IE20080952U1 - Thuricin cd, an antimicrobial for specifically targeting clostridium difficile - Google Patents

Abstract

ABSTRACT The present invention relates to a new baeteriocin. to microbial strains which can produce it and to uses of the bacteriocin and the strains. The bacteriocin is effective uganist Clnstridium difticile and Listeria monoeytogenes amongst other oi'ganisins.

Description

FIELD of the Invention: Thc present invention relates to a microbial strain. which produces a bacteriocin having a narrow spectrum ofinhibition but being effective, particularly against ('. diffici/c and I,/sierra monocymgenes and to the bacteriocin produced by the strain.

Background to the Invention With the upsurge in antibiotic resistance among pathogens and the increase in hospital acquired infections such as MRSA and C. drfficile there is a renewed urgency in discovering novel antimicrobial compounds to combat these diseases. First described in 1935. C. dz"/ficfle was not recognised as the causative agent of nosocomial diarrhoea until the 1070s (George et al., 1978; Hall & O'Toole. 1935). However. ('/().\'lrI'a'iIm1 r./t"/ffici/e associated disease (CDAD) is the now most common hospital acquired diarrhoea and is a major problem of gastroenteritis infection and antibiotic associated diarrhoea in nursing homes and care facilities for the elderly. Indeed, the health protection agency in UK reported 32,189 cases of CDAD for the first 6 months of 2007 in UK ~ (http://www.hpa.org.uk/infections/topics az/hai/ tablesforwebsite/Cdiff Quarterly Nov 2007.xls). The main predisposing factor for the acquisition ofCD/\D is antibiotic therapy. In the 1970s the administration of clindamycin followed by ampicillin and amoxicillin were implicated as the inducing agents of CI)/\l): these were replaced by cephalosporins in the 1980s and more recently by tlouroquinolones (Aronsson el al., 1985; Bartlett, 2006; Winstrom e! u/.. 200]). There is also the added problem ofthe hyper-virulent strain of(.‘. dip/fici/e PCR ribotype 027. the incidence of which is increasing in US, Canada and Europe (Bartlett. 2006). Antimicrobial peptides produced by bacteria. now designated as bacteriocins. first came to prominence ~80 years ago with the discovery by Rogers & Whittier (1928) of nisin by Lactococcus lactis subsp. /ac/is which demonstrated a broad spectrum of activity against other lactic acid bacteria (LAB) and other Gram positive organisms. While the bacteriocins produced by LAB are the most widely studied and tend in the main to have a broad spectrum of activity, antimicrobial compounds are produced by many other bacterial species including Gram positive organisms Bczci/lu.s‘ (Ahern at c//.. 2003; Bizani e/ al.. 2005: Cherifel a/.. 2003; Cherif er a/., 2001; Scibi cl Cl/. 2007. "leo & Tan. 2005); ('l0sIr1'dium (Kemperman at al.. 2003). Gram negative aicafigfii Work carried out previously on various strains of B. rhuringiensis have yielded a variety ofbacterioeins (Ahern el al. 2003. Chehimi et a/. 2007, Cherifet a/, 2001. liavret and Yousten 1989, Gray el al. 2006a. Gray er al. 2006b.) demonstrating bactericidal properties against B. Ihuringiensis strains. B. cereus strains. and Listeria m(2nr)eyrr)gcrrc.r strains. However, these baeteriocins do not exhibit two—component activity.

Previous work by Yudina el al. describes proteins of parasporal crystals (Cry proteins) from entomopathogcnic bacterium B. thuringiemis (subsp. Kurstaki. ga//eriuc, /cnebriois) as well as some fragments thereof, obtained by limited proteolysis which are capable of antimicrobial action against anaerobic bacteria and C. bu/yriczurz. ( '. ace/0/911/y/iczmz and Methanosarcina bur/reri. US Patent No. 7,247,299 describes antimicrobial heat-stable compounds isolated from a novel strain of B. sub/i/i.s' (deposited 8.5.05) isolated from the GIT ofpoultry, which are effective against ('. per/i‘1'ngem', ('. difficile, Campylobaclerjejzmi, Camp. calf, and S. pnezmmniac. US Patent No. 7.144.858 describes the synthesis ofnew antibiotic compounds for use Gram positive bacteria such as Bacillus (including B. thuringienm), (i‘/osmdizmi (including C. dif/ic'ile), Streptococcus, Mycobacterium, and Slap/zy/ococczis. US Application 20080213430 describes the artificial synthesis and recombinant expression of antibacterial peptides against bacteria such as B. sub///is, ( '. dz"/(‘fici/e. la‘. co/i. Staphylococcus. and the like. llowever. these peptides have a broad spectrum ofinhibition against a wide range ofGram positive organisms.

Previous work using the naturally occurring lantibioties lacticin and nisin have shown that these microbially derived peptides are effective in killing C. di)‘/‘ic'ile at concentrations that compare well with commonly used antibiotics such as vancomycin and metronida/yole (Bartoloni el al., 2004; Rea er a/., 2007).

However, these lantibioties have a broad spectrum of inhibition against a wide range of(}rain positive organisms including those which would be considered beneficial to human gut health such as Lact0bac‘fllus and Bi/idrrbuclerium. Indeed. previous work in this laboratory has demonstrated that lacticin 3147 negatively affects the levels of l.ucmbaci//its and Br"/idobaclcrium in faecal fermentation (Rea et al., 2007).

The aim of this study was therefore to isolate bacteria which produce narrow spectrum antimicrobial compounds which target C. diflicile. To this end spore forming bacteria in the human gut were targeted; this would not be an obvious source ofantimicrobials against (Y dr'_/'/ici/e.

[Q L1: Object of the Invention: The object ofthe invention is to provide an agent effective against L. f1Z()I’I()('yI()g€I7€.\' and (7. c/i/fici/c but not against organisms considered beneficial to human or animal health. A further object is to provide compositions comprising such an agent. which can be used as disinfectants or antiseptics. as probiotic components in foodstuffs or as pharmaceutical compositions.

Summary of the Invention: According to the present invention there is provided a bacterial strain Bacillus //’1lII‘I‘l1gl'€I7.\'1'.\' 6431 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. 41490 and strains which are substantially similar thereto. also encoding a bacteriocin effective against Listeria rn0n0Cymgene.s' and ('/ox/i'ia’izmz difficile. but not against Bi/idobacterium and Lactobacil/zi.s' species.

The strain was deposited on 9th July 2007.

Suitably the strain produces a bacteriocin which is not effective against Gram positive llora ofthe gastro—intestinal tract.

The invention also provides a bacteriocin effective against Listeria nzoimcytogenes and (.'/as/ridimn difficile, produced by this bacterial strain.

In a still further aspect the invention provides a bacteriocin designated Thuriein C1) comprising 2 peptides, Trn—a and Trn—B. Trn—ot having a molecular mass of about 2763 and 'l'rn—[3 having a molecular mass ofabout 2861. The bacteriocin is heat~stable up to about 85° Centigrade, with a reduction of activity at about 90° Centigrade and a loss of activity at about 100° Centigrade after 15 minutes‘ incubation. By heat~stable we mean that the bacteriocin is not readily subject to destruction or alteration by heat.

Thuricin CD has the ability to inhibit (.’/0s/ricizmt diffieile and Listeria mrmoet'I()gei2es.

Thuriein CD is active in the pH range 2-10.

Suitably. the bacteriocin is not effective against Bi/idobacterium and [,aclobaci//its species. The bacteriocin may not be effective against Gram positive organisms found iii the gastro—intestinal tract. Suitably the bacteriocin is also effective against Bacillus cerezts. other Baci//its I/iuringieivsis strains. C‘/omidium per_fringens. B. m_vcoic./es". and 13. _firnm.s'. fl/().S'/I”l'dI.l.ll77 clifficile ribolype 027, C. tyrobutyricum, C. /itlzzmcbz/i'ei'z.s'c and ( '. im./0/is. By not effective we mean that the bacteriocin does not affect the viability of these organisms.

