. difficile carries a protective proteinaeceous S-layer that encases the cell (60). The S-layer coating may not provide a complete covering to the cell wall or, alternatively, the arrays of self-assembled S-layer proteins may be broken down or denatured by AmyCide. Pores formed by ordered arrays of S-layer proteins are 30 in diameter (61) so it is possible that AmyCidem could permeate this barrier.
CONCLUSIONS
[0376] In conclusion, the inventors have shown that that B. amyloliquefaciens and B. subtilis strains carry surprising activity against C. difficile, which can be attributed to biosurfactants and Chlorotetataine and their ability to i) form micelles, ii) act synergistically and iii) concentrate or stabilise other antibiotics. These biosurfactants include different isoforms of surfactins, iturins, fengycins and potentially others (see
[0377] A role of extracellular polysaccharide in stabilising the complex must be considered since the highest levels of activity always correspond to the SEC fractions and individual components have the lower activities. C15 iturin A is a water-soluble lipopeptide that can form micelles (7 nm diameter) and plays a role in solubilising other lipopeptides with the formation of mixed micelles (3 nm diameter). The profile of SG297 is different, but the inventors assume the same concept applies.
[0378] The inventors assume that different strains produce different activities dependent upon the concentration of lipopeptides and micelles thus produced. To explain the B. subtilis strains that have lower activity, but no C15 iturin A, the inventors propose that there are other lipopeptides that play a role in solubilising lipopeptides, but C15 iturin is effective.
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