Field of the invention

The current invention is in the field of inhibiting C. difficile sporulation with cephamycins, and optionally with an antibiotic that inhibits vegetative C. difficile cells for the purpose of treating C. difficile infections, the symptoms thereof, and reducing the rates of transmission of C. difficile infections.

Related application

This application claims priority from Australian provisional application AU 2017903760, the entire contents of which are hereby incorporated by reference.

Background of the invention

Clostridium difficile (recently renamed as Clostridioides difficile) is a Gram positive, spore-forming anaerobic bacterium that causes severe gastrointestinal illness that can be fatal. In particular, C. difficile causes disease when the normal gastrointestinal microbiota is suppressed by antibiotics that are used to treat other, unrelated, medical conditions. It is the most significant cause of hospital-acquired diarrhoea (Clostridium c//7 /c//e-associated-diarrhea: CDAD) in many countries, including Australia, with hypervirulent strains that result in major epidemics emerging since 2000. Use of fluoroquinolones, all 3 generations of cephalosporins, macrolides, clindamycin, and β lactam/p-lactamase inhibitors have all been independently associated with an increased risk of CDAD.

C. difficile is known to produce highly resistant and persistent spores that contaminate hospital environments. While the vegetative cells of C. difficile cannot survive in an aerobic environment for very long, the spores can, thereby permitting C. difficile to survive in aerobic environments outside of the host and acting as a reservoir for further infections. Moreover, the spores are very resistant to commonly used disinfectants. The spore form of C. difficile is critical for the initiation of disease because it is the infective particle that is ingested by patients. Spores may also contribute to bacterial survival in the host during antibiotic therapy and to subsequent recurrence following the cessation of therapy. Spores are therefore critical in the C. difficile infection cycle. In all likelihood, spore ingestion and germination once it reaches the intestine of the host is followed by vegetative cell colonisation and proliferation, after which toxin and further spore production occur. Current treatments for C. difficile disease primarily involve the administration of antibiotics, usually metronidazole or vancomycin or fidaxomicin, which act against vegetative cells. Although these antibiotics are effective at inhibiting C. difficile, they also prevent the re-establishment of the normal bowel microbiota. Consequently, approximately 15-30% of patients experience relapses in C. difficile infection after treatment ceases, with many patients suffering multiple relapses, probably resulting from spore ingestion or persistence. Moreover, metronidazole and vancomycin both enhance sporulation, which increases the environmental spore load.

Even though many research efforts have been directed toward prevention and treatment strategies, the most effective therapy remains the administration of metronidazole and vancomycin. There are currently no effective compounds available that reduce or prevent C. difficile sporulation. Clearly, new strategies are required to manage the increasing incidence, severity and recurrence of C. difficile disease and infection.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the invention

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

The present invention provides a method of preventing, inhibiting, minimising or reducing sporulation of C. difficile, the method comprising contacting C. difficile vegetative cells or spores with an inhibitor of SpoVD. The present invention also provides a method of preventing, inhibiting, minimising or reducing sporulation of C. difficile, the method comprising contacting C. difficile vegetative cells or spores with an inhibitor of PGT (01085).

The present invention also provides a method of treating C. difficile infection comprising the steps of: administering a therapeutically effective amount of an inhibitor of SpoVD; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of preventing infection of a subject with C. difficile, or reducing or minimising the likelihood of becoming infected with C. difficile comprising the steps of: administering a therapeutically effective amount of an inhibitor of SpoVD and/or PGT; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of preventing, inhibiting, reducing or minimising C. difficile sporulation comprising the step of administering a therapeutically effective amount of a SpoVD inhibitor to a subject infected with C. difficile. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing the transmission rate of infection by C. difficile comprising the step of administering a therapeutically effective amount of SpoVD inhibitor to a subject that is infected with C. difficile, wherein the SpoVD inhibitor minimises or reduces sporulation. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing recurrence, or reducing or minimising the likelihood of recurrence, of C. difficile infection, following the cessation of an antibiotic treatment targeting vegetative C. difficile cells, the method comprising the step of administering a therapeutically effective amount of a SpoVD inhibitor.

In any embodiment of the above methods, the method may also include inhibiting PGT in addition to inhibiting SpoVD.

In any embodiment, the inhibitor of SpoVD and the inhibitor of PGT are the same molecule.

The inhibitor of SpoVD and/ or of PGT may be selected from the group consisting of a small molecule, an antibody, a peptide or an interfering RNA. In any aspect of the invention, the inhibitor of SpoVD or of PGT inhibits the activity of the protein. Preferably, the inhibitor binds to the active site of SpoVD or PGT. More preferably, the inhibitor of SpoVD or PGT competes with, or prevents the binding of a substrate of SpoVD or PGT for binding to SpoVD or PGT.

In any embodiment, the inhibitor of SpoVD is a cephamycin. Preferably, the cephamycin is selected from cefoxitin, cefmetazole and cefotetan. More preferably, the cephamycin is cefotetan.

In any embodiment, the inhibitor of PGT (01085) is a cephamycin. Preferably, the cephamycin is selected from cefoxitin, cefmetazole and cefotetan. More preferably, the cephamycin is cefotetan.

In any embodiment of the invention, the subject to whom the SpoVD and/or PGT inhibitor is administered to, is a human subject. Alternatively, the subject is a farm or domestic animal, including a pig, cattle, a horse, poultry or a companion animal (such as a cat, dog, mouse or rat). The subject may be juvenile or adult.

The present invention provides a method of treating C. difficile infection comprising the steps of: administering a therapeutically effective amount of a cephamycin; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells. The present invention also provides a method of preventing infection of a subject with C. difficile, or reducing or minimising the likelihood of becoming infected with C. difficile comprising the steps of: administering a therapeutically effective amount of a cephamycin; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing or minimising C. difficile sporulation comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing or minimising the severity of symptoms associated with C. difficile infection, comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing or minimising C. difficile associated diarrhea, comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing the transmission rate of infection by C. difficile comprising the step of administering a therapeutically effective amount of cephamycin to a subject that is infected with C. difficile, wherein the cephamycin minimises or reduces sporulation. Optionally the method also comprises administration of a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

The present invention also provides a method of reducing recurrence, or reducing the likelihood of recurrence, of C. difficile infection, following the cessation of an antibiotic treatment targeting vegetative C. difficile cells, the method comprising the step of administering a therapeutically effective amount of a cephamycin.

In any method of the invention, the cephamycin is preferably selected from cefoxitin, cefmetazole and cefotetan; and the antibiotic that targets vegetative C. difficile cells is preferably metronidazole or vancomycin or fidaxomicin.

In any method of the invention, where it would assist to know if the subject already has a C. difficile infection before administering a cephamycin, the method further comprises the step of diagnosing the C. difficile infection.

The present invention also provides a use of therapeutically effective amount of a cephamycin, and a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells in the preparation of a medicament for:

• for treating C. difficile infection.

• to prevent infection of a subject with C. difficile, or reducing or minimising the likelihood of becoming infected with C. difficile.

The present invention also provides a use of therapeutically effective amount of a cephamycin, and optionally a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells, in the preparation of a medicament for:

• for reducing or minimising C. difficile sporulation in a subject infected with C. difficile.

• for reducing or minimising the severity of symptoms associated with C. difficile infection in a subject infected with C. difficile.

• for reducing or minimising C. difficile associated diarrhea in a subject infected with C. difficile.

• for reducing the transmission rate of infection by C. difficile.

The present invention also provides a use of therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing recurrence, or reducing the likelihood of recurrence, of C. difficile infection following the cessation of antibiotic treatment targeting vegetative C. difficile cells. In any use of the invention the cephamycin is preferably selected from cefoxitin, cefmetazole and cefotetan; and the antibiotic that targets vegetative C. difficile cells is preferably metronidazole or vancomycin or fidaxomicin.

The present invention also provides an inhibitor of SpoVD and/or PGT:

• for inhibition, prevention or minimising sporulation of C. difficile;

• for treatment of C. difficile infection.

• to prevent infection of a subject with C. difficile, or reduce the likelihood of becoming infected with C. difficile;

• to minimise the severity of C. difficile infection.

The present invention also provides a cephamycin, and an antibiotic that targets vegetative C. difficile cells, for use in a therapeutically effective amount:

• for treatment of C. difficile infection.

• to prevent infection of a subject with C. difficile, or reduce the likelihood of becoming infected with C. difficile.

The present invention also provides a cephamycin, and optionally an antibiotic that targets vegetative C. difficile cells, for use in a therapeutically effective amount:

• to reduce or minimise C. difficile sporulation in a subject infected with C. difficile.

• to reduce or minimise the severity of symptoms associated with C. difficile infection in a subject infected with Clostridium difficile.

• to reduce or minimise C. difficile associated diarrhea in a subject infected with C. difficile.

• to reduce or minimise the transmission rate of infection by C. difficile wherein cephamycin minimises or reduces sporulation in a subject infected with C. difficile.

The present invention also provides a cephamycin for use in a therapeutically effective amount to reduce recurrence, or reduce the likelihood of recurrence, of C. difficile infection following the cessation of antibiotic treatment targeting vegetative C. difficile cells. When the invention provides a cephamycin, or a cephamycin and an antibiotic that targets vegetative C. difficile cells, or cephamycin and optionally an antibiotic that targets vegetative C. difficile cells, the cephamycin is preferably selected from cefoxitin, cefmetazole and cefotetan; and the antibiotic that targets vegetative C. difficile cells is preferably selected from metronidazole or vancomycin or fidaxomicin.

In a further aspect of the invention there is provided a pharmaceutical composition comprising a cephamycin or a pharmaceutically acceptable salt thereof, and an antibiotic for treating vegetative C. difficile cells or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable carriers or diluents.

The compositions may be a single formulation of both actives, or a separate formulation of each active but formulated for simultaneous administration, or sequential administration. Alternatively, the administration of the first active and the second active may be separated by a predetermined period of time.

The present invention also provides a non-pharmaceutical composition, for use in any method of the invention. For example, where the inhibition, reduction or minimising of C. difficile sporulation is required in a non-human subject, the SpoVD inhibitor, or PGT inhibitor may be included in the subject's drinking water or feed. Accordingly, the present invention provides an animal feed for inhibiting, reducing, minimising or preventing C. difficile sporulation, the feed comprising a therapeutically effective amount of a SpoVD inhibitor.