FJ ‘J: The bacteriocin may have a bacteriocidal effect against C cliflici/e of approximately 5 x l0l’Cl“U of C. diflicile per ml. being killed within 60 minutes and 180 minutes when thuricin CD is present at a concentration of5uM and 200 AU/ml respectively.

The bacteriocin is effective at nanomolecular concentrations.

The bacteriocin has been shown not to effect the viability ofthe probiotie strains l.aeI0/aacil/us‘ casei 338 or Bifidobacte/‘izmz laclis BbI2.

The bacteriocin may have an inhibition spectrum as shown in Table 2.

Preferably the bacteriocin is one in which the component thuricin Trn—o has an N- terminal amino acid sequence GNAACVIGClGSCVISEGIGSLVGT/\l7TLG and thuricin Cl) component Trn-[3 has the N—terminal amino acid sequence (iWV/\VV(i/\(7(}TVCl.,ASGGVG'l‘lil*AAASYl*‘l.. Preferably, the bacteriocin is one in which the Trn—ot and Trn-[3 components have the amino-acid sequences as shown in l’igure 3, with or without the leader peptide sequence, or sequences which are substantially similar thereto and which also exhibit bacteriocin activity.

In a still further aspect the invention provides a host cell comprising the Thuricin Cl) component Trn—0t encoding gene or the Trn-B encoding gene. The host cell may also comprise the thuricin CD component Trn—o and Trn-[3 —encoding gene. Preferably. the genes have the nucleic acid sequences as shown in Figure 3, or sequences which are substantially similar thereto and which also encode bacteriocin activity.

By “substantially similar” is meant sequences which because of degeneracy of the genetic code, substitution ofone amino-acid for another, or changes in regions ofthe amino—acid sequence which are not critical to bacteriocin activity, still result in a bacteriocin molecule having the properties defined herein.

The invention also provides Thuricin CD component Trn—oi, and Thuricin CD component ’l'rn—B.

Also provided is a disinfectant composition comprising the bacterial strain. a host cell. a bacteriocin or a Thuricin CD component Trn-u or Trn-B as defined above.

The invention provides a probiotic culture comprising vegetative cells or spores ofa strain or a host cell as defined above. The strain or cell may be inactivated so that the strain is no longer viable.

Also provided is a sporicidal composition comprising the bacterial strain. a host cell. a bacteriocin or a Thuricin CD component Trn-u or Tm-B as defined above.

R) LII Also provided is a pharmaceutical composition comprising the bacterial strain, a host cell. a bacteriocin or a Thuricin CD component Trn-or or Trn-[3 as defined above. together with pharmaeeutically effective carriers or excipeients. The pharmaceutical composition may be formulated as an enema preparation. as an encapsulated peptide with targeted delivery to the colon, as an encapsulated probiotic for targeted delivery to the colon. as an animal or veterinary preparation for use or as a probiotic or purified peptide.

The Trn-or and/or Trn-B peptide may be used without the presence ofa live organism as food ingredient for control ofL mrmocytogenes in food. The invention also finds use in the control of C. perfringens in poultry.

The disinfectant. pharmaceutical, sporieidal. food. or other compositions may be formulated together with appropriate carriers or exeipients.

Brief Description ofthe Drawings: Figure 1: Inhibition of C‘. difficile AT CC 43593 by cell free supernatant of B.

I/7zII‘ingiem'i.s' DPC 643 1 (A), and demonstration ofits proteinaeeous nature through the effect of Proteinase K (B).

Figure 2. RP-HPLC chromatogram of thuricin 6431 showing separation of l’ l and Trn-B (A): MALDI-TOF MS chromatograph Trn-or (B) and Trn—[3 (C) showing molecular mass and WDA of both peptides showing the effect ofequimolar concentrations of peptides over a range ofconeentrations (D). l"igure 3 'l‘1‘n—li.

The orientation of the genes encoding the thuricin peptides Tm-or and lrigure 4 Concentration of thuricin CD Tm-oi and Trn-[3 required to inhibit growth of(’. difficile ATCC 43493 by 50%. figure 5 The effect of Thuricin CD (200/\U/ml) on the growth of('. di//iei/e R027. L. monocylogenes, L. paracasei 338 and B. /actis 85712 at 37C. (0 Control; O l2()t)AU/ml thuricin). lrigure 6 The effect of 500pg/ml thuricin CD on the growth oft‘. diffici/e ribotype 001 in a model faeeal fermentation when added at 0, 8 and l6h (A). Activity ofthuriein CD during the course ofthe fermenatation (B). Detection by Rl’—l lPI,C of thuricin peptides Trn—o. and Tm-[3 at Oh (black line) and after 4h fermentation in the model faeeal environment (C). lE0a0952 Figure 7 Stability of thuricin CD in simulated gastric juice (A) after 2h, simulated ilcal juice (B) after 5h, simulated colon juice (C) after 9h, porcine gastric juice (D) after 2h and porcine ileal juice (B) after 5h incubation at 37°C.

Ix) ‘J1 Detailed Description ofthe invention Bacterial strains used ('. t/i///‘oi/u A'l‘(‘(.‘ 42639 was used as target strain for Well Diffusion Assays (WDA). ('. c/if“/ic'i/ti R027 NAPlwas used for bacteriocin sensitivities in time kill studies. A full list oltarget organisms and their sources which were used for determination ofthe spectrum ol‘ inhibition ofthe bacteriocin producing cultures is outlined, together with the media and growth conditions in Table l. B. cereus NCIMB 700577 and B.

Iltztriitg1‘cnsis NCIMB 701157 were used as positive controls for the PCR reaction using git/‘l3 primers.

Isolation Q/'l3acIeri0cin Producing Cultures. l“aecal samples from both diseased and healthy individuals were received in the laboratory and frozen at - 80°C. On the day of analysis samples were thawed at room temperature and mixed with equal volumes ofethanol, and allowed to stand at room temperature for ~ 30 min. Samples were subsequently serially diluted in anaerobic diluent. l00 ul spread on the surface ol‘ Wilkens Chargrin Anaerobic Agar (WCAA) and grown for 5 days at 37 °C in an anaerobic chamber. Colonies which developed were overlaid with ~10 ml of Reinforced Clostridium Agar (RCA) inoculated at 1.25% with a log phase culture of C lostridizmt diff/ici'le ATCC 43593. The plates were incubated for a further 18b and inspected for zones of inhibition of the overlaid culture. Colonies showing a clear zone ofinhibition were sub-cultured onto lresh WCAA having lirst removed the agar overlay using a sterile scalpel. Approximately ,000 colonies were screened and one colony showing potent antimicrobial activity against the overlaid C. dtfficile strain was purified and stocked at -80 "C on Mierobank Beads and designated as DPC643l and the inhibitory substance produced was designated thuricin CD.

Genotypic characterisation /051‘/)i'\lx’l sequencing ofDPC 6431 Genomic DNA was isolated from overnight broth cultures of B. Ihuringiensfs DPC 6431 and amplified by PCR as described by (Simpson et al., 2003). Comparisons ol‘ the l6S rDNA sequences were obtained using the BLAST programme that allowed the assignment ofa strain to a particular species.

R) L11 tEce0952 Identification to species level using gyrB primers Speeies—specific oligonucleotide primers for the 918 gene for B. eereus, B.

I/mringiensi.s and B. anthracis were purchased from MWG with sequences as described by Yamada et al., (I 999). PCR products were analysed on 1.5% agarose gel with 100 bp ladder as molecular marker and visualised using an Alphalmager 3400. /3. ee/‘ens NCIMB 700577 and B. I/iziringiensis NCIMB 701157 were used as positive controls. Because ofthe pathogenic status of /3. anlhracis there was no positive control for B. cmthracis.

