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US 2017.007 1884A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0071884 A1 Page et al. (43) Pub. Date: Mar. 16, 2017

(54) METHODS FOR TREATING BACTERIAL (30) Foreign Application Priority Data May 1, 2013 (AU) ...... 201390.1517 (71) Applicant: Neoculi Pty Ltd., Burwood (AU) Publication Classification (72) Inventors: Stephen Page, Newton (AU); Sanjay (51) Int. Cl Garg, Adelaide (NZ) A6II 3/55 (2006.01) A6IR 9/00 (2006.01) (21) Appl. No.: 15/363.523 (52) U.S. Cl. CPC ...... A61K 31/155 (2013.01); A61K 9/0014 (22) Filed: Nov. 29, 2016 (2013.01); A61K 9/0053 (2013.01) (57) ABSTRACT Related U.S. Application Data The invention comprises methods for treating and prevent (63) Continuation of application No. 14/888.289, filed on ing a bacterial in a Subject, methods for preparing Oct. 30, 2015, now Pat. No. 9,539,223, filed as a for use in treating and preventing a bacterial application No. PCT/AU2014/000480 on May 1, infection in a subject, and pharmaceutical and Veterinary 2014. antibacterial compositions when used therein. Patent Application Publication Mar. 16, 2017. Sheet 1 of 31 US 2017/0071884 A1

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METHODS FOR TREATING BACTERIAL Soft tissue lesions as well as a rapid, fulminating, necrotizing INFECTIONS with significant associated mortality. Recently MRSA strains have become host-adapted in several key TECHNICAL FIELD animal including livestock, horses and companion 0001. This invention relates to methods of treating and animals and regular cases of human-to-animal and animal preventing a bacterial infection in a subject, methods for to-human transfer are being documented. This has important preparing a medicament for use in treating and preventing a consequences for strain transmission and public health. A bacterial infection in a Subject, and pharmaceutical and recent survey of 751 Australian veterinarians for MRSA nasal carriage found that a remarkable 21.4% of equine veterinary antibacterial compositions when used therein. veterinarians were MRSA-positive compared to 4.9% of BACKGROUND ART small animal veterinarians and 0.9% of veterinarians with little animal contact. These ecological shifts of MRSA 0002. A marked increase in prevalence of multi-drug together with the emergence of resistance to new drugs resistance in disease-causing Gram-positive (G+ve) developed specifically for MRSA such as confirm ( aureus, Enterococcus spp. and Streptococ that new MRSA anti-infectives are urgently needed. Fur cus pneumoniae) and Gram negative (G-ve) thermore, hospitals that use for treating MRSA (, Enterobacter spp., Salmonella spp., then have to contend with outbreaks of Vancomycin-resistant Acinetobacter baumanni, and enterococci (VRE) infections in their patients, once again ) has coincided with an unprec with limited alternative choices. edented global decline in investment in new anti-infective drugs. There are few currently registered alternatives for 0006. The global emergence and spread within the com multidrug resistant (MDR) bacterial infections, forcing cli munity of highly virulent MDR Gram-negative (G-ve) nicians to consider older generation drugs such as bacteria such as E. coli O25b:ST131 confirms that bacterial with narrow spectrum and considerable potential for toxic pathogens can simultaneously evolve both virulence and side-effects. In addition, there are fewer novel classes of resistance determinants. Echoing recent MRSA epidemiol antiinfective therapeutics moving through the drug devel ogy, E. coli O25b:ST131, a major cause of urinary tract and opment pipeline. bloodstream infections in humans, has now been isolated 0003. Since the year 2000, a period of almost 15 years, from extraintestinal infections in companion animals, and only 5 novel mode of action (MOA) antibacterial agents poultry. The increasing significance of E. coli O25b:ST131 have been approved by the US FDA linezolid (an oxazo and other MDR with combined resis lidinone) in 2000, (a lipopeptide) in 2003, tance to fluoroquinolones and extended spectrum beta (a ) in 2007, (a lactams and is another worrying trend, espe macrollide tiacumicin) in 2011, and bedaquiline (a diaryldui cially considering there have been few recent breakthroughs noline) in 2012. Notably, none of these agents has significant in the development of G-ve spectrum anti-infectives apart activity against gram negative bacteria. No novel MOA from incremental advances in the family. antibacterial agents were approved in 2013 and to date in 0007. The World Health Organisation has identified anti 2014 only and , both analogs of exist biotic resistance as one of the three major future threats to ing classes, have been recommended for approval in the US. global health. A recent report from the US Centers for While there are more than 300 anti-infective medicines in Disease Control and Prevention (CDC) estimated that “in various stages of development, the large