In any embodiment, the SpoVD inhibitor in the animal feed is a cephamycin. Preferably, the cephamycin is selected from cefoxitin, cefmetazole and cefotetan. More preferably, the cephamycin is cefotetan.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the figures

Figure 1 : C. difficile infection and flow chart of sporulation effects on all aspects of the infection pathway. Figure 2: The in vitro effect of cephamycins on sporulation. Total heat-shocked spore numbers were determined for each day of the sporulation assay with independent triplicate or quadruplicate untreated (UT) cultures of C. difficile 027 strains M7404, R20291 , DLL3109 and CD196 in comparison to cefoxitin, cefotetan or cefmetazole treated cultures. Vegetative cells were not affected by cefoxitin, cefotetan or cefmetazole at the sub-inhibitory concentration used for the sporulation assays. Fold inhibition as follows; Cefoxitin M7404 300 - 1500, R020291 200-600, DLL3109 100-200, CD196 500-700. Cefotetan: M7404 5000 - 10,000, R020291 600-5000, DLL3109 3000 - 22,500, CD196 100 - 1000. Cefmetazole: M7404 2000 - 16,000, R020291 200, DLL3109 340-1700, CD196 120-800.

Figure 3: The in vivo effect of cephamycin treatment on spore shedding of C. c//7 /c//e-infected mice. (A) The effect of 50 pg/mL cefoxitin, cefotetan and cefmetazole on spore shedding in vivo. Spore shedding for untreated and cephamycin-treated mice was monitored at 24 hrs and 36 hrs post-infection. N = 5 mice per group; Error bars represent the SEM and P values were determined using a one-way Anova test ^** P<0.005, ^*** P<0.001 . (B) The effect of lower cefotetan doses (30 pg/mL, 25 pg/mL and 20 pg/mL) on spore shedding compared to untreated mice at 24 hrs and 36 hrs post-infection. N = 3-5 mice per group; Error bars represent the SEM and P values were determined using a two-way Anova test ^** P<0.005. (C) Total viable cell counts at 24 hours and 36 hours for mice either untreated or treated with 50 pg/mL of cefoxitin, cefotetan or cefmetazole or (D) untreated or treated with 30, 25 or 20 pg/mL of cefotetan. Error bars represent the SEM with P values determined via a one-way Anova test. No statistical significance in vegetative cell numbers was observed at 24 hrs or 36 hrs (P > 0.200).

Figure 4: Visualization of C. difficile sporulation morphology of M7404 untreated and cefoxitin treated cells. TEM images of untreated and cefoxitin treated cultures (A and B) on day 1 , (C and D) day 2, (E and F) day 3 and (G and H) day 6. Images are representative of the most common morphological phenotype observed. Red arrows indicate the asymmetric sporulation septum and spores. Green arrows show mislocalised proteins and irregular shaped vegetative cells.

Figure 5: Identification of the molecular targets of the cephamycins. (A) Detection of Penicillin binding proteins (PBPs) in untreated (UT) and cefoxitin (+CF), cefotetan (+CT) or cefmetazole (+CZ) treated C. difficile M7404 strains with Boc-FL. Membrane proteins from UT and +CF treated cultures were isolated on days 1 -3 of a sporulation assay and their PBPs labeled with Boc-FL. The labeled proteins were separated by SDS-PAGE and their PBP profiles visualized using a Fluorlmager. Proteins present in UT but not in +CF, +CT or +CZ treated cultures are indicated by asterisks. Two -1 1 1 kDa PBPs, Cd_M7404_01339, a putative cell surface PBP and Cd_M7404_01003, a putative class B PBP, were found to be absent in the cefotetan and cefmetazole day 2 and 3 treated samples. Mass spectrometry was used to identify the proteins denoted by asterisks. (B) Identification of SpoVD and 01085 (hereafter, PGT) as targets of cefoxitin. Boc-FL labeled PBPs were separated by SDS-PAGE and visualized using a Fluorlmager. Asterisk denotes SpoVD or PGT. (C) Sporulation assays to determine role of targets in sporulation. Total spore counts were performed with triplicate M7404 untreated, cefoxitin treated, spoVD complement, spoVD mutant, pgt complement and pgt mutant cultures. Data represent the mean SEM. P values were determined using a Student's t-test ^*** P<0.01 , ^*** P<0.0001 ).

Figure 6: Binding affinity between SpoVD and three cephamycin antibiotics measured in a competition assay with Boc-FL. Cefotetan showed the highest affinity for SpoVD.

Figure 7: Crystal structure of 01085. High resolution X-ray crystal structure to

2.1A.

Figure 8: Human PLG (hPLG) binding to C. difficile spores. Immunofluorescence (IF) using hPLG primary antibodies and an anti-mouse Alexa 488 secondary antibody (green) was used to visualise hPLG binding to M7404 spores. The bright field image (A), hPLG labeled image (hPLG Ab) (green) (B) and the overlapped images shows the association of hPLG with M7404 spores (C). Stimulated emission depletion (STED) microscopy of horizontal (D, E, F) and vertical (G, H, I) transverse sections of M7404 spores stained using an anti-spore antibody (green) and an anti-hPLG antibody (red) shows a more defined association of hPLG with M7404 spores. Western blot showing hPLG binding to C. difficile spores from diverse geographical locations and origins (M7404 Canadian human epidemic isolate (E1 ); R20291 UK human epidemic isolate (E2); JGS6133 US animal isolate (A-US); A135 Australian animal isolate (A-AU); DLL3109 Australian human epidemic isolate (E-AU); VPI10463 US human reference isolate (R) and CD37 US non-toxigenic isolate (NT) detected using an anti-human PLG Ab (J). Figure 9: Enhanced virulence effects of hPLG on C. difficile M7404 infected mice. (A) Survival time of C57BL/6J mice (expressing mouse PLG) and HPLG mice (expressing both human and mouse PLG) that were either uninfected or infected with C. difficile M7404. (B) Survival time of C57BL/6J mice (expressing mouse PLG) and PLG KO (not expressing mouse PLG) that were either uninfected or infected with C. difficile M7404. Data is pooled from three independent mouse trials. Error bars represent the mean ± SEM of n=5-20 mice. ^*** indicates P < .0001 .

Figure 10: Co-administration of cefotetan and vancomycin prevents recurrent CDI. Infected mice were orally treated with vancomycin, fidaxomicin or a combination of vancomycin and cefotetan (30 pg/mL or 50 pg/mL) until spore shedding could no longer be detected. After treatments ceased (Day 0), mice were monitored daily for (A) survival and (B) weight loss. Weight loss is presented as the % weight relative to the day before, with each point representing a single mouse. (C) Faecal spore load was determined daily after cessation of treatments and is presented as cfu/gram faeces (Iog10). N = 9- 10 mice per group; The Kaplan-Meier survival curve was assessed using a log-rank (Mantel-Cox) test ^**** P < 0.0001.

Detailed description of the embodiments

Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

Clostridium difficile, recently renamed as Clostridioides difficile, (C. difficile) is a spore-forming obligate anaerobe. Due to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate C. difficile infections. During this infection cycle, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients. It is therefore desirable to be able to treat C. difficile infection by administering a compound that targets sporulation, together with a compound that targets C. difficile vegetative cells.

The present inventors have identified factors which are critical to the ability of C. difficile to form spores. In particular, the inventors have confirmed a major role for the protein SpoVD in the C. difficile sporulation cycle. In addition, the inventors showed that 01085 (PGT) also contributes to the sporulation ability of C. difficile.

The present invention thus provides a method of inhibiting, minimising or reducing sporulation of C. difficile, the method comprising contacting C. difficile spores or vegetative cells with an inhibitor of SpoVD.

The present invention also provides a method of inhibiting, minimising or reducing sporulation of C. difficile, the method comprising contacting C. difficile spores or vegetative cells with an inhibitor of PGT (01085).

The present invention thus provides a method of inhibiting, minimising or reducing sporulation of C. difficile, the method comprising contacting C. difficile spores or vegetative cells with an inhibitor of SpoVD and an inhibitor of PGT.

In any embodiment, the inhibitor of SpoVD and the inhibitor of PGT are the same molecule.

The present invention also provides a method of treating C. difficile infection comprising the steps of: administering a therapeutically effective amount of an inhibitor of SpoVD; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

In any embodiment, the inhibitor of SpoVD is a cephamycin. Preferably, the cephamycin is selected from cefoxitin, cefmetazole and cefotetan.

As used herein, "SpoVD" refers to the C. difficile protein with Uniprot accession numbers C9YPN0 and A0A031 WAQ0 (annotated locus Cd_M7404_02549). SpoVD is also known as 'Stage V sporulation protein D', 'sporulation-specific penicillin binding protein' or 'PASTA domain containing protein'.

SpoVD from C. difficile is a 73.28 kDa protein having the amino acid sequence:

MRKVKRISKKRLVLVLILACALFFCLVIRTGYLQLMKGNWLSTKALEQQTRDIPIE PKRGTIYDRNMKELAVSVTKYTVWCKPVEVEDKKEAAEKVAEILDEDYKDIYALISKKN MALVKVKRWIDDDKASQIRDAKLSGIWVAEDNQRYYPYGNFAPYVLGHTSSDATGISG VEMQYDKKLKGKPGKLIVSTDASGREIPQGMEKYYEPVQGNGLVLSIDEVIQHYTEKA VQ KAYE L N N AKKVTAI AM N P KTG D I LALAS KP D YD P N D S RTP I YP YYQ E E L E KYN D KD K IKGYYQMWRNPAVSDTYEPGSTFKLITSSSALEEGVIKDGEKFTCTGSVTVGGRKIKC WRHYRPHGTQEFKQAVQNSCNPVFVELGSRLGVGKMYDYIESFGLMDKTGIDLPGEA KGILYNEKNVGPVELATISFGQSISVTPIQLITAISSIANGGDLMQPRWKSYTDNKGNIT ETVKPKKVRSVISKETSKKMLEIAESVVTEGGGKIAYIPGYRLGGKTGTAQKVIDGKYAP GKYICSFVGIAPCDDPQIWLAIVDEPTGVSAFGSTTAGPIVKEIMNDSLKYLGVKPVYK EEEKAEYEKKQVKVPDVRNLKIGDAVKALEDAKLKPDLDADIELPEDTKVKDIFPKPGV KVNEDSSITLYFEN (SEQ ID NO: 1 1 )

Reference herein to a SpoVD protein refers to the C. difficile protein having the above sequence, including any functional derivatives or fragments thereof. A functional derivative or fragment includes any homolog or sequence variant of SpoVD that has the same function as SpoVD including in relation to facilitating sporulation of C. difficile.