Production of thuricin CD from cell free supernatants: B. //1zu'/'ngiensis DPC 6431 was grown aerobically from stock for ~ 6h in Bill broth and sub—cultured into fresh BHI at 0.1% for 18b in BHI. Following growth, the culture was eeritrifuged twice at 8200 g for 10 min. Activity was determined using the well dillusion assay (WDA) as described by Ryan et al., (1996). Activity against a range of target organisms was determined by W DA. Cultures were grown over—night in various broth media and temperatures as outlined in Table l. Twenty ml of the appropriate agar medium was inoculated with 100 pl of target organism and, once solidified. 50 ul of the cell free supernatant (CFS) of B. thurmgiensis DPC 6431 was added to a well made in the agar. Plates were incubated under conditions appropriate for the various target organisms as outlined in Table l. Zones of inhibition (mm) were measured and relative sensitivity determined by measuring the diameter ofthe zone.

Zones olsize 3 9 mm were designated +; of 10-15mm were designated 1 +1 of 16- Zlmm were designated +++; of: 22mm were designated ++++; Sensitivity of thuricin CD to enzymatic degradation, heat and pH.

The cell free supernatant was tested for sensitivity to the following enzymes at a concentration of25mg/ml: pepsin, trypsin, peptidase, or-chymotrypsin type Vl l l. Ot- chymotrypsin type 11 and proteinase K. All enzymes were purchased from Sigma.

Cell free supernatants were incubated with the enzymes for lh at 37°C. Activity post enzyme treatment was determined using the well diffusion method using (T di['/iei/e ATC,‘C 43593 as the target organism. Sensitivity ofthe antimicrobial to Proteinase K was also determined by applying 5 pl ofProteinase K (6.5mg/ml) to the edge of the well containing the sample.

Activity over a range of pH was determined in CFS by adjusting pH from 2 to 9 using either 0.5M llCl or IM NaOH. The effect ofacid or base on the target organism was IQ L11 lEaae952 determined using uninoculated broth medium adjusted through the p11 range. Activity was determined using WDA assay using C. dz’/}”1c'i/e ATC C 43593 as the target strain.

Heat sensitivity was determined by heating the CFS for 15 min at a range of temperatures from 37°C to 100 °C; the control was incubated at 37 °C. Activity was determined using WDA with C. diflici/e ATCC 43593 as the target organism.

Determination of the thuriein CD production during growth. /3. //zuringierz.s'i.s‘ DPC 6431 was sub-cultured twice in B111 broth and then inoculated again at 1% into Blll broth; growth was followed by measuring absorbanee at 600.,.,, Samples. taken at intervals during growth for determination ofantimicrobial production. were centrifuged twice at 8200 g for 10 min. serially diluted and 50 ul of each dilution inoculated into wells in agar plates seeded with C. d('/f/”Ic'ile /\'l‘CC 43593. Activity units were determined by WDA.

Purification and molecular mass determination of thuricin CD.

Pmdz/clion ofllrzrricin (71): Tryptone Yeast Broth (TYB) was made up as follows: Tryptone (Oxoid) 2.5 g; Yeast extract (Oxoid) 5.0 g; MgSO4 7H3O 0.25 g; MnS().; 41130 0.05 g were dissolved in 900 ml distilled 1120. The media was clarified. before autoclaving at 121°C for 15 minutes. by passing through a column Containing propan- 2—o1 washed XAD beads (Sigma—Aldrich). Before use, filter sterilised glucose and 0- glycerophosphate were added to give a final concentration of 10 g and 19 g /1 respectively and a final volume of 11. ls’ac.'i//us I/mringiensis DPC 6431 was sub—cultured twice in B111 broth at 37°C before use. Two litres ofTYB were inoculated with the culture at 0.1% and incubated shaking at 37°C over—night. The culture was centrifuged at 8.280 g for 15 minutes.

The cell pellet and supernatant were retained. The cells were rcsuspended in 200 ml of % propan-2—ol pH 2.0 per litre of broth and stirred at 4°C for 4h. The culture supernatant was passed through XAD beads, pre—washed with 11 of distilled 1130. The column was washed with 500 ml of30 % ethanol and the inhibitory activity eluted in 400 ml of 70% propanol pH 2.0 and retained (S1). The cells that had been resuspended in 70% propan01 pll 2.0 were centrifuged at 8.280 g for 15 minutes and the supernatant (S30) retained; S1 and S2 were combined. The propan—2-ol was evaporated using a rotary evaporator (Buchi) and the sample applied to a 6 g (20 ml) Phenomenex C-18 column pre-equilibrated with methanol and water. The column was washed with 2 column volumes of30 % ethanol and the inhibitory activity was eluted lEoeo952 in 1.5 column volumes of70 % propan—2—ol pll 2.0. This preparation was concentrated using rotary evaporation before separation of peptides using lll’l.C as follows: aliquots of approximately 2ml were applied to a Phenomencx (Phenomenex.

Cheshire. UK) C1g1'CVCI'S€ phase (RP)—llPLC column (Primesphere l0tt Cl8-MC 30. 250x 1 0.0 mm. 10 pm) previously equilibrated with 45% acetonitrile, 0.1% trifluoroacetric acid TFA. The column was subsequently developed in a gradient of 45% acetonitrile containing 0.1% TFA to 65% acetonitrile containing 0.1% 'l‘l<‘A from 4 — 40 minutes at a flow rate of9.9 ml/min. Biologically active fractions were identified using C. difficile as target organism in WDA. Fractions containing the active peptides were pooled, freeze dried and reconstituted at the required concentration in 70% propan—2—ol pll 2.0 and frozen at -20°C until use. Subsequent dilutions were made in sterile 50mM phosphate buffer pll 6.5.

Molecular mass determination of thuricin CD: Mass spectrometry was performed on biologically active fractions with an Axima CFR plus MALDI TOF mass spectrometer (Shimadzu Biotech, Manchester. UK). A 0.5-pl aliquot of matrix solution (-cyano 4-bydroxy einnamic acid. 10 mg/ml in acetonitrile—0.l % (v/v) tritluoroacetic acid) was deposited onto the target and left for 5 seconds before being removed. Any residual solution was allowed to air-dry and the sample solution deposited onto the pre-coated sample spot; 0.5 pl ofmatrix solution was added to the deposited sample and allowed air—dry, The sample was subsequently analysed in positivc—ion rellectron mode to determine molecular mass.

Determination of amino acid sequence of biologically active peptides.

N—terminal amino acid determination of biologically active fractions was carried out by lidman degradation at Aberdeen Proteome Facility. University of Aberdeen.

Aberdeen. Scotland, UK. l)etermination of nucleotide sequence of thuricin Cl) Degenerate primers. based on the partial amino acid sequences ofthe 2 peptides. were designed with the following sequences: Tm-ot —F/FC 5” GGT TGG GTA GC A GTA GTA GGT GCA TGT GGW ACA GTW ACC CAWCC; Trn-ct -R/FC 5'CG'l‘ AAA CAT ACT GTA CCA CAT GCA CCT ACT ACW GCW ACC CAW CC; Trn-[3 1’/l3 C 5‘ G01‘ AAT GC A TGT GTA WTW GGW TGT WTW GG; Trn-[3 ~R/FC 5’ CCA A'l‘A CGA CCA ATT ACA CAW GCW GCW TTW CC. Chromosomal DNA was extracted from B. I/iuringenisis using the Qiagen QiAamp DNA Mini Kit. PCR tieaetfiz was performed on extracted DNA. using the following conditions: 94°C x 5 min; 94°C x 1 min. o4.5“C x 1 min. 72°C X 1 min 25 cycles with temperature gradient of64.5°C— 695°C: final extension step 72°C x 7 min. The Trn~ot ~—F/FC/ Trn~t3 —R/FC primer combination resulted in a PCR product of~22() bp. This product was purified using Qiagen Qiaquick PCR purification Kit and cloned using the Invitrogen T()l’() TA Cloning kit (A). The presence ofthe cloned fragment was confirmed by restriction analysis of recombinant plasmids with 1fe0Rl (N EB), used according to the manufacturers instructions. Recombinant plasmid DNA was then sequenced commercially by Lark (Windmill Road llcadington OX3 7BN Oxford, UK) using the T7 and T3 priming sites. Sequence assembly and analysis was performed using the Seqlluildcr and Seqman programmes from the Lasergenc software package.