majority of these the United States, more than two million people are sickened medicines are previously approved antibacterial compounds every year with -resistant infections, with at least or their derivatives that are undergoing studies for new 23,000 dying as a result. The extra medical costs, in the Indications. USA alone, associated with treating and managing a single 0004 Furthermore, the prevalence of multidrug-resis case of antibiotic-resistant infection are estimated to be tance in animal-specific pathogens together with greater between USS18,588 and US$29,069 per year resulting in an regulation of the registration and usage of in overall direct cost to the US health system of over USS20 animals, has caused veterinarians to become increasingly billion annually. In addition, the cost to US households in reliant on the traditional classes of antimicrobial agents. The terms of lost productivity is estimated at over USS35 billion risk of transfer of MDR Zoonotic organisms from animals to per annum. Twenty five thousand patients in the European humans has also led to calls for further restrictions on the Union (EU) still die annually from infection with MDR usage of some recently registered antibacterial drugs such as bacteria despite many EU countries having world’s best the fluoroquinolones and the third and fourth generation practice hospital Surveillance and infection control strate . gies. The EU costs from health care expenses and lost productivity associated with MDR infections are estimated Epidemiology of Antibacterial Resistance Development in to be at least € 1.5 billion per year. Pathogens of Humans and Animals 0008. There is an unmet clinical need for antibacterial 0005 Much of the evolution in resistance development is agents with novel mechanisms of action to supplement and driven by changes in the epidemiology of key MDR organ replace currently available antibacterial agents, the efficacy isms. Once only restricted to human hospitals and aged care of which is increasingly undermined by antibacterial resis facilities, resistant tance mechanisms. There remains a need for alternative (MRSA) strains are now being isolated from the community antibacterials in the treatment of infection by multi-resistant in alarming proportions. Furthermore, community-acquired bacteria. However, as reported by the Infectious Diseases MRSA strains are more likely to carry the Panton-Valentine Society of America and the European Centre for Disease leukocidin (PVL) toxin, a linked to skin and Control and Prevention, few new drugs are being developed US 2017/007 1884 A1 Mar. 16, 2017

that offer promising results over existing treatments (Infec not limited to: less than 200 ug/mL after 12 hours; less than tious Diseases Society of America 2010, Clinical Infectious 5ug/mL after 24 hours; less than 1 ug/L after 48 hours and Diseases, 50(8): 1081-1083). less than 0.5ug/mL after 72 hours. 0009. It is an object of the present invention to overcome 0017. The agent causing the bacterial infection is a bac at least one of the failings of the prior art. terial agent. In one preferred embodiment, the agent is not a 0010. The discussion of the background art set out above protozoan species. In one preferred embodiment, the agent is intended to facilitate an understanding of the present is not a coccidian protozoan. More preferably, the agent is invention only. The discussion is not an acknowledgement not Clostridium perfringens nor a heterotrophic bacterial or admission that any of the material referred to is or was species present in Soil samples collected by Hansen et al part of the common general knowledge as at the priority date from Jyndevad Denmark as discussed in the following of the application. papers: Hansen et al. 2012, Chemosphere, 86:212-215; and Hansen et al. 2009, Environmental Pollution 157:474-480. SUMMARY OF INVENTION 0018. In another embodiment, the bacterial agent is gram 0011. According to one aspect of the invention, there is negative. In another embodiment, the bacterial agent is gram provided a method of treating or preventing a bacterial positive. In another embodiment, the bacterial agent has no colonisation or infection in a Subject, the method comprising . In another embodiment, the bacterial infection is the step of administering a therapeutically effective amount caused by a mixture of at least two agents selected from the of robenidine, or a therapeutically acceptable salt thereof, to group consisting of gram negative, gram positive and the Subject. In this aspect, the bacterial colonisation or bacterial agents with no cell wall. infection is caused by a bacterial agent. 