The present invention provides a method of treating C. difficile infection comprising the steps of: administering a therapeutically effective amount of an inhibitor of PGT; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

In any embodiment, the inhibitor of PGT is a cephamycin. Preferably, the cephamycin is selected from cefoxitin, cefmetazole and cefotetan.

As used herein, "PGT" or 01085, refers to the C. difficile protein having the accession numbers Cd_M7404_01085, or Uniprot accession numbers: A0A160VZ04, Q18B92 or A0A0H3N5P7. PGT is also known as a 'putative peptidoglycan glycosyltransferase', 'peptidoglycan glycosyltransferase', 'penicillin binding protein 2' or 'penicillin binding transpeptidase domain protein'.

The C. difficile PGT protein is a 62.63 kDa protein having the amino acid sequence:

MSKKKTPFLKKVGKRSWCIFTIILIIYSVLIYRLVDIQVLKGDKYKQSVESQSVEKV ELNSGRGIIYDRNNKKLTDTSKSQVLIVEKEKLNNNYKILELIKKATKMNDLDIYKAVQE Q LTRPIIQIQTKNIDKSMKKELEKNGIMVEEKTMRYAKDGLLSHTIGYIKEDDKSGQSGIE K SMDSVLRNSNEKYISAFKAGDAGNEKSLNILKGSVKTVDNKDKDRHLKTTIDYNIQKKL EQILNKEENPTAAIISEASTGEILAMCSRPNFDQNDISKSLKGKNGEFENRVIKATYPPG SVFKMWLFSALENGVIDENYTYNCTGKTKVGNTNEILRCNKRDGHGFQNLRQAFSNS CNPAFLDIAMKLGKEKILKSAEKLHLFEKVDIGLDEEKIREAPKNISIRNLAIGQENIEF TP LQINQMTQIIANNGTFKPLYLYKSLVDNNMNTIKTYKSSKKEELISPYVCTQVKEYMKSV SRIGTAKDLKDIEGGCGVKTGTAQSSLNKKAIDHGWITGFYPEERPKYVITVLVEGTQK GNKSATPIFKEICESIK (SEQ ID NO: 12)

Reference herein to a PGT protein refers to the C. difficile protein having the above sequence, including any functional derivatives or fragments thereof. A functional derivative or fragment includes any homolog or sequence variant of PGT that has the same function as PGT including in relation to facilitating sporulation of C. difficile.

The present invention also provides a method of preventing infection of a subject with C. difficile, or reducing or minimising the likelihood of becoming infected with C. difficile comprising the steps of: administering a therapeutically effective amount of an inhibitor of SpoVD and/or PGT; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

As used herein, minimising, reducing or preventing sporulation of C. difficile refers to reducing the number of spores formed or preventing the formation of any spores from C. difficile. The minimising, reducing or preventing sporulation may occur in the context where C. difficile spores have already formed, and the aim is to prevent or reduce the amount of any further sporulation of C. difficile. Alternatively, the minimising, reducing or preventing sporuiation may occur in the context where the C. difficile cells exist purely in a vegetative state, and the aim is to prevent or reduce the levels of any spores being formed.

The skilled person will be able to determine whether a candidate agent is capable of inhibiting sporuiation of C. difficile, including by performing routine in vitro or ex vivo assays for determining the presence of C. difficile spores.

Moreover, the skilled person will be able to determine whether a candidate agent is capable of inhibiting SpoVD or PGT including by conducting simple binding assays to determine changes in

As used herein, a SpoVD or PGT inhibitor may be any molecule that inhibits the activity of SpoVD or PGT. The inhibitor may be a direct inhibitor of the active site of the proteins, may act allosterically to inhibit activity, inhibit interaction of SpoVD or PGT with its substrate, or may reduce the level of SpoVD or PGT by reducing the transcriptional activity of the spoVd or pgt genes, or reducing the amount of SpoVd or PGT mRNA or protein present in the cell.

The inhibitor of SpoVD or of PGT may be selected from the group consisting of a small molecule, an antibody, a peptide or an interfering RNA. In any aspect of the invention, the inhibitor of SpoVD or of PGT inhibits the activity of the protein. Preferably, the inhibitor binds to the active site of SpoVD or PGT. More preferably, the inhibitor of SpoVD or PGT competes with, or prevents the binding of a substrate of SpoVD or PGT for binding to SpoVD or PGT.

As used herein, reference to a SpoVD inhibitor or inhibitor of SpoVD also includes a pharmaceutically acceptable salt, solvate, polymorph or prodrug thereof.

The inventors have made the surprising discovery that cephamycins can be utilised to target sporuiation. Cephamycins are very similar to cephalosporins, and are often classified as second generation cephalosporins. Cephamycins have a methoxy group at the 7-alpha position, which imparts resistance to extended-spectrum β- lactamases. They include cefoxitin, cefmetazole, cefotetan, defprozil, defuroxime, cefuroxime axetil, cefamandole, cefminox, cefonicid, ceforanide, cefotiam, cefbuperazone and cefuzonam. The ability of cephamycins to reduce or minimise sporulation was highly surprising and very unexpected as C. difficile is resistant to most cephalosporins. In light of this, exposure to cephalosporins was known to be a strong risk factor for C. difficile outbreaks. By the 1990s, cephalosporins, particularly cefuroxime, cefotaxime, ceftazidime and ceftriaxone, were the antibiotics with the highest relative risk of C. difficile disease, because of their frequent use in hospitals. Strategies to combat C. difficile associated diarrhea (CDAD) relied upon sparing use of particular antibiotics including all 3 generations of cephalosporins. It was therefore unanticipated when the inventors determined that cephamycins could reduce sporulation. With reduced sporulation comes the flow on effects illustrated in Figure 1 and detailed throughout this specification. That is:

• fewer spores to give rise to vegetative cells; fewer vegetative cells equate to lower toxin production and lessening of symptoms and fewer new spores

• fewer spores shed so lower rates of transmission

• fewer spores to recruit other factors which augment the severity of the symptoms and the infection

• fewer spores to become dormant or latent in the gut, so reduced risk of reactivation of infection upon cessation of antibiotic therapy.

As can be seen from Figure 1 , controlling sporulation is key to the methods and uses of the invention.

Accordingly, in one aspect of the invention, there is provided a method of treating C. difficile infection comprising the steps of: administering a therapeutically effective amount of a cephamycin; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

There is also provided use of therapeutically effective amount of a cephamycin, and a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells, in the preparation of a medicament for treating C. difficile infection.

There is further provided a cephamycin, and an antibiotic that targets vegetative C. difficile cells, for use in a therapeutically effective amount to treat C. difficile infection. The terms 'treatment' or 'treating' of a subject includes the administration of a compound or composition, as described herein, to a subject with the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the C. difficile infection or the symptom of the infection, or the risk of (or susceptibility to) the infection. The term "treating" refers to any indication of success in the treatment including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the infection more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.

In addition to being responsible for the persistence of infection in a subject, the spores are shed from infected subjects in high volumes in faecal matter. The spores are highly resistant and contaminate hospital environments, thereby playing a critical role in the infection process and facilitating efficient disease transmission. Accordingly, in one aspect of the invention, there is provided a method of reducing or minimising C. difficile sporulation comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile.

There is also provided use of therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing or minimising C. difficile sporulation in a subject infected with C. difficile.

There is further provided a cephamycin for use in a therapeutically effective amount to reduce or minimise C. difficile sporulation in a subject infected with C. difficile.

"Reducing or minimising C. difficile sporulation" refers to reducing or minimising levels of C. difficile sporulation relative to the level of C. difficile sporulation in the absence of administering a therapeutically effective amount of cephamycin. The skilled person is able to make such as assessment via observation of patient symptoms, laboratory analysis of spore load in stool samples and toxin production, and epidemiological evidence of fewer new infections. In the above aspects of the invention, associated with reducing or minimising C. difficile sporulation in a subject infected with C. difficile, it may be advantageous to also administer, or include in the medicament, or use, an antibiotic that targets vegetative C. difficile cells. Without being bound by any mode of action or mechanism, by targeting the vegetative cells, fewer cells will be available to sporulate, and toxin production will be reduced, thereby also seeking to reduce or minimise symptoms. Preferably the antibiotic is metronidazole or vancomycin or fidaxomicin.

The inventors have also determined that C. difficile spores are involved in recruiting other factors which augment the severity of infection. Minimising or reducing sporulation can therefore help reduce or minimise the severity of symptoms associated with C. difficile infection. Accordingly in another aspect of the invention, there is provided a method of reducing or minimising the severity of symptoms associated with C. difficile infection, comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile.

There is also provided use of therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing or minimising the severity of symptoms associated with C. difficile infection in a subject infected with C. difficile.

There is further provided a cephamycin for use in a therapeutically effective amount to reduce or minimise the severity of symptoms associated with C. difficile infection in a subject infected with C. difficile.

"Reducing or minimising the severity of symptoms" refers to reducing or minimising the severity of symptoms of a subject, relative to the severity of symptoms before the subject received a therapeutically effective amount of cephamycin, and may include delaying, slowing, stabilising, curing, healing, alleviating, eliminating relieving, altering, remedying, ameliorating and improving the symptoms. The assessment of a reduction of the severity of symptoms includes any objective or subjective parameter.

In the above aspects of the invention, associated with reducing or minimising the severity of symptoms associated with C. difficile infection, it may be advantageous to also administer, or include in the medicament, or use, an antibiotic that targets vegetative C. difficile cells. By targeting the vegetative cells, fewer cells will be available to sporulate, thereby decreasing the recruitment of factors which augment the severity of infection.

Symptoms of C. difficile infection may begin during antibiotic therapy or 5 to 10 days after the antibiotic is stopped; less commonly, symptoms do not develop until as late as 10 weeks later. Symptoms can vary in severity, the most common symptom being C. difficile associated diarrhea (CDAD). Accordingly in another aspect of the invention, there is provided a method of reducing or minimising C. difficile associated diarrhea, comprising the step of administering a therapeutically effective amount of a cephamycin to a subject infected with C. difficile.