(DNASTA R. Madison, WI). The consensus sequence was further analysed by database searches using the Blastn. Blastp and tlilastx programmes available on http://www.ncbinlm.nih.gov.

Inverse PCR to obtain the surrounding DNA regions lnverse PC R primers, based on the DNA sequence of the fragment encoding the two peptides that was isolated by degenerate PCR (above) were designed with the following sequences: Primer FCinl: 5’ CAT GCA CCT ACT GCT ACC CAA CC 3‘ and Primer l’(‘in2: 5’ CAG AGT TTG CAG CTG CAT CTT ATT TCC 3'.

Chromosoinal DNA was extracted from B. Ihuringenisis using the Qiagen QiAamp DNA Mini Kit and digested with the restriction enzyme Hindlll (NEB) according to manufacturers instructions. Digested DNA was then relegated at concentrations known to encourage the formation of monomeric molecules. Inverse PC R was performed using Expand Long Template DNA polymerase (Roche) according to the manufacturer’s instructions. This resulted in amplification of a product of 4500 bp.

This product was purified using Qiagen Qiaquick PCR purification Kit and cloned using the lnvitrogen TOPO TA Cloning kit (A). The presence ofthe cloned fragment was contirmed by restriction analysis of recombinant plasmids with E00181 (NEB). used according to the manufacturer’s instructions. Recombinant plasmid DNA was then sequenced commercially by Lark (Windmill Road Headington OX3 7BN Oxford.

UK) using the T7 and T3 priming sites and primer walking. Sequence assembly and analysis was performed using the SeqBuilder and Seqman programmes from the Lasergene software package. (DNASTAR. Madison, WI). The consensus sequence {Q A \.z to L1‘: was further analysed by database searches using the Blastn, Blastp and tBlastx programmes available on http://www.ncbi.nlm.nih.gov.

Specific activity determination.

Ninct_v—six well microtiter plates were used to determine the MIC5o ofthuricin CD.

MI(‘_;() was delined as the concentration of peptides at which 50% inhibition ofgrowth ol‘(‘. d[ffic1'[e ATCC 43493 occurred. One hundred and fifty microlitres ol‘3 replicate overnight cultures, diluted 1:10 in reinforced clostridium medium (RCM) which had been previously boiled and cooled, were inoculated into triplicate into wells ol‘96 well microtitre plates in an anaerobic chamber. Thuricin CD Trn—ot and Trn-B were added to the wells at varying concentrations both singly and in combination and linal volume made up to 200ul with sterile 50mM phosphate buffer. Control wells contained l50ul culture and 50 pl buffer (positive control) or l50pl uninoeulated broth medium and 50 pl buffer (blank). The optical density at 600nm was recorded atler 5h anaerobic incubation at 37°C. Triplicate readings were averaged and the ()l)(,imnm values for the uninoeulated medium were subtracted from each value. A 50% growth inhibition was determined as halfthe final OD6o0,,m +/- 0.()5 otthe control culture. The concentrations of Trn-or in combination with Trn-[3 which caused 50% inhibition were plotted to generate an isobologram. The specific activities and optimum ratios of the 2 peptides were determined at the point of intersection ofthc x- an i _y—axis. l)cm0ns/ration 0/iactivily oflhuricin ('1) using kf/I curves.

The ellcct olithuricin against (1. drflicilc R027 NAPl, 1.. monocytogenes NCTC 5348.

U7. case/' 338 and B. lactis Bbl2 was determined in Reinforced Clostridium Medium (RCM Merck). Brain Heart Infusion broth (Merck), MRS (de Mann-Rogosa-Sharpe) medium (Difco) and MRS containing 0.05% eystein respectively. Three independent cultures were prepared for each strain and grown overnight at 37°C. Three replicate one ml volumes of sterile double strength broth medium was prepared for each strain and inoculated with the test organisms to give initial cell numbers of l0’<~l0°/ml.

Thuricin was added to give the required concentration and the volume made up to Zml with sterile distilled water. The bacteriocin was omitted from the control and volume substituted with sterile water. Samples were removed at intervals, serially diluted and plated on RCA. Blll agar, MRS agar or MRS agar containing 0.05% eystein depending on the strain. Plates were counted after 24 h (L. monocy/ogwm‘). or 48h (('. tiff’/ic‘i/c’. Lb. cusci 338 and B. [aclis Bbl2) incubation at 37°C.

I\) LI: '4) ex ca tEea0952 /)c/crminu/ion rifiriirzimzim inhibiloiji eorzceivtrations C. difficile ribotypes 001, 106 and 027 were taken from ~80°C stock and grown on Fastidious Anaerobic Agar containing 7% defibrinated horse blood. Before use a colony was transferred into each ofthree 10ml volumes of RCM and grown over- night at 37°C. The cultures were then sub-cultured into 10ml fresh RCM at 1%.

Three replicates were set up for each strain. Cultures were grown for 6h anaerobically at 37°C. Solutions of vancomyein and metronidazole were prepared in water and solutions of thuricin diluted from the stock solution in 50mM phosphate buffer pll 6.5.

Triplicate serial two-fold dilutions ofthe antimicrobial compounds (100ul) were prepared in mierotitre plates for each compound. C. diflicile strains were diluted 1:10 in double strength RCM and 100p] was added to each well. Controls for each strain were set up without the addition of antimicrobials. The microtitre plates were incubated for 16h at 37°C anaerobically and the ()1) of the plates read after incubation using a microtitre plate reader. The MIC was defined as the concentration at which there was no evidence ofgrowth.

I)emon.sIrufioi1 op/'l_vsi.s' of C. di[fic'i/e by I/mricin CD.

The lytie effect of thuricin CD on C. dif/ic'ile was carried out as described by Rea cl ul (2007).

Determination of thuricin stability in faeeal fermentation ’reparaIim7 op/C. difficile inoculumz C. difficile DPC 6537 (PCR Ribotype 001) was taken from —8()°C stock and streaked on Fastidious Anaerobic Agar and incubated anaerobically at 37°C. ()n the night before the experiment ~ 1 colony was inoculated into 10ml of RCM which has been previously boiled and cooled and incubated anaerobically at 37°C.

Preparation ofFaeca/ Medium: Faecal growth medium was prepared as described by Fooks and Gibson (2003) with minor modifications as follows. The ingredients were made up to 800ml. the pH adjusted to 6.8. One hundred and sixty ml was added to each fermentation vessel and autoclaved at 121°C for 15 min. Prior to inoculation the faeeal medium was sparged with ()3—free nitrogen for ~1h. A 20% faeeal slurry was made from a fresh faeeal sample in 50mM phosphate buffer containing 0.05% eystein which has been previously boiled and cooled just prior to use and mixed using a stomacher for no longer than 1 min. Two fermentor vessels were inoculated with 35ml of the slurry preparation and 2ml of the overnight culture of C. di/_}’/‘oi/c. To the test vessel lml of 100 mg/ml thuricin was added at 0, 8 and 16h incubation and both E0aa$52 vessels sampled at 0. 4, 8, 12, 16, 20 and 24h for both microbiological analyses of C. difiici/e and Bifidobacferia sp. Samples were also taken for analysis of thuricin activity using WDA and MALDI-T017 MS.

Srcrbi/iry o/’//mricin: The stability ofthuricin during fermentation was measured using WDA using RCM agar plates seeded with (I. ciifficile as described previously. One ml samples were also centrifuged and passed through activated lml CH; SPIS columns and the peptides eluted with 70% propan0l. The presence of individual peptides was measured using RP-HPLC as described previously in this docu1nent..

Microbiological analyses: C. difficile was enumerated on CCEY agar (il.abM) and Bifia’0bacIcriu/72 sp. on modified MRS agar containing 0.05% cystein and 50mg mupirocin/l alter 48b and 72h at 37°C incubation respectively.