There is also provided use of therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing or minimising C. difficile associated diarrhea in a subject infected with C. difficile.

There is further provided a cephamycin for use in a therapeutically effective amount to reduce or minimise C. difficile associated diarrhea in a subject infected with C. difficile.

By "reducing or minimising C. difficile associated diarrhea" it is meant reducing the severity and frequency of diarrheal episodes relative to the severity and frequency before the subject received a therapeutically effective amount of cephamycin. The diarrhea may also change in composition, and be less water, or have less blood.

Other symptoms of C. difficile infection include abdominal cramping and tenderness, dehydration, fever, nausea, loss of appetite, and weight loss. In severe cases, subjects may experience abdominal distension and potentially a ruptured bowel and associated sepsis. The severity and extent of these symptoms may also be minimised or reduced by administration of a therapeutically effective amount of a cephamycin.

In the above aspects of the invention, associated with reducing or minimising the severity of symptoms associated with C. difficile infection and reducing or minimising C. difficile associated diarrhea, it may be advantageous to also administer, or include in the medicament, or use, an antibiotic that targets vegetative C. difficile cells. By targeting the vegetative cells, fewer cells will be available to sporulate. Without being bound by any theory of action, fewer spores means a lower level of recruitment of other cellular factors which augment the severity of infection.

As noted, C. difficile adapts to changing environmental conditions by producing highly resistant and persistent spores which are very resistant to commonly used disinfectants. The spores act as a reservoir for new transmissions. Reducing or minimising sporulation therefore reduces the number of spores shed in to the environment. This in turn reduces or minimises the risk of transmission of infection with C. difficile, especially nosocomial transmission. As such, in yet another aspect of the invention, there is provided a method of reducing the transmission rate of infection by C. difficile comprising the step of administering a therapeutically effective amount of a cephamycin to a subject that is infected with C. difficile, wherein the cephamycin minimises or reduces sporulation.

There is also provided use of therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing the transmission rate of infection by C. difficile wherein the cephamycin minimises or reduces sporulation in a subject infected with C. difficile.

There is further provided a cephamycin for use in a therapeutically effective amount to reduce or minimise the transmission rate of infection by C. difficile wherein the cephamycin minimises or reduces sporulation in a subject infected with C. difficile.

An assessment of a reduction of the transmission rate of infection is typically a subjective one, but includes historical and epidemiological data. The assessment can made via agent-based simulation models (see for example Rubin et al., (2013), PloS One. 8 (1 1 ) 1 -1 1 ).

In the above aspects of the invention, associated with reducing or minimising the transmission rate of infection by C. difficile it may be advantageous to also administer, or include in the medicament, or use, an antibiotic that targets vegetative C. difficile cells. Targeting vegetative C. difficile cells as well as spores assists with reducing the transmission rate on the basis that fewer cells are present to sporulate.

The in vivo production of spores during infection is also likely to be an important cause of recurrent infection or 'rebound' infection. Treatment of patients suffering with C. difficile infections using antibiotics such as metronidazole, vancomycin and fidaxomicin leads to clearance of vegetative C. difficile cells, but has little effect against the spores. The spores persist within the gut, and then germinate following cessation of antibiotic treatment, resulting in a rebound or recurrent infection. Administration of cephamycins as well as antibiotics that are targeting the vegetative C. difficile cells, such as metronidazole, vancomycin and fidaxomicin, seeks to reduce or minimise the in vivo production of spores during antibiotic therapy and should therefore reduce the risk of subsequent rebound infections. Accordingly, in another aspect of the invention, there is provided a method of reducing recurrence, or reducing the likelihood of recurrence, of C. difficile infection, following the cessation of antibiotic treatment targeting vegetative C. difficile cells, the method comprising the step of administering a therapeutically effective amount of a cephamycin. The cephamycin can be administered together with, or upon cessation of, the antibiotic treatment targeting vegetative C. difficile cells.

There is also provided use of a therapeutically effective amount of a cephamycin in the preparation of a medicament for reducing recurrence or reduce the likelihood of recurrence, of C. difficile infection following the cessation of antibiotic treatment targeting vegetative C. difficile cells.

There is further provided a cephamycin for use in a therapeutically effective amount to reduce recurrence, or reduce the likelihood of recurrence, of C. difficile infection following the cessation of antibiotic treatment targeting vegetative C. difficile cells.

For subjects in hospital for other reasons, particularly those highly susceptible to nosocomial infections for whatever reason (e.g. elderly, immunosuppressed) there is the option to administer a cephamycin together with an antibiotic that targets vegetative C. difficile cells for the purposes of preventing transmission of C. difficile to a subject. Accordingly, there is also provided a method of preventing infection of a subject with C. difficile, or reducing the likelihood of becoming infected with C. difficile comprising the steps of: administering a therapeutically effective amount of a cephamycin; and administering a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells.

As used herein, 'preventing' or 'prevention' is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) becoming infected with C. difficile.

In any embodiment of the invention, 'inhibiting' or 'reducing' sporulation of C. difficile may also include preventing sporulation of C. difficile.

There is also provided use of therapeutically effective amount of a cephamycin, and a therapeutically effective amount of an antibiotic that targets vegetative C. difficile cells, in the preparation of a medicament for preventing infection of a subject with Clostridium difficile, or reducing the likelihood of becoming infected with Clostridium difficile.

There is further provided a cephamycin, and an antibiotic that targets vegetative C. difficile cells, for use in a therapeutically effective amount to preventing infection of a subject with C. difficile, or reducing the likelihood of becoming infected with C. difficile.

The term 'administered' in each aspect of the invention described herein means administration of a therapeutically effective dose of a cephamycin, or in relevant embodiments, a cephamycin and another antibiotic treatment targeting the vegetative C. difficile cells such as vancomycin or metronidazole or fidaxomicin, or composition(s) thereof to an individual. By 'therapeutically effective amount' is means a dose that produces the effects for which it is administered. That is, the amount that (i) treats a C. difficile infection; (ii) reduces or minimises C. difficile sporulation; (iii) reduces or minimises one or more symptoms of a C. difficile infection including diarrhea; (iv) reduces the recurrence or likelihood of recurrence of C. difficile infection; and (v) reduces the rate of transmission of infection by C. difficile.

The exact dose to be administered will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art and described above, adjustments for systemic versus localized delivery, age, body weight, general health, gender, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art. The phrase 'therapeutically effective amount' generally refers to an amount of one or more inhibitors, or, if a small molecule inhibitor, a pharmaceutically acceptable salt, polymorph or prodrug thereof of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In the context of the present invention, a therapeutically effective amount generally refers to an amount of one or more inhibitors, including cephamycins, which are effective for inhibiting or preventing sporulation of C. difficile, thereby treating, attenuating or preventing symptoms associated with C. difficile infection.

In each of the methods and uses of the invention, the method optionally comprises a first step of diagnosing the C. difficile infection via means known in the art including, but not limited to, observation of patient symptoms, laboratory analysis of C. difficile spore load in stool samples or toxin detection.

Compounds, formulations, compositions and treatment regimens

A "therapeutic composition", "pharmaceutical composition", "composition for treating" and other like terms refers to a composition including a cephamycin combined with appropriate pharmaceutically acceptable salts, carriers or diluents. Optionally the composition may also include an antibiotic that targets vegetative C. difficile cells.

In each aspect of the invention described herein the cephamycin that is used, or administered in a method, or used in the preparation of a medicament is preferably selected from cefoxitin, cefmetazole and cefotetan. In each aspect of the invention described herein the antibiotic that targets vegetative C. difficile cells that is used, administered in a method, or used in the preparation of a medicament, is preferably selected from metronidazole or vancomycin or fidaxomicin.

In one aspect of the invention there is provided a pharmaceutical composition comprising a cephamycin or a pharmaceutically acceptable salt thereof, together with pharmaceutically acceptable carriers or diluents.

In aspects of the invention whereby the cephamycin is administered with an antibiotic treatment targeting vegetative C. difficile cells, the administration may be: • a single formulation including a cephamycin and the antibiotic treating vegetative C. difficile cells

• a separate formulation of a cephamycin and a separate formulation of the antibiotic treating vegetative C. difficile cells, but administered at the same time i.e. simultaneous or concurrent administration

• a separate formulation of a cephamycin and a separate formulation of the antibiotic treating vegetative C. difficile cells, administered one after the other i.e. sequential administration

• a separate formulation of a cephamycin and a separate formulation of the antibiotic treating vegetative C. difficile cells, administered at different, predetermined times.

For the first time, there is provided a pharmaceutical composition comprising

• a cephamycin or a pharmaceutically acceptable salt thereof; and

• an antibiotic that targets vegetative C. difficile cells or a pharmaceutically acceptable salt thereof;

together with pharmaceutically acceptable carriers or diluents.

Additional actives may optionally be included for treating some of the symptoms whilst also targeting C. difficile cells and spores. For example, an antidiarrheal may be included. Alternatively the composition may consist of the cephamycin and the antibiotic as the only 2 actives.

It has not previously been known to formulate these two antibiotics in the same pharmaceutical composition. Exemplary combinations of actives include one cephamycin with one antibiotic that targets vegetative C. difficile cells as per the combinations listed below:

The antibiotic that targets vegetative C.

The cephamycin

difficile metronidazole

cefoxitin

vancomycin fidaxomicin

metronidazole

cefmetazole vancomycin

fidaxomicin

metronidazole

cefotetan vancomycin

fidaxomicin

The term "pharmaceutically acceptable salt" as used herein refers to one or more salts of a given compound which possesses the desired pharmacological activity of the free compound and which are suitable for use in contact with the tissues of human and animals without undue toxicity, irritation or adverse response. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. J. Pharmaceutical Sciences, 66: 1 - 19 (1977) and P. H. Stahl and C. G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich:Wiley-VCH/VHCA, 2002). Either or both of the cephamycin and the antibiotics that treat vegetative C. difficile cells may be included in the composition in derivative form. Non-limiting examples of such suitable derivatives include pro-drugs, metabolites, esters, hydrates, polymorphs, solvates, stereoisomers, complexes and enantiomers.

Pharmaceutically acceptable carriers or diluents contemplated by the invention include any diluents, carriers, excipients, and stabilizers that are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as plasma albumin, gelatine, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The pharmaceutical compositions may be formulated for administration by any appropriate method. Non-limiting examples of administration methods for which the composition may be formulated include oral, intravenous, topical, intraperitoneal, intramuscular, parenteral, sublingual, transdermal and intranasal; preferably the cephamycin and the antibiotics that treat vegetative C. difficile cells are formulated for oral delivery or for intravenous administration; more preferably oral delivery.

In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and if necessary, shaping the product. Formulation may be conducted by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed.

In certain embodiments the pharmaceutical composition may be provided in the form of a device, disposable or reusable, including a receptacle for holding the pharmaceutical composition. In one embodiment, the device is a syringe. The device may hold 1 -2 ml_ of the pharmaceutical composition. The pharmaceutical composition may be provided in the device in a state that is ready for use or in a state requiring mixing or addition of further components.

It will be understood that the specific dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination (i.e. other drugs being used to treat the patient), and the severity of the particular disorder undergoing therapy. The dose to be administered may be in the form of single doses of higher concentration, or divided doses of lower concentration. The concentration of any given dose will depend on the frequency of administration.

In any embodiment of the invention, the dose of cephamycin administered to an individual for inhibiting the activity of SpoVD and/or of PGT in C. difficile, for preventing, minimising or inhibiting C. difficile sporulation, for treatment of C. difficile infection, for preventing infection of a subject with C. difficile, or reducing the likelihood of becoming infected with C. difficile, for reducing or minimising C. difficile sporulation in a subject infected with C. difficile, for reducing or minimising the severity of symptoms associated with C. difficile infection in a subject infected with C. difficile, for reducing or minimising C. difficile associated diarrhea in a subject infected with C. difficile, for reducing or minimising the transmission rate of infection by C. difficile, may include a dose of cephamycin ranging from 0.2 mg to 0.5 mg. The dose may be administered once or more times daily, preferably at least twice daily. Accordingly, in certain embodiments, the total daily dose of cephamycin may be at least 0.4 mg, at least 0.8 mg, at least 1 mg, at least 10 mg, at least 100 mg, at least 500 mg, at least 1 g, at least 2 g, at least 3 g or at least 4 g. The skilled person will be familiar with altering the dose administered in accordance with the intended mode of administration (for example, if the dose is intended to be an oral dose as compared with intravenous, intramuscular or alternative dosing regimens which may impact on the bioavailability of the drug).

A single oral formulation of the pharmaceutical composition of the invention may comprise active ingredients of 0.2 - 0.5 mg of a cephamycin and 400 - 700 of metronidazole, formulated for a treatment regimen of twice or 3 times daily.

Alternatively, a single oral formulation of the pharmaceutical composition of the invention may comprise active ingredients of 0.2 - 0.5 mg of a cephamycin and 50 - 800 of vancomycin formulated for a treatment regimen of twice or 3 times daily.

Further still, a single oral formulation may comprise active ingredients of 0.2 - 0.5 mg of a cephamycin and 200 mg of fidaxomicin formulated for a treatment regimen of twice daily.

In the above embodiments directed to single oral formulations, the formulation also includes pharmaceutically acceptable carriers or diluents. As noted above the pharmaceutical composition of the invention may be a separate formulation of a cephamycin and a separate formulation of the antibiotic treating vegetative C.difficile cells. These formulations and treatment regimens include:

The separate formulations may be administered simultaneously or concurrently, sequentially, or at different predetermined times (especially in the case of a treatment regimen involving oral and IV, oral and IM, IV and IM administration of the 2 formulations).

In each embodiment the formulation also includes pharmaceutically acceptable carriers or diluents.

Subjects

The subject to which the cephamycin is being administered is preferably a mammal. It will be clearly understood that, although this specification refers specifically to applications in humans, the invention is also useful for veterinary purposes. C. difficile is an established human and animal pathogen and there is considerable overlap among some animal and human strains. Thus in all aspects the invention is useful for domestic animals such as cattle, sheep, horses, pigs, goats and poultry; for companion animals such as cats and dogs; and for zoo animals. Therefore, the general term "subject" or "subject to be / being treated" is understood to include all animals. Kits

In other embodiments there is provided a kit for use in a method, or for a use, mentioned above, the kit including:

- a container holding a pharmaceutical composition of cephamycin; and

- a label or package insert with instructions for use.

In certain embodiments the kit may contain one or more further active principles or ingredients. For example, the kit may also include a container holding a pharmaceutical composition of an antibiotic that targets vegetative C. difficile cells. Alternatively, the kit may include a container holding both a pharmaceutical composition of cephamycin and an antibiotic that targets vegetative C. difficile cells as per the new formulation of both actives as described herein.

In any kits of the invention, the cephamycin is preferably selected from cefoxitin, cefmetazole and cefotetan; and the antibiotic that targets vegetative C. difficile cells is preferably metronidazole or vancomycin or fidaxomicin.

The kit or "article of manufacture" may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a therapeutic composition which is effective for the methods and uses described herein, and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates the intended use for the therapeutic composition, and may include instructions for use.

The kit may further comprise a container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

EXAMPLES

It will be understood that these examples are intended to demonstrate these and other aspects of the invention and although the examples describe certain embodiments of the invention, it will be understood that the examples do not limit these embodiments to these things. Various changes can be made and equivalents can be substituted and modifications made without departing from the aspects and/or principles of the invention mentioned above. All such changes, equivalents and modifications are intended to be within the scope of the claims set forth herein.

1 . The in vitro and in vivo effect of cephamvcins on sporulation: sporulation is minimised or reduced

Different antibiotics were tested for their ability to inhibit or reduce C. difficile sporulation.

In vitro sporulation assay

Triplicate HIS broth cultures (Heart Infusion powder, Yeast extract, 0.1 % L- cysteine, 25% glucose) were grown overnight and adjusted to an OD ₆ oo of 0.1 in pre- reduced HIS broth and incubated until reaching mid-exponential phase (OD ₆ oo between 0.45-0.5). Once mid-exponential 50 μΙ_ of OD ₆ oo=0.5 was added to 15 mL of pre- reduced TY broth (Tryptone, Yeast extract, Sodium thioglycolate). To three cultures 30 μί of 20 pg/mL cefoxitin, cefotetan or cefmetazole (Sigma) was added. 30 μί of 20 pg/mL of the cephalosporins cefaclor, cefuroxime and cefotaxime was also added to a M7404 culture. An aliquot of 1 ml was immediately removed for TEM imaging studies. Another 1 mL was used for serial dilutions and samples were plated onto HIS agar containing 1 % (w/v) sodium taurocholate (New Zealand Pharmaceuticals) to determine total viable counts. Concurrently, a sample was heat-shocked at 65°C for 30 minutes, and then plated onto HIS agar containing sodium taurocholate to obtain heat-resistant viable counts. This was repeated for Day 1 , Day 2 and Day 3. Three or four biological replicates were tested for each strain. Data were analysed using GraphPad Prism 5 and statistical significance assessed using the student's t-test, Mann-Whitney or one-way Anova. Results

Total heat-shocked spore numbers were determined for each day of the sporulation assay with independent triplicate untreated (UT) cultures of C. difficile 027 strains M7404, R20291 , Kl and CD196 in comparison to (Figure 2A) cefoxitin treated or (Figure 2B) cefotetan or cefmetazole treated cultures (i.e. the cephamycin treated cultures). Three cephalosporins cefaclor, cefuroxime and cefotaxime was also tested against M7404 (not shown in figures; data in table). Vegetative cells were not affected by cefoxitin, cefotetan or cefmetazole. Fold inhibition as follows:

The cephamycins were unexpectedly able to reduce sporulation whereas the cephalosporins were not. In the in vitro experiments, the cephamycins were able to reduce sporulation whereas the tested cephalosporins were not able to reduce sporulation to any significant level.

Spore shedding using Murine Model of Clostridum difficile infection

Cefoxitin, cefotetan and cefmetazole inhibition of sporulation in vivo was examined using a mouse model of C. difficile infection (CDI) (Figure 3A). Animal handling and experimentation were performed in accordance with Victorian State Government regulations and approved by the Monash University Animal Ethics Committee (Monash University AEC no. MARP/2014/142).

Male, 6 week old, C57BL/6J mice (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) were pre-treated with an antibiotic cocktail in the drinking water for seven days as described in Sorg and Dineen (2009, Current protocols in microbiology. Chapter 9:Unit9A 1 ), followed by two days of cefaclor alone. Antibiotic treatment ceased the day of infection with C. difficile M7404 (10 ⁵ spores/mouse) by oral gavage. For cephamycin treatment spore shedding studies, mice received 100 μΙ_ of 50 pg/mL cephamycin (cefoxitin, cefotetan or cefmetazole) or 100 μΙ_ 30, 25 or 20 pg/mL cefotetan by oral gavage 8 hours post-infection, followed by immediate access to the cephamycin in the drinking water (50 pg/mL cephamycin or 30, 25 or 20 pg/mL cefotetan) or left untreated for the duration of the trial. Mice were monitored twice daily for signs of disease (weight loss, behavioural and physical changes, and diarrhoea) and faeces were collected in order to enumerate C. difficile spore load. Faecal pellets were collected 24 hours and 36 hours post-infection, resuspended in PBS (100 mg/ml), heat shocked for 30 minutes at 65 °C and plated for spore enumeration as described (Lyon, et al., (2016), PLoS Pathog 12, e1005758). Animals were humanely killed by C0 ₂ overdose or cervical dislocation when defined endpoints were met as previously defined (Carter et al., (2015) mBio 6, e00551 ).

Results:

The effect of 50 pg/mL cefoxitin, cefotetan and cefmetazole on spore shedding in vivo was examined using a mouse model of CDI. Spore shedding for untreated and cephamycin-treated mice was monitored at 24 hrs and 36 hrs post-infection. N = 5 mice per group; Error bars represent the SEM and P values were determined using a oneway Anova test ^** P<0.005, ^*** P<0.001 (Figure 3A). A significant reduction in spore shedding was seen at the 36 hr time-point for cefoxitin, cefotetan and cefmetazole.

While treatment with cefotetan resulted in the greatest reduction in in vivo spore shedding, in vivo vegetative cell growth was abolished at this concentration. To confirm that cefotetan was specifically affecting sporulation in vivo, we examined the effect of lower cefotetan doses (30 pg/mL, 25 pg/mL and 20 pg/mL) on spore shedding compared to untreated mice at 24 hrs and 36 hrs post-infection. N = 3-5 mice per group; Error bars represent the SEM and P values were determined using a two-way Anova test ^** P<0.005 (Figure 3B).