These experiments were carried out in duplicate Stability of thuricin C1) in simulated and porcine gastric juices /'._'_/feel 0/'simu/area’ gastric, ileal and colon juice on stability oflhuricm ( '1) C difficile 64539 was grown overnight and inoculated at 1.25% into Reinforced Clostridium Agar (RCA). Simulated gastric, ileal and colon juice were prepared as outlined by Breumer el ai (1992). Purified thuricin CD was made up to ll)0IT1g/I111 in 70% ll’A. Seventy ul (lmg/ml final concentration) ofthuricin was added to 70()0 ul of porcine gastric and ileumjuice and incubated at 37°C. At intervals samples were taken and activity was measured with the WDA using C. di/ficile seeded plates. The samples were also assayed for the presence of the Trn—ot and Trn—[i peptides using MAl.l)l—’l‘()l“ MS /«;_//m of ex vivo porcine gastric and i/ealjuice on the stability ofthe I/izrriein ( '1) (Y cli//‘ici/c 64539 was grown overnight and inoculated at 1.25% into Reinforced Clostridium Agar (RCA). Purified Thuricin CD was prepared as described above.

Seven ml of porcine ileal and gastric juice was centrifuged for 15 min at 12.000 rpm to remove debris. Seventy ul (lmg/ml) ofthuricin was added to 7000 it] olporeine gastric and ileum juice and incubated at 37°C. At intervals samples were taken and activity was measured using the WDA and checked for the presence of the Trn—ot and 'l‘rn—[% peptides using MALD1—TOF MS.

RESULTS iEQ80952 The aim otithis work was to isolates narrow spectrum bacteriocin producing organisms. from within the GI tract, with high activity against C. difficile. which would cause the least perturbation of the resident flora of GIT.

Initial screening for bacteriocin producers l"r()m ~ 30,000 colonies screened from a range olifaecal samples from both healthy and diseased adults, one colony was shown to produce a large zone ofinhibition of the t’. (Ii/K"/ic'i[e overlay culture (Figure I). This colony was isolated from the taecal sample ola patient with IBS. Purification of this colony and growth in BIII broth showed that a potent antimicrobial compound. active against C. dz‘/“fiei[e, was produced into the fermentation medium; activity was lost on treatment with Proteinase K indicating that the antimicrobial substance was proteinaeeous in nature (liig I). This culture was stocked in the culture collection ol‘l\/Ioorepark Food Research Centre and designated as DPC 6431; the bacteriocin was designated thuriein CD.

Identification of DPC 643] to species level S rDN/\ sequencing ofDPC 6431 indicated highest homology (96%) ofthe strain to B. ee/‘cits/B. I/vzzringiens/.5‘/’B. anlhmcis. La Due el al (2004) have stated that /f. mt//traeis. /3. cerezrs and B. lhuringiensis all cluster together within a very tight clade ( /3. cerciis group) phylogenetically and are thus indistinguishable from one another \ ia los rl)l\'*/\ sequencing. DPC 6431 was subsequently identified as B. /huringiem/ls" using gyrll primers. PCR products corresponding to the correct size for B. eerczis or /3.

I/72irmgieiisis (365 and 368 respectively) were obtained with positive controls tor each oi‘ these organisms. No PCR product was obtained when gyrB primers for B. eerezrs or /3. an/lirucz'.s' were tested with DNA from B. lhztringiensis DPC 6431. Due to the pathogenic nature of B. anthracis there was no positive control for that primer.

Characterisation of bacteriocin from DPC 6431 Highest concentration of the thuriein CD was produced during the late log phase and stationary phase of growth probably coinciding with the onset of sporulation. Activity remained stable during the stationary phase of growth. The pH decreased during the exponential growth phase to ~58 from an initial pH of~7.5; during the stationary phase the pll rose again to close to its starting value (data not shown).

The incubation ofthe cell free extract with 25mg/ml ofot—chymotrypsin and proteinase K resulted in complete loss ofaetivity: incubation with pepsin or trypsin showed a 50% or 20% reduction in activity respectively alter lh incubation at 37°C. lEoe095Z Cell free supernatants of thuricin were active throughout the pH range 2-10 and heat stable up to 850C. there was a reduction in activity at 90°C and activity was lost at ()()"C alter 15 minutes incubation at the respective temperatures.

Inhibition spectrum of B. t/zuringiensis 6431 Cell free supernatant of B. t}7urz'ngie77.s'is 6431, when tested against a range of Gram positive and Gram negative bacteria using the WDA method, showed a narrow spectrum ol‘ inhibition inhibiting closely related Bacillus species such as B. cereus. other B. //mriiigiensis strains, B. mycoides and B. firmus; no inhibition was detected against /3. snbiilis or B. Coagulans. Within the Clostridium sp. all C. difficile isolates tested. including C’. difficile ribotype 027 (NCTC 13366), were very sensitive to the culture supernatants ofDPC 6431 exhibiting large zones ofinhibition. ('. /ym/777/3'riCum, ('. /ithu.s'eburen.s'€, and (‘. i77d0/is were also inhibited whereas other ('/as/7'7'd7'z77v77 species tested were not inhibited (( sporogenes) or only weakly inhibited ((7. /iislolyticum and C. perfririgens), Among the other Gram positive pathogens tested only Listeria m0770cyl0gene.s' was sensitive to thuricin Cl). Of the lactobacillus species tested only L. fermenlzmz was strongly inhibited by thuricin CD with all other species being only very weakly inhibited (L. crispatus and 1.. jo/mmmii) or not at all. No bifidobacteria strains tested were sensitive to thuricin 6431. Thuriein Cl) showed no activity against any Gram negative organisms tested. The complete inhibition spectrum of B. thuringien.w‘.s' 6431 is outlined in Table 1.

Table l Gram positive and Gram negative bacteria using the well diffusion assay.

Spectrum of inhibition ot‘B. rhztringiensis DPC 6431 against a range oi‘ largcl Strain /far I//It.\‘ Cum/.\‘ /fut 1//74.3‘ /5’tm//H3‘ mugu/(777.3 Inn‘;//77.3‘ /Il‘I}IH.\‘ /mt///17.3 /lltltt/I(jL’.\ Hut 1//1/A‘ .317/7///7.3‘ /$174 I//l(.\' Sll/"ll//A />’tl¢'7//1/.\' //711/'n7_t3IcI7.\'/\‘ b‘u /fut IL‘l‘UIt/8.1’/l'u_2//IA‘ I)’///1/ol7t7CIu7‘z77777 (ldr//k’.s'L‘€II.\'/S B//it/uhuu/«rtum umnzu//.3 Hl/l(l(J}lt/t'/t’/ /um [7/"aw [$1/it/o/mCrw'zzl777 /7l‘c‘\‘L’ Ht"/It/"nl7<1t'/urizrlri /(7l’lg1I/It /it// /it/It/oh:7t'/e7'71rn7 /zsez/do/0/7gzu77 ('1/777;u'/u/tat tar./u/u/7! ('/oslrlriilt/77 u’/f/in/e ('/(writ/I77/77 t/7/‘/it I/e t '/us//‘/t//z//77 7////icl/e Growth Stram No medium lncubation '1‘emp:@9.tv . .

NCIMB 700577 BHl' 30°C .\‘C1.\4i370057s Blll 30°C IMG 6326"‘ BHI 30°C IMG 71257 B111 .10“(‘ DPC 0335 B111 30°C LMG 3192: B111 30°C DPC 3344 B111 30°C ms 14 BHI 37°C LMG 71323 13111 37°C LMU 10253 BA 37°C DPC 6169 mMRs~‘ 37°C DPC S420 mMl{S 37°C DPC 6160 mMRS 37°C BB12 mMRs 37°C DSM7. 20007 mMRS 37°C DSMZ 0393 mMRS 37“C osmz 20092 mMRS 37°C Cl 120 CA‘ 37°C ArCC 000 RCA‘ 37°C ATCC 43504 RC/\ 37°C R027 NCIC 13366 RCA 37°C Sensituit) ++4 +++ ++++ +++ ++++ ++++ ++++ $080952 '/11.\'I1‘11/111111 1/1//11 1/1» ('/115'/1'/17111111 171/7111/11 ('/11.1’!/‘11li11111 1/1‘//71':/1' C/11sIr11/111171 171/f‘/1111/e C/115'!/'11i1'11117 1/117161/1' C/11.1‘!/711'/1/111 (I'M/7('(°/L’ ('/115/1711111171 171’//'11‘!/1' ('/115'/1'11/111/11 d1//Ic‘//1* (‘/115'11‘11/711171 1/If/717/1’ C/11.1'II‘11//11111 1//[/117/5 (3/11.\‘I1'11/111111 1/11/111/1' ('/11.1‘/r11/111111 1/1/71111/1' ('/1111‘//111/111111 11°17"/1111/1' (V/11.1/1'11//111711/1//11'1/1' ('/11.1‘/1'11/1111111/1//111/1' 1'/11.51171/11/111 1/1_///1'1/1' ('/11.1‘/r11/1111111/1{/117/c C/11.1‘/171//11111 1/1/‘/11‘!/1* (' '/11.911‘/1011111 /715/11/>1‘/161/m C/11.1‘//‘I1/111111 1/11/0/1.1‘ C/11.1‘Ir11/111711 /1111511111/rense ( '/11.11/*1:/111111 .v,11111'11g».I11c.1' (711.1/1'11//1/111 11'1'11/11111‘1'11'z1/71 /:71/1'1 11/1111'/1'1‘ .111/111:1:/1/ /[11/1'1‘11/1111‘/1'1‘X11/111:1IkI /:17I1'r111'111'1'11.v 1'11.1'.1'1*/1//111'11.1' /:11/u1‘111'111'1’115‘ 1/11r1111.1' /:11/e1'111111'1‘11.« /111'1‘11/15 E11/1'/‘111 111 1-111/111*1‘/11117 /:11/1'1‘111'1111'11.1 /1/1111‘ /L°.\(‘/It'I‘71'lLl U1/I /7111'/11/11117//11.1 11111/11/111//115 l.111111/71111//11.1 /111/gu1‘11/.1‘ l,1111/11/11111//115111.114 l,111'/11/111111//1111' (‘I'I.\'/117/II.\' l,171‘I1>/7111///11.1 1111111/11.1" l.111‘/11/11111//11.1‘/1*/71111111111/1 l.111I11/11111//1/5' ‘L111//1111u'111r1 /1111‘/11/711131//21.1 111'/1‘eI11‘11.1' l,111 /117711171//1111 y/11/1111111711 /.(l(‘I(Jb(IL'I//I/X /711r111.'c1.s'1*1 /.111I11/11151//11.1‘1'e11/W1 I_111 /11/7111'://11.1 1'/1111n1111.vu.v /.111'I11/71111//11.1 r111111ni.1 l_11cl11b11c11l11.s 51111111111115 l.111‘I111'11cc11.\' /11111.1 [.111/111‘111'1‘1/5'/11177.1‘ I,c11c11111>511,1c l4cuc1111115111c I.1.s'Ieri11 11711111 1111 (1219 ] Cr 15 ' 31: UC 313 CR 79 : 47 CR11 ' 29 '11 112 :1‘ 59 UC 27 B547 NCIMB 5113 NCIMB 9731 NCIMB 111537 NCIMB 95114 NCIMB 13243 ATCC 121359 NCTC 8155 [MO 10745‘ LMG 10741; 1.1v111 79737 1.1/111 7973' [M11 6399“ 3786 NCl)() 113117 1..v111 119111’ ATCC 334 LMU 9479*’ [MG 1211119 [MG 5902 1.MG 9435"" 1.111 DSM 10533‘ 3313 NCIMB 11951 GO DSM 2114113‘ UCC 1 113 11111‘ 3157 HP DPC 139 1311c 2411 11111‘ 3572 DPC 171113 DPC 3437 Sco11A ‘ 6275 IMG 3293 LMG 1 141111 LMG NCFB 565 NCFB 11511‘ ATCC 331115 ATCC 17522 DPC 51145 DPC 6049 ATCC 25923 111111‘ 3767 11111‘ 152119 l,M(j 141194‘ I)l’C 5244 DPC 5345 DPC 11159 DPC 1155 DPC 11143 RCA RC.A RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA RCA 15A‘ 'l‘SA TSA 'l‘SA ‘ISA ‘ISA TSA BHI MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS MRS [M17 [M17 MRS MRS BHI BHI BHI BHI RH] BHI MRS SLA“ SLA SLA SI.A BHI BHI BH] BHI TSA TS./\ BHI TSA B1 II BHI TSA + sucrose° ’l'SA 7 sucrose TSA 1 sucrose °C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C °C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C °C °C °C °C 37°C 37°C 37°C 37°C 37°C °C °C 37°C 37°C °C °C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C 37°C ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ [\.) U1 DJ CD lEeaet52 'Brain heart infusion broth‘ :Modilied MRS containing 0.05% Cystein; 3Campylobacter agar ‘Reinforced clostridium agar; 5Trypticase soya agar; “Sodium lactate agar; 7Trypticase soya agar containing 25mM sucrose. (/.ones of inhibition (mm) were measured and relative sensitivity determined by measuring the diameter olthe zone. Zones ofsize 5 9 mm were designated +; of l()—l 5mm were designated t l; of 16- 2lmm were designated +++; off 22mm were designated l l ‘ l) Characterisation ofthuricin CD.

Puritication olithuricin using XAD beads and separation ofactive components using RP-lll’I.C resulted in 2 well-separated peaks at 26 minutes and 34 minutes (Fig 2 A) with molecular masses of2786 and 2883 respectively. These hydrophobic peptides were designated thuricin CD Trn-or (mol mass 2883) and Trn—B (mol mass 2786) (l‘ig B and C). Activity was present in both the cell free supernatant and also by propan- 2-ol extraction of the cell pellet indicating that thuricin is also attached to the cell wall.

WDA studies of both peptides show that it is a two component bacteriocin: 'l‘rn—ot has activity when present alone. however its activity is enhanced by the presence ot"l'rn-[’>.

At low concentrations (~ 2.5uM) both peptides are required for activity using the WDA(l’ig. 2 D).

Elucidation of amino acid sequence. lidman degradation identified the amino acid sequence of the first 22 amino acids for thuricin Cl) 'l‘rn—ot (G-N—A—A—C—V—[I/L|—G—C-[I/L]—G-S—C—V—[I/L]—S—E—G— l/Ll-C-N- li) and 22 amino acids for thuricin CD Trn—[3 (G—W—V—A—V—V-G—A~C—(}—'l‘—V—C-L-A- S(}—V —C—l:I-C-14‘). Despite repeated efforts it was not possible to determine further sequence data using this technique. No homologous sequences were identitied when the above sequences were compared to the NC Bl database llilpi/'i[_\N’W'\\/'.nCbi.l1lm .nih.gov Initial determination of nucleotide sequence The orientation ofthe genes encoding the two peptides is shown in Figure 3. The PCR product obtained contains the C’— terminal end of the T rn—B peptide and the ribosomal binding site and promoter sequences for Trn—B. as well as its start eodon methionine and ;\l—terminal amino acid sequence.

Sequence analysis of the inverse PCR product lhe complete nucleotide sequence of the two peptides was determined together with the start codon methionine and leader peptide sequence as outlined below IEo3o952 Trn—ot DNA sequence A’IGGAAG'l‘TATGAACAATGCTTTAA'l‘TACAAAAGTAGATGAGGACEATTG UAGCEAAACGCTGCTTGTG'l‘/\/\'l"l‘GGTTGTAT 'l‘(lG(L‘AG’!"l‘GCG'l‘AATTAGTGAAG GAATT GGTTC AC TTGTAGGAACAGC A Tl"l'AC'l‘"l"l‘AGGTTA Trn—[l DNA sequence Al‘GGAAG'l'"l‘T'l‘AAACAAACAAAATG"l‘AAA'l‘A'I"l‘ATTCCAGAATCTGAAG AAGl‘A(iG]‘GG'l"l‘GGG'l‘AGCA(}'l‘AG'l‘AGGTGC Al‘(}'l'G(}'l'ACAG'l‘A'li'GCTTAGC'l‘AGTGGTGG'l'G'l"l'GGAACAGAG'l"l"l‘(}(s‘A (}("l‘(l(fAl‘C'l"l'A'l"l"l‘CC'l‘ATAA The complete amino acid sequences of both peptides was determined and is shown below together with the leader peptide. The leader peptides are cleaved at GG 'I‘rn-ot Protein sequence Ml-ZVMNNAIJTKVDEEIGGNAACVIGCIGSCVISE(3lGSLVG'l‘AF'l‘LG 'l‘rn—|i Protein sequence Ml{Vl NKQN VNIIPESEEVGGWVAVVGACGTVCI,ASGGVG'l‘EFA/\AS\r’l"l, Sequence analysis of the surrounding regions reveals an upstream putative promoter located be lore a hypothetical protein followed by two ABC transporter systems and then the 'l‘rn~ot and Trn—B peptides. Three unusual proteins are located downstream of‘ the peptides, two radical S-adenosylmcthionine SAM proteins and a C-terminal protease.