Total viable cell counts at 24 hours and 36 hours were determined for mice either (Figure 3C) untreated or treated with 50 pg/mL of cefoxitin, cefotetan or cefmetazole or (Figure 3D) untreated or treated with 30, 25 or 20 pg/mL of cefotetan. Vegetative cell numbers were determined by subtracting heat-shocked spore counts from total viable cell counts. Error bars represent the SEM with P values determined via a one-way Anova test. No statistical significance in vegetative cell numbers was observed at 24 hrs or 36 hrs (P > 0.200) confirming that cefotetan specifically targets sporulation in vivo.

With reduced sporulation comes a reduction in spore shedding. When spore shedding is reduced, transmission of C. difficile infection is likewise reduced as spores are key to transmission.

2. Visualization of C. difficile sporulation morphology of M7404 untreated and cefoxitin treated cells: cephamvcin treatment causes sporulation dvsregulation.

During normal sporulation, the first stage of sporulation is asymmetric septum formation. Asymmetric cell division generates a smaller forespore compartment and a larger mother cell after which the mother cell engulfs the forespore. The forespore develops into the spore through the production of the spore cortex, inner and outer coats and the exosporium. The mother cell then lyses and releases the mature dormant spore.

The inventors however noted and investigated mislocalised proteins and irregular shaped vegetative cells following treatment with the cephamycin, cefoxitin.

For TEM analysis, 1 ml_ of cells of the wild type and cefoxitin treated strains were collected during sporulation assays at the various time points and centrifuged for 3 mins at 13,000rpm. Cells were initially fixed with 2.5% glutaraldehyde in cacodylate buffer 0.1 M pH 7 and post-fixed in 1 % osmium tetroxide in water. Samples were pelleted and embedded in low melting point 2% agar. Blocks of agar containing samples were transferred in 0.5% uranyl acetate/H20 and then dehydrated through a series of ethanol washes. Finally, samples were embedded in epoxy resin. Thin sections (50nm) were cut and stained with 2.5% lead citrate and 10% uranyl acetate before viewing under a Phillips CM120 electron microscope at 80kV. Results:

Transmission electron microscopy (TEM) images of untreated and cefoxitin treated cultures (Figure 4A and B) on day 1 , (Figure 4C and D) day 2, (Figure 4E and F) day 3 and (Figure 4G and H) day 6. Images are representative of the most common morphological phenotype observed. Red arrows indicate the asymmetric sporulation septum and spores. Green arrows show mislocalised proteins and irregular shaped vegetative cells.

Without being bound by any theory, the disruption of the sporulation process by cephamycins appears to result in a build-up of protein aggregates (electron dense bodies) resulting from sporulation dysregulation i.e. proteins involved in the sporulation process are unable to function due to the blocking of the sporulation process, resulting in a "traffic jam" of proteins. This build-up of protein aggregates may also contribute to the irregular shaped vegetative cells. This combined effect on sporulation leads to a reduction or minimisation of spores.

3. Identification of the molecular targets of the cephamycins: cephamycins target

PBPs.

Cephalosporins have the same mode of action as other beta-lactam antibiotics; they disrupt the synthesis of cell wall peptidoglycan, which is important for cellular integrity. The final step in the synthesis of peptidoglycan is catalysed by transpeptidases, also known as penicillin binding proteins or PBPs, which bind to the D- Ala-D-Ala at the end of peptidoglycan precursors to crosslink the peptidoglycan. Beta- lactams mimic this site and competitively inhibit PBP-mediated crosslinking.

This experiment identified at least 2 molecular targets of the cephamycins and confirmed their role in sporulation.

In vivo labeling of C. difficile with Boc-FL for gel-based analysis

C. difficile M7404 cells were grown as detailed in the sporulation assays method. Fluorescent labeling was carried out as detailed in (Kyne (2010) N Engl J Med 362, 264-265.) with a few changes. Briefly, 1 ml_ of culture was harvested by centrifugation (16, 100 x g for 1 min at RT) and washed with phosphate buffered saline (PBS; pH 7.4). Cell pellets were resuspended in 50 μΙ_ of PBS containing 5 g / ml_ of Bocillin-FL, a penicillin flurophore-conjugate (ThermoFisher Scientific). After incubation for 10 min with Boc-FL at RT, the cells were washed and resuspended in 500 μΙ_ of PBS containing 1 mg mL ^-1 of lysozyme and were incubated for 30 min at 37 °C. The cells were lyzed by Branson Sonifier 250 (power setting 3, 30% duty cycle for 5 χ 6 s intervals) and membrane proteome was isolated by centrifugation at 16,000 χ g (25 min), 4 °C. Membrane proteome was resuspended in 100 μΙ_ PBS and protein concentration was determined by NanoDrop 1000 Spectrophotometer (Thermo

Scientific). Proteome sample was diluted to 2.5 mg mL ^-1 in PBS. Following the addition of 10 μί of 4 x SDS-PAGE loading buffer to 40 μΙ_ of proteome, the sample was heated for 5 min at 90°C, cooled to RT and the proteins separated on a 10% SDS-PAGE. Labeled proteins were visualized in gel using a Typhoon 9210 gel scanner (Amersham Biosciences). All gel images were analyzed using ImageJ software (NIH).

Protein identification

Bands of interest were separated by SDS PAGE gel, excised, and subjected to tandem mass spectrometry at the Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia.

Construction of TargeTron mutants

Gene target sequences of spoVD and 01085 were identified by searching the annotated genome of C. difficile strain M7404 (Wasabi). The ClosTron online tool (ClosTron.com) was used to find TargeTron insertion sites. Construction of the TargeTron plasm ids was performed as previously described, with some modifications (Carter et al., (2014) Int J Med Microbiol 304, 1 147-1 159). Gene block fragments of 350bps (SEQ ID NO: 1 and 2, pgt and SpoVd, respectively) containing retargeted regions were digested with Hindlll and BsrGI and the digested products ligated into the pDLL46 vector backbone. The resulting plasmids, pDLL46-spo\ D and pDLL46-07085, were then digested with Hindlll and StuI and sub-cloned into vector pDLL56. The pDLL56-spo\ D pDLL56-07085 plasmids were introduced into the B. subtilis conjugative donor strain BS34A as previously described (Anagnostopoulos, and Spizizen, (1961 ) Journal of bacteriology 81 , 741 -746). The resulting strain was used as the donor for the conjugative transfer of plasm id DNA into C. difficile strain M7404 as before (Mackin et al., (2013) PloS one 8, e79666). Mutants were isolated using the method previously described (Kocaoglu and Carlson (2013), Current protocols in chemical biology 5, 239- 250) and the insertion of the intron into spoVD and 01085 was confirmed by PCR using primer pairs SpoVDFIISpoVDR (SEQ ID NO: 3 and 4) and 1085F1/1085R3 (SEQ ID NO: 5 and 6).

The CdspoVD and Cdpgt complementation plasmids were constructed by PCR amplifying the CdspoVD gene and Cdpgt gene from C. difficile M7404 using the primer pair spoVD_complementF_Sphl and spoVD_complementR_Aatll for spoVD and 01085t_complementF_Sphl and 01085_complementR_Aatll for 01085 (pgt). The resulting 3.3 kb and 2.1 kb fragments were purified using a PCR purification kit (Qiagen) following the manufacturer's instructions, digested with SphI and Aatll and cloned into the corresponding sites of pJIR3566, resulting in the CdspovD or Cdpgt complemented plasmid. Complementation of the spoVD mutation was achieved using the spoVD complementation plasmid as detailed above.

AAAAAAGCTTATAATTATCCTTAGATAACGATAGAG TGCGCCCAGATAGGGTGTTAAGTCAAGTAG I I I AA GGTACTACTCTGTAAGATAACACAGAAAACAGCCA AC CTAAC C G AAAAG C G AAAG CTG ATAC G G G AAC A

01085 (PGT) (SEQ ID NO: GAG C AC G GTTG G AAAG C G ATG AGTTAC CTAAAG A 1 ) CAATCGGGTACGACTGAGTCGCAATGTTAATCAGA

TATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTT CTAATTTCGATTTTATCTCGATAGAGGAAAGTGTCT GAAACCTCTAGTAC AAAG AAAG GTAAGTTATGTCT ATC G ACTTGTAC AC C AG AT

AAAAAAG CTTATAATTATC CTTAG AAGC C G C C G AA GTGCGCCCAGATAGGGTGTTAAGTCAAGTAG I I I A AGGTACTACTCTGTAAGATAACACAGAAAACAGCC AAC CTAAC C G AAAAG C G AAAG CTG ATAC G G G AAC A

SpoVD (SEQ ID NO: 2)

GAG C AC G GTTG G AAAG C G ATG AGTTAC CTAAAG A CAATCGGGTACGACTGAGTCGCAATGTTAATCAGA TATAAGGTATAAGTTGTGTTTACTGAACGCAAGTTT CTAA I I I CGATTGCTTCTCGATAGAGGAAAGTGTCT GAAAC CTCTAGTAC AAAG AAAG GTAAGTTA I I I I CG GCGACTTGTACACCAGAT spoVDFI (SEQ ID NO: 3) AAAAAAG CTTATAATTATC CTTA spoVDR (SEQ ID NO: 4) CAGATTGTACAAATGTGGTGATAACAGATAAGTC

01085F1 (pgfF1 ) (SEQ ID

GGAATTGAAAAGAGTATGGATAG NO: 5)

01085R3 (pgtR3) (SEQ ID

TTTTATACTTTCACATATTTC NO: 6) spo VD_complementF_Sphl acatgacgcatgcCATCAGATTGTATATGTCCACAGCAT (SEQ ID NO: 7) TTACCT spoVD_complementR_Aatll

acatgcagacgtcCTCCAACTATGGCTACAAATGCAT (SEQ ID NO: 8) pgt_complementF_Sphl AC ATG AC G C ATG CTAG AAG GTAG AATG AAG ACTG C (SEQ ID NO: 9) ATACTATGT pgt_complementR_Aatll ACATGCAGACGTCATCCTGTTATCCATCCGTGGTC (SEQ ID NO: 10) TAT

Results:

Detection of penicillin binding proteins (PBPs) in untreated (UT) and cefoxitin treated (+CF) C. difficile M7404 strains with Boc-FL is shown in Figure 5A. Membrane proteins from UT and +CF treated cultures were isolated on days 1 -3 of a sporulation assay and their PBPs labeled with Boc-FL. The labeled proteins were separated by SDS-PAGE and their PBP profiles visualized using a Fluorlmager. Proteins present in UT but not in +CF treated cultures are indicated by asterisks. Mass spectrometry was used to identify the proteins denoted by asterisks, which led to the identification of Stage V sporulation protein D (SpoVD) and 01085 as targets of cefoxitin (Figure 5B). Boc-FL labeled PBPs were separated by SDS-PAGE and visualized using a Fluorlmager. Asterisk denotes SpoVD or 01085. SpoVD is a PBP with an unknown catalytic activity. 01085 is a sporulation specific, Class B PBP and is a putative peptidoglycan glycosyltransferase.