Specific activity of thuriein.

The isobolgram (Fig. 3) shows that the l\/llC50ofthuricin 'l‘rn—B (O.5ttl\/l) is 10 told lower than lrn-ct (5uM) when present as individual peptides, however when the peptides are combined the MlC50 oflrn-[5 is reduced to 0.05pM when combined with 0.025 ulvl 'l‘i"n— or indicating that thuriein Trn-or can be made 100 fold more active when low concentrations of Trn- or are added. These results show that the 2 peptides when combined at low concentrations (< luM) are very inhibitory to C difficile when combined at a ratio of2:l Trn—B: Trn-ct. lhc bactericidal nature ofthuricin CD is demonstrated in Fig 5; initial experiments determined the concentration ofthuricin required to kill C diflicile using kill curves. lwo hundred All thuriein/ml reduced the viable cells of('. diflicile PCR ribotype (J27 trom ~ 10° /ml to zero within 2h. The same concentration ofthuricin reduced the cell (.11 [Q KJI IE08093? numbers of L. monocytogenes by 1.5 log cycles and had no effect on the viability of the probiotics I./7. easel" 338 and B. /actis BB12 in the same time period (Fig 5). The addition ofthuriein CD to logarithmically growing C. dtzfficile caused a gradual reduction of ()D60()nrn; this decrease in OD was paralleled with a concomitant release ofthe intracellular enzyme acetate kinase into the growth medium. In contrast there was no increase in the concentration of acetate kinase in the control sample without thuricin.

Thuriein CD is also effective against C. diflicile ribotype 001 in a model faecal environment when added at 0, 8, and 16hr (Fig. 6A). Faecal fermentations spiked with l0“ cfu (7. difficile ribotype 001/ml showed that when 500ttg thuricin was added at 0. 8. and 16hr. C difficile was reduced over 1000-fold when compared with the control alter 16hr incubation. The activity ofthuriein CD was mapped during the course of fermentation as demonstrated in Fig. 6B. As can be clearly seen, the addition of 500ug ofthuricin at 0, 8, and 16hr resulted in reduction of growth of (T c/iflici/c. Thuricin peptides Trn-ot and Trn—[3 were detected by RP—HPl.C after Ohr (black line) and 4hr fermentation in the model faecal fermentation (Fig. 6C). The presence of thuricin did not affect the numbers ofBi'fid0baclerI'a relative to control up to lohr incubation.

Studies on the stability ofthe thuricin peptides Trn—ot and Trn—t3 in simulated gastric (Fig. 7/\). ileal (Fig. 7B) and eolonjuice (Fig. 7C) demonstrated that there was no reduction of activity in any of the simulated gut environments when thuricin (‘D was incubated for 2. 4, or 9 hours in gastric, ileal and colonjuice, respectively. l-‘urther1nore_ incubation ofthuricin peptides Trn—ot and Trn-B for 2hr and 5hr in ex vim proeine gastriejuice (Fig. 7D) and ilealjuice (Fig. 7E), respectively. also demonstrated no reduction in activity. The results for determining the minimum inhibitory concentration ofthuriein shows that clinically significant ribotypes of ( '. c/r'['ficile are more sensitive to thuricin when compared to the antibiotics vancomycin and metronidazole, which are currently used for treatment, as shown in the Table 2 below. Ribotype 001 and 106 are commonly associated with outbreaks of(.‘l)Al) in lrish and UK hospitals respectively, while ribotype 027 is associated with increased severity of symptoms and increased morbidity due to the elavated toxin production resulting in disease that is more refractory to treatment.

K J ‘J1 lEOM95Z Table 2. Comparison of minimum inhibitory concentrations (MIC) for thuricin. vancomycin and metronidazole against a range ofclinically significant ribotypes of (7. ctiflici/ca.

T: timMaw_ MKXuM) ? g .Ribiot_y_pem_» Thuricin \/“ancomycin Metronidazolem 001W _g 0.097 0.39 H 3.125 _ g 0.125 0.39 1.56 H _gj 027"» 0.012 0.39 3.125 2..- J Discussion Due to the high incidence of C. drffici/e worldwide in hospitals and facilities for the elderly radical approaches have to be considered for the treatment of this disease.

The two component lantibiotic lacticin 3147 has been shown to be very effective in killing ('. c/ifificile at low concentrations. however. as it is a broad spectrum antibiotic it also affected the Lactobacillus and Bi/idobczcicrium populations (by 3 log cycles) in simple faeeal fermentations at concentrations required to kill (‘. di/fici/e (Rea er a/.. 2007) The work reported here focused on searching within the GI tract for sources of antimicrobial producing bacteria to address this problem. The aim was to isolate a narrow spectrum bacteriocin producer. which would have potent activity against (T dz/fie/'/e while perturbing the gut microbiota as little as possible. As a result of screening ~30.000 colonies from laecal samples one colony was detected that showed inhibition ofthe C difficile overlay. The faecal samples had been prc-treated with ethanol to facilitate the isolation of spore forming bacteria. The fact that just one antimicrobial producing colony was isolated from just one sample at a low dilution would suggest that the B. I/turingienm strain DPC 6431 isolated was not a major constituent of the gut microbiota. (‘haracterisation of the antimicrobial peptide produced by DPC 6431, thuricin (T1). demonstrated that its antimicrobial inhibition spectrum (using WDA) is narrow and while very effective against C. difficile isolates including the PCR ribotype 027 has little or no activity against the beneficial microflora such as the Laclobaci//us and /31/idobaelerimn populations.

IQ ’.Jv B. I/71/ringiensi.s‘ is a sport? forming Gram positive insect pathogen which has been used extensively for many years in biological pest control. Bacteriocins have been identified previously from a number of B. /hm‘ingiensis strains (Ahern cl ul.. 2003: Barboza-Corona et al., 2007; Chechimi el al. 2007; Cherif et a/., 2003; Cherifez a/.. 2001; liavret & Yousten, 1989; Gray el al, 2006a; Gray et al. 2006b: Kamoun 91 a/.. 2005). Ahern at (112003 characterised a BLIS substance from a strain of B.

I/71rringiensi.s' which produced 2 active peptides designated thuriein 439a and thuriein 43%; both peptides showed antimicrobial activity however two component activity was not reported. From a survey ofthe literature the sequence and molecular mass of thuriein 6431 is most similar to that produced by B. thuringiensis 439. Ahern el al (2003) reported that the amino acid sequences of the two peptides 439a and 439b were identical but the peptides have different molecular weights. The sequence reported for thuriein 439a/b has 2 unidentitied amino acids (X) which the authors suggxst are likely to be cystein, therefore, there is just 2 amino acid distinguishing (a cystein instead ofa valine and a glutamic acid instead of valine) in the first 19 amino acids of the peptides from B. thzzringicnsis 6431 and 439. However, 'l‘rn—ot from thuriein Cl) is significantly different from thuriein 439 peptide. lhuricin Cl) was shown to be very active against a range of (.‘/mtridium species while no anti- clostridium activity was reported for thuriein 439 (Ahern et al.. 2003). A comparison between the amino acid sequences and molecular masses and spectrum of activity of lhuricin Cl) and 439 is shown below in Table 3.