In order to then determine the role of SpoVD and 01085 in sporulation, total spore counts were performed with triplicate M7404 untreated, cefoxitin treated, spoVD complement, spoVD mutant, 01085 complement and 01085 mutant cultures (Figure 5C). Data represent the mean SEM. P values were determined using a Student's t-test ^*** P<0.01 , ^*** P<0.0001 ).

The sporulation capacity of the spoVD and 01085 mutants were assayed in comparison to the wild-type M7404 strain. It was found that the spoVD mutant was completely impaired in its ability to produce spores, with no spores detected from day 1 - 3. No effect on vegetative cell numbers was observed. Sporulation assays with a complemented spoVD mutant showed restoration of sporulation ability. The results confirm a major role for spoVD in the C. difficile sporulation cycle and support the hypothesis that SpoVD is an anti-sporulation target of cephamycins. The 01085 mutant's sporulation ability was inhibited 21 -123 fold, with no effect on vegetative cells observed. Sporulation was not completely attenuated compared to the spoVD mutant, possibly due to the functional redundancy of other sporulation factors.

4. Binding affinity between SpoVD and three cephamvcin antibiotics measured in a competition assay with Boc-FL: cephamycins competitively bind the PBP SpoVD to minimise or inhibit sporulation.

The ability of cephamycin antibiotics to outcompete the fluorescently-labelled penicillin (Boc-FL) for binding to SpoVD was assessed by fluorescence polarization assays using a spectrofluorimeter (PHERAstar FSX, BMG).

Recombinant SpoVD Protein Production and Purification

The expression construct, pET22b-Spo\ D, encoding residues 34-659 (with a C- terminal hexa-histidine tag) was used to transform Escherichia coli strain C41 (DE3). The transformed cells were grown in auto-induction media containing ampicillin at 28°C for 24 hours. Cells were lysed by sonication in 50 mM Phosphate-Buffered Saline (PBS) pH 7.4; 250 mM NaCI; 10 % Glycerol; 0.01 % Triton X-100 and 0.05 mM EDTA. His- tagged SpoVD was purified using a two-step process of Ni-NTA-agarose chromatography (binding buffer PBS pH 7.4, 0.25 M NaCI, 10% (v/v) glycerol and 10 mM imidazole, elution buffer PBS pH 7.4, 0.25 M NaCI, 10% (v/v) glycerol and 0.25 M imidazole) and followed by size exclusion chromatography on a Superdex 200 16/60 (20 mM Tris-HCI pH 7.5, 0.15 M NaCI). Fractions containing purified SpoVD were concentrated to 5-10 mg/ml_ and stored at -80°C before thawing for assays as required.

Competition Assay of Cephamycin Antibiotics with Boc-FL for SpoVD

The reactions were carried out in 384-well plates, 60 μΙ_ total volume, in PBS pH 7.4, 0.01 % Triton-X-100 at ambient temperature. Assays contained 60 nM SpoVD, 40 nM Boc-FL, and appropriate concentrations of cefoxitin, cefotetan or cefmetazole (established in 1 :4 dilution series). The reactions were started by addition of SpoVD. Excitation and emission wavelengths of 490 and 520 nm respectively, and a focal height of 7.2 mm were used. Fluorescence polarization was calculated from parallel and perpendicular fluorescence intensities measured simultaneously over the course of the reaction (25s intervals). Each assay was performed in triplicate. IC50 values for each of the cephamycin antibiotics were calculated using PRISM software (one site binding) by plotting initial reaction velocities against antibiotic concentration, and presented ± the standard deviation.

Results:

All 3 cephamycin antibotics showed good affinity for SpoVD; cefotetan showed the highest affinity for SpoVD (Figure 6) with an IC50 of 56 nM.

Antibiotics IC ₅₀ (nM) ± standard error

Cephamycins CcfSpoVD CdPGT

Cefoxitin 873 ± 1 .0 685 ± 1 .0

Cefmetazole 3 189 ± 1 .0 473 ± 1 .0

Cefotetan 56.0 ± 1 .2 618 ± 1 .0 Binding of cephamycins to PBPs such as SpoVD therefore minimises or inhibits sporulation, thereby reducing spore shedding and in turn treating the primary C. difficile infection and reducing transmission.

5. Determination of the crystal structure of 01085: structure - activity relationship of cephamycins with the PBP 01085 (PGT).

Determination of the crystal structure of 01085 helps to characterise structure-activity relationships of cephamycins with spore-specific PBPs.

Figure 7 illustrates the crystal structure of the cephamycin target 01085 (also referred to herein as "PGT"). It was determined to be a two domain protein: o N-terminal is elongated with several long ©-strands and a subdomain of shorter strands and small helices (residues x-y). Role of N-terminal is unknown. Note the position of 1085 N-terminal is completely different to 3EQU and has a significant structure extension. This may facilitate binding to other proteins, or may project the transpeptidase domain from toward peptidoglycan. o C-terminal domain is the carboxy-transpeptidase (CTP) domain that is common to all PBPs.

The active site of PBPs (CTP) contains three conserved sequence motifs. o By homology with 3EQU, active site residues are S268 & K271 (motif 1

SXXK); S325 & N327 (motif 2 SXN); R306 & C307 (note strange Cys in comparison to 3EQU); K465, Thr467 & G468.

o S268 should be the serine nucleophile that is acylated by both peptide substrate and ©-lactam antibiotics,

o Regions adjacent to the active site have a significant impact on the penicillin acylation rate.

Protein purification, crystallization, data collection, structure solution, and refinement

The expression construct, pET22b-01085, encoding residues 32-554 (with a C-terminal hexa-histidine tag) was used to transform Escherichia coli strain C41 (DE3). pET22b- 01085 was used to over-express 1085 in BL21 (C41 ) cells grown in Autolnduction Media. Over-expressed cells were lyzed in 50 mM Phosphate-Buffered Saline (PBS) pH 7.4; 250 mM NaCI; 10 % Glycerol; 0.01 % Triton X-100 and 0.05 mM EDTA. Preliminary purification of the hexa-Histidine tagged 1085 was purified using a two-step purification process of Ni-NTA-agarose column (using PBS pH 7.4; 250 mM NaCI; 10 % Glycerol) followed by size exclusion chromatography on a Superdex 200 16/60 (in 20 mM Tris pH 7.5; 150 mM NaCI) using a AKTAxpress high throughput chromatography system

(http://proteinexpress.med.monash.eud.au/index.htm).

Fractions containing purified 1085 were concentrated to 7 mg/ml_ prior to crystallization. Crystals of 01085 were obtained by the hanging drop method 0.2M tri-sodium citrate and 20 % PEG3350. Single crystals were flash cooled in liquid nitrogen. Diffraction data was collected at 100 K using synchrotron radiation on the MX2 beamline (3ID1 ) at the Australian synchrotron. Data was processed using XDS (Kabsch W (2010) XDS. Acta Crystallogr D66: 125-132) and scaled using AIMLESS (Evans (201 1 ) Acta Crystallogr D Biol Crystallogr 67: 282-292) from the CCP4 suite (CCP4 (1994) Acta Crystallogr D50: 760-763). Crystallographic parameters and data collection statistics are provided in Table 1 . Initial phases were obtained by the molecular replacement method using the program PHASER (McCoy et al., (2005) Acta Crystallogr D Biol Crystallogr 61 : 458- 464) using chain A of 3EQU as a search model (Bompard-Gilles et al. (2000) Structure 8: 153-162). The molecular replacement search model was prepared from the C- terminal domain from the structure of the penicillin binding protein 2 from Neisseria gonorrhoeae (PDB ID 3EQU, chain A) using chainsaw (Stein (2008) Journal of Applied Crystallography 41 : 641 -643.) to prune non-conserved residues (maintained all atoms common to the target and model residues). A single peak in the rotation and translation function was evident. Early structure refinement and model building was performed using REFMAC and Coot (Emsleyand Cowtan K (2004) Acta Crystallogr D Biol Crystallogr 60: 2126-2132.). After the initial C-a backbone trace was completed, maximum likelihood refinement using Phenix (Krissinel and, Henrick K (2007) J Mol Biol 372: 774-797.), incorporating translation, liberation and screw-rotation displacement (TLS) refinement was carried out. All model building and structural validation was performed using COOT (Chung et al. (201 1 ). PLoS ONE., 6(3): e181 19.). Solvent molecules were added only if they had acceptable hydrogen-bonding geometry contacts of 2.5 to 3.5 A with protein atoms or with existing solvent and were in good 2F ₀ -F _C and Fo-Fc electron density.

The protein-protein interface analysis was performed using PDBePISA (Sorg and Dineen (2009) Current protocols in microbiology. Chapter 9:Unit9A 1 ). All crystal structure figures were prepared in the Pymol Molecular Graphics System, version 1.3r2 (Schrodinger, LLC).

6. Human plasminogen (hPLG) binding to C. difficile spores: hPLG exacerbates C difficile infection and symptoms.

Disease is exacerbated in the C. difficile model when human PLG is also present in vivo.

Without being bound by any theory, human PLG in the context of a C. difficile infection alters the immune response and degrades tissue components to potentiate bacterial spread. Accordingly, reduction of spores equates to a reduction in disease severity, transmission and recurrence.

Immunofluorescence (IF) using hPLG primary antibodies and an anti-mouse Alexa 488 secondary antibody (green) was used to visualise hPLG binding to M7404 spores. The bright field image (Figure 8A), hPLG labeled image (hPLG Ab) (green) (Figure 8B) and the overlapped images shows the association of hPLG with M7404 spores (Figure 8C). Stimulated emission depletion (STED) microscopy of horizontal (Figure 8D, E, F) and vertical (Figure 8G, H, I) transverse sections of M7404 spores stained using an anti-spore antibody (green) and an anti-hPLG antibody (red) shows an association of hPLG with M7404 spores. Western blot showing hPLG binding to C. difficile spores from diverse geographical locations and origins (M7404 Canadian human epidemic isolate (E1 ); R20291 UK human epidemic isolate (E2); JGS6133 US animal isolate (A-US); A135 Australian animal isolate (A-AU); DLL3109 Australian human epidemic isolate (E-AU); VPI10463 US human reference isolate (R) and CD37 US non-toxigenic isolate (NT) detected using an anti-human PLG Ab (J).

Method

PLG binding via Immunofluorescence

1 x 10 ⁹ C. difficile M7404 spores (isolated as per Law et al., (2012), Cell Rep., 1 (3): 185-90) were incubated for 1 .5 hours at room temperature (RT) with 10 g of commercially acquired hPLG (Bansia Scientific) or hPLG that was purified from human plasma using previously described methods (Carter et al., (2015) mBio 8, e00551 ). Spores were then washed 5 times in binding buffer (50 mM Tris, pH 7.5, 100 mM NaCI, and 2 mM MgC ) as previously described by Chung et al, 201 1 . Spores were then incubated with 5% skim milk in TBS (5 mM Tris-HCI, 15 mM NaCI, pH 7.4) for 1 - 2 hrs and then washed three times in TBS buffer. Washed spores were then incubated with 2 pg/ml a-human Plasmin(ogen) antibody (MAB2596; R&D systems) overnight at 4°C. The spores were again washed 3 times in TBS buffer, followed by incubation in the dark with a 1 :50 dilution of 2 mg/ml Alexa Flour 488 goat a-mouse IgG (H + L) (Life Technologies) for 45 mins at RT. After a final 3 washes in TBS buffer the spores were resuspended in TBS buffer, mounted onto poly-L-lysine slides and imaged on an Olympus BX-51 attached to an Olympus power unit U-RFL-T.

PLG binding via super resolution (STED)

Human PLG was labelled with Alexa Fluor 647 NHS ester (Invitrogen, A20006) as per manufacturer's instructions and eluted using a NAP-5 column (GE Healthcare, 17-0853-02). C. difficile spores were then bound with 10 g of the fluorescently labelled hPLG, as described above, washed 3 times with binding buffer, and then incubated overnight at 4 °C with an anti-C. difficile whole spore antibody (produced in house) that had been labelled with either Atto-488 NHS ester (Invitrogen, 41698) or Alexa Fluor 568 NHS ester (Invitrogen, A20003). The labelled spores were again washed 3 times in binding buffer, mounted onto poly-L-lysine coated coverslips (No.1.5H, Zeiss) and imaged using a stimulated emission depletion (STED; Abberior Instruments GmbH, Gottingen, Germany) microscope equipped with an Olympus 100x oil objective (UPlanSApo NA = 1.4) with a 1 watt 775 nm pulsed STED laser. Image acquisition was performed in RESCue (REduction of State transition Cycles) mode with a 775 STED laser power of 6% and confocal laser power for 640 nm, 561 nm and 488 nm of 25%, 60% and 20% respectively.

Unlabel!ed spores and spores bound to unlabelled hPLG were included as negative controls.

PLG binding via Western blot analysis

Detection of hPLG bound to M7404 spores was performed as described in [36] with the following modification. Following binding of hPLG to 1 x 10 ⁶ spores and 5 washes in binding buffer (as described above), bound proteins were eluted using 3 M potassium thiocyanate (Sigma). The 5 ^th wash was kept as a wash control to show that only bound hPLG was present in our test samples. Eluted hPLG was then separated on a 12% SDS-PAGE gel and analysed via Western blot analysis using 1 - 2 pg/ml a- human Plasmin(ogen) antibody and detected using a goat a-mouse IgG (H + L) HRP conjugate (Millipore).

Results:

C. difficile spores are important for disease transmission and recurrences. The data presented here shows that spores interact with the human protein plasminogen. This is the first time that C. difficile spores have been shown to directly interact with any host proteins, in this case by the acquisition of a host protease onto their surface. Reducing C. difficile sporulation within the infected host therefore reduces interaction with the host protein plasminogen and in turn seeks to counter the exacerbation of C. difficile infection regulated by hPLG.

8. Enhanced virulence effects of hPLG on C. difficile M7404 infected mice.

The survival time of C57BL/6J mice (expressing mouse PLG) and HPLG mice (expressing both human and mouse PLG) that were either uninfected or infected with C. difficile M7404 was determined (Figure 9A). Survival time of C57BL/6J mice (expressing mouse PLG) and PLG KO (not expressing mouse PLG) that were either uninfected or infected with C. difficile M7404 was also determined (Figure 9B). Data is pooled from three independent mouse trials. Error bars represent the mean ± SEM of n=5-20 mice. ^**** indicates P < .0001 .

Method

Animal handling and experimentation was performed in accordance with Victorian State Government regulations and approved by the Monash University Animal Ethics Committee (AEC no. SOBSB/M/2010/25 and MARP/2014/136). Male, 6-7-week- old, C57BL/6J mice (Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia) were pre-treated with an antibiotic cocktail in the drinking water containing kanamycin (0.4 mg/ml; Amresco), gentamicin (0.035 mg/ml; Sigma), colistin (850 U/ml; Sigma), metronidazole (0.215 mg/ml; Sigma), vancomycin (0.045 mg/ml; Sigma) and cefaclor (0.3 mg/ml; Sigma) for 7 days, followed by three days of cefaclor alone. On the day of infection, antibiotic treatment was ceased and mice were infected with C. difficile M7404 (10 ⁶ spores/mouse) by oral gavage. Mice were monitored twice daily for disease signs (weight loss, behavioural and physical changes, and diarrhea). Faecal pellets were collected 24 hours post-infection and resuspended in PBS (100 mg/ml_), heat shocked (30 minutes, 65°C) and plated for spore enumeration. Animals were humanely killed by CO ₂ overdose or cervical dislocation when defined humane endpoints were met. Survival curves were assessed for statistical significance using a log-rank (Mantel-Cox) test. Differences in data values were considered significant at a P value of < .05.

Results

It was shown that spores from C. difficile bind the human host protein, plasminogen (hPLG), a potent protease, to likely invade host tissues and alter the immune response as a potential host evasion mechanism. The data clearly shows that when hPLG is present in the context of a C. difficile infection, disease is exacerbated. Furthermore proteins required to maintain tissue integrity are markedly reduced and the immune response altered under these conditions.

Spores are traditionally thought of as dormant particles responsible only for disease initiation. Here a new virulence mechanism is provided for spores indicating that they play an active role in virulence during infection and are likely to contribute to disease transmission and recurrence. These data very importantly also show that targeting C. difficile spores is envisaged to help to reduce disease symptoms and severity, transmission and recurrence in the hospital and community setting.

9. Co-administration of cefotetan and vancomycin prevents recurrent CDI.

C. difficile spores likely play an important in disease recurrences. The C. difficile recurrence model was adapted from (Hutton et al., (2017) Sci Rep 7, 3665 (2017).

Method

Mice were pre-treated with antibiotics and then infected with 108 M7404 vegetative cells. Eight hours following infection, all mice were administered 100 μΙ of vancomycin (6 mg/ml_) by oral gavage and then given access ad libitum to either vancomycin alone (0.4 mg/ml_), a combination of vancomycin (0.4 mg/ml_) and cefotetan (30 Mg/mL), or a combination of vancomycin (0.4 mg/ml_) and cefotetan (50 pg/mL), administered in the drinking water. Another group of mice were orally gavaged with 100 μΙ of fidaxomicin (0.6 mg/ml_), twice daily beginning 8 hrs post-infection and administered every 12 hours thereafter.

Mice were monitored daily for signs of infection and faecal samples were enumerated for the presence of spores. All treatments were administered until spore shedding could no longer be detected. Once levels of C. difficile in the faeces reached undetectable levels, vancomycin treatment ceased (day 10 or day 1 1 ), mice were separated and housed individually and administered plain drinking water daily for the trial duration (up to 21 days). Mice were weighed and faecal samples collected daily to detect spore shedding and disease relapse. Mice were humanely euthanised according to animal ethics guidelines if they lost 10% body weight in 24 hours or met other disease criteria as previously defined (Carter et aL , (2015) mBio 6, e00551 ).

Results

Infected mice were orally treated with vancomycin, fidaxomicin or a combination of vancomycin and cefotetan (30 pg/mL or 50 pg/mL) until spore shedding could no longer be detected. After treatments ceased (Day 0), mice were monitored daily for (Figure 10A) survival and (Figure 10B) weight loss. Weight loss is presented as the % weight relative to the day before, with each point representing a single mouse.

Faecal spore load (Figure 10C) was determined daily after cessation of treatments and is presented as cfu/gram faeces (Iog10). N = 9-10 mice per group. The Kaplan-Meier survival curve was assessed using a log-rank (Mantel-Cox) test ^**** P < 0.0001.

Discussion

Mice receiving vancomycin alone succumbed to recurrent disease 4-5 days after vancomycin cessation (Fig. 10). By contrast, mice co-treated with 30 pg/mL cefotetan showed a significant delay in disease recurrence and succumbed to disease 7.5-8.5 days after treatment cessation (P<0.0001 ). Severe diarrhoea and weight loss was seen in both these groups on the day that the mice relapsed (Fig. 10B). Importantly, all mice that were co-treated with vancomycin and 50 pg/mL cefotetan did not relapse and survived to the end of the trial (21 days antibiotic cessation) (Fig. 10a; P<0.0001 ). A similar result was observed for fidaxomicin treated mice (Fig. 10a; P<0.0001 ). In both treatment groups weights remained stable (Fig. 10b).

This is the first time a prevention in CDI recurrence, with the co-administration of two-antibiotics or fidaxomicin treatment, has been shown. Interestingly, mice from both surviving groups began shedding spores in the faeces 7 days post-antibiotic cessation (Fig. 10c), ), however, neither group developed any other signs of disease, suggesting that the disease susceptibility period induced by antibiotic treatment had passed, perhaps through the restoration of the gut microbiota, despite the mice being colonised with C. difficile.