Table 3. Comparison ofthuricin identified in this study with thuriein 439 of/Xherne at ul (2003) Amino Acid Sequence Mol Mass Inhibitory activity The nucleic acid sequence and the orientation of the genes encoding the thuricin peptides peptide 1 and peptide 2 are shown in Figure 3. A search of the NCBI database showed no homologous sequences to the sequences identified here.

The discrepancies between the mo] masses as determined by MALDI-'l‘()l* MS (2763 and 2861) and from the amino acid sequence (2770 and 2864) ofthuriein CD would appear to be the result of post translational modification. The predicted mass from the DNA sequence of Trn-or and Trn-[3 peptides, 2769 Da and 2876 Da. respectively. differ by 6 mass units from what was obtained from MS (mass spectrometry). For 'l'rn-or. the differences in mass indicate a loss of two hydrogen atoms from each of Ser 21. 'l'hr 25. and 'l‘hr 28, and for Tm-B is suggestive that Thr 21, Ala 25, and 'l‘yr 28 are all two mass units lighter than expected. Cys residues for both peptides are in the same positions (residues 5. 9. and 13), while the post-translational modifications occur at the same positions (residues 21. 25, and 28). . thuricin (fl) is active over a wide pll range and moderately heat stable retaining actix ity up to 95°C for 15 minutes. MlC5i; studies show it to be a very potent inhibitor of ( '. (fl:/fIiL'I‘/L’ at concentrations as low as 0.5 and 5uM (Trn-on and Trn—B respectively) when present as single peptides but the MIC50 is reduced to 0.05uM when both peptides are present indicating that thuricin is a 2 component bacteriocin highly active against ('. clr')f/"ic'i/e at low concentrations. Kill curves demonstrated that thuricin Cl) is very effective in reducing cell numbers of(.'. riifficile ribotype 027 (NAP l ) at low concentrations and is also lytic in nature. The efficacy ofthuricin against ('. cliflici/e 027 is signilicant as this strain has been shown to be hyper virulent. the incidence of which is increasing worldwide resulting in increased severity, high relapse rate and significant mortality (Kuiper et al 2007). Comparisons of MIC values for thuricin with those obtained for vancomyein and metronidazole. which are the current antibiotics used to treat C. dz’/jicile infections are clinically significant. Interestingly. similar concentration ofthuricin Cl) did not affect the viability of1.. cusei 338 or /3’. /aclis Bbl 2 in contrast to the effect oflacticin (Rea er (112007) which would indicate that beneficial flora in the gut would not be perturbed by this antimicrobial.

When assessing microbially derived peptides for the treatment or prevention of disease the issue ofbio-availability needs to be addressed. The demonstrated Ix) 'Ji IEo80952 degradation of the antimicrobial activity of thuriein CD in vim) with ot—chymotrypsin and pepsin and trypsin would suggest that this bacteriocin would not survive gastric transit without protection such as encapsulation. llowever, the alternative strategy of feeding spores or vegetative cells ofthis organism as probiotics could be investigated as a method to target the delivery of this peptide within the GIT. Probiotic cultures are usually associated with species of bacteria which are normal inhabitants ofthe Gl'l' such as Luctolvacillus and Bzfidobacreriznn species. However, S. boulardii which is not a normal constituent of the human gut microbiota is currently used a probiotic in the treatment ofCDAD. Bacillus species are currently being used as probiotic cultures for both human and animal use (for reviews see Hong el al 2005 and Sanders cl :1! 2003). Because spores can survive hostile environments the question has been raised as to their true habitat? While it would have been assumed that they arrive in the human gut as a consequence of ingestion of the spore from the environment, Hong er al (2005) state however, that there is the possibility that Bacillus species exist in an endosymbiotic relationship with their host being able temporarily to survive and proliferate in the gut. The advantage of administering spores over vegetative cells is their stability and ability to pass through the hostile environment of the stomach. In mouse studies it has been shown that while vegetative cells ofB. sublilus did not survive passage through the stomach almost all of the administered spores survived gastric transit and were recovered in the small intestine (Due el al 2003).

In a study of greenhouse workers excreting B. Ilmringiensis due to occupational exposure to B. Il1uringien.s'is—based pesticides no gastrointestinal symptoms correlated with the presence of B. /lruringiensis in the faecal samples (Jensen et al 2002). A study of B. //rzmhgiensis in the gut ofhuman—llora—associated rats which had been fed 8. //721/'/n_giensi.s' spores and vegetative cells detected no adverse effects on the composition ofthe indigenous gut flora or no cytotoxic effect in gut samples by Vero cell assay (Wilcks et (11,, 2006).

Although there is a discrepancy between the results using the purified proteolytic enzymes and the result in the various GI environments, an explanation for this may be that the concentration of the purified enzymes used is much greater than that present in the (31 environments. Note that these results refer to the thuriein peptides only and not the \ egetative cells or spores. lEoa0%52 In conclusion this work has shown that the B. lhuringi7ensis' strain DPC 6431 produces a potent heat stable two—component bacteriocin which has potential as a novel therapeutic agent CDAD either in peptide form or as a probiotic in either a vegetative cell or spore format. the bacterioein of the invention has a number of advantages. The antimicrobial substance designated thuricin CD was produced into the fermentation medium (1 litre of medium yielded 350mg thuricin) and the cell free supernatant showed a narrow spectrum ofinhibition inhibiting Bacillus species, C difficile including PCR ribotype 027. C’. per/i'i'ngens and Listeria monocylogenes. Bi/idobacterium and Laclobucil/u.s' species were not inhibited with the exception of Lb. fermemum and Lb. c'rispat21.s' and Hi. /‘olirisonii. which were very weakly inhibited. The baeteriocin is heat stable, active over 21 wide pll range and is sensitive to a range ofproteolytic enzymes. It is a tvvo— component bacteriocin with the peptides having molecular masses ot‘2763 (lrn-ot) and 2861 ('l'rn—B). lhuricin CD exhibited an MlC5oof0.5 pM and 5pM for 'l'rn—[t and 'l‘rn—or. respectively, when both peptides were present alone. When the peptides were present together the MlC5o was 50 nM Trn-[3 in combination with 25nM o1"l‘rn—u.: a ratio 012: 1 .'l‘he bactericidal effect ofthuricin CD was demonstrated through time kill experiments in which ~5 x 106 ctu ot’(.'. cliffici/e per ml were killed within 180 min at concentration of20()AU/ml. Fhuricin CD is a two component bacterioein active at nano molar concentrations.

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Claims (5)

Claims

1. B. Thurirzgiensis 6431 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. 41490 and strains which are 3 substantially similar thereto, also encoding a bacteriocin effective against (7l0.s‘Iria’ium (1'i_/lic'z'le and Listeria manacytogenes.

2. A bacteriocin effective against 1.isre/‘ia manocyfogenes and C lo.s‘I/‘idizmi cfi/_‘fic‘ile, produced by a bacterial strain as claimed in claim 1.

3. A bacteriocin designated Thuricin CD comprising 2 peptides. ’l‘rn-or and Trn—[3, 'l‘m-or having at least one of the characteristsics of: (a) a molecular mass of about 2763 and Trn—B having a molecular mass of about 2861; (b) being heat~stable up to about 85° Centigrade and having a reduction of activity at about 90° Centigrade and a loss of activity at about 100° 15 Centigrade after 15 minutes’ incubation; (c) having the ability to inhibit Bacillus cereus, other Bacillus tlzuringiensis Clostridium difflcile, Listeria monocytogenes, B mycaides, B firmus, C. diflicile ribotypc 027, C. lyrobutyricum, C. lillzuseburerzse, C. indolis and C. perfringens; and 20 (d) being active in the pH range 2-10. or an individual component thereof.

4. A composition comprising the bacterial strain of claim 1, a bacteriocin as claimed in claim 2 or 3, or a component thereof.

5. A bacteriocin substantially as described herein with reference to the Examples 25 or the accompanying drawings.