0019. The bacterial agent causing the bacterial infection 0012. According to another aspect of the invention, there may be a gram positive bacterial agent selected from the is provided the use of robenidine, or a therapeutically group comprising, but not limited to, Staphylococcus spp. acceptable salt thereof, in the manufacture of medicament Streptococci, Enterococcus spp., Leuconostoc spp., Coryne for the treatment of a bacterial colonisation or infection in a bacterium spp., Arcanobacteria spp., Trueperella spp., Rho Subject. In this aspect, the bacterial colonisation or infection dococcus spp., spp., Anaerobic Cocci, Anaerobic is caused by a bacterial agent. Gram-Positive Nonsporulating , Actinomyces spp. 0013 The subject may be any subject capable of coloni Clostridium spp., Nocardia spp., Erysipelothrix spp., Listeria sation or infection by bacteria. The Subject may be mam spp. Kytococcus spp., spp. Ureaplasma spp. malian, or may be piscine or avian. Preferably, the subject is and Mycobacterium spp. selected from the group comprising, but not limited to, 0020. In one embodiment, the bacterial agent is gram human, canine, feline, bovine, ovine, caprine, other rumi positive and selected from the group comprising, but not nant species, porcine, equine, avian, or piscine. limited to Staphylococcus spp. Examples of Staphylococcus 0014. As used herein, the term robenidine, (also known spp include Staphylococcus epidermidis, Staphylococcus as 1,2-bis(E)-(4-chlorophenyl)methylideneaminoguani haemolyticus, Staphylococcus lugdunensis, Staphylococcus dine, or, as described by this specification, NCL812) refers Saprophyticus, Staphylococcus auricularis, Staphylococcus to a compound having the following chemical structure: capitis, Staphylococcus Caprae, Staphylococcus Carnosus, Staphylococcus cohnii, Staphylococcus hominis, Staphyllo coccus pasteuri, Staphylococcus pettenkoferi, Staphylococ H cus pulvereri, Staphylococcus saccharolyticus, Staphylococ S- N n 4 cus simulans, Staphylococcus Schleiferi, Staphylococcus warneri, Staphylococcus xylosus, Staphylococcus arlettae, NH Staphylococcus caseolyticus, Staphylococcus chromogenes, C C Staphylococcus condimenti, Staphylococcus delphini, Staphylococcus equorum, Staphylococcus felis, Staphyllo 0015 The robenidine may be administered to the subject coccus fleurettii, Staphylococcus gallinarum, Staphylococ in a dose selected from the group comprising 0.1 mg/kg to cus hyicus, Staphylococcus intermedius, Staphylococcus 250 mg/kg body weight, preferably 1 mg/kg to 100 mg/kg kloosii, Staphylococcus lentus, Staphylococcus lutrae, body weight, and more preferably 5 mg/kg to 50 mg/kg body Staphylococcus muscae, Staphylococcus nepalensis, weight. The robenidine may be administered to the subject Staphylococcus piscifermentans, Staphylococcus pseudin using a dosing schedule selected from the group consisting termedius, Staphylococcus Sciuri, Staphylococcus simiae, of hourly, 3 times daily; twice daily; daily; every second Staphylococcus succinus, and Staphylococcus vitulinus. day; twice weekly; once weekly; once fortnightly; once 0021. In another embodiment, the bacterial agent is gram monthly; once every two months or by constant rate or positive and is selected from the group comprising, but not variable rate infusion. Preferably, the robenidine is admin limited to, Streptococcus spp. Examples of Streptococcus istered until colonisation or the signs and symptoms of spp include Streptococcus agalactiae, Streptococcus alac infection have at least been partially treated or alleviated. tolyticus, Streptococcus anginosus, Streptococcus canis, 0016. In one embodiment, the concentration of robeni Streptococcus constellatus, Streptococcus Cricetus, Strepto dine (or a robenidine metabolite) in the subjects blood after coccus cristatus, Streptococcus downed, Streptococcus dys treatment is within a range selected from the group com galactiae Subsp. dysgalactiae, Streptococcus dysgalactiae prising, but not limited to: between 0.1 and 10 ug/mL at 2 Subsp. equisimilis, Streptococcus equi Subsp. equi, Strepto hours, 1 and 200 ug/mL after 12 hours; between 0.1 and 5 coccus equi Subsp. Zooepidemicus, Streptococcus ferus, ug/mL after 24 hrs; between 0.01 and 2 ug/mL after 48 Streptococcus gallolyticus Subsp. gallolyticus (formerly hours; between 0.0001 and 1 ug/mL after 72 hrs. Preferably, Streptococcus bovis biotype i), Streptococcus gallolyticus the concentration is selected from the group comprising, but Subsp. pasteurianus (formerly Streptococcus bovis biotype US 2017/007 1884 A1 Mar. 16, 2017

ii/2), Streptococcus gordonii, Streptococcus hyointestinalis, 0025. In another embodiment, the bacterial agent is gram Streptococcus hyovaginalis, Streptococcus infantarius, positive and selected from the group comprising, but not Streptococcus infantarius Subsp infantarius, Streptococcus limited to, Arcanobacteria spp. Examples of Arcanobacteria infantis, Streptococcus iniae, Streptococcus intermedius, spp include A. haemolyticum, A. pyogenes (now known as Streptococcus lutetiensis (formerly Streptococcus bovis bio Trueperella pyogenes, originally known as Actinomyces type ii.1), Streptococcus macaccae, Streptococcus mitis, pyogenes), and A. bemardiae. Streptococcus mutans, Streptococcus oralis, Streptococcus 0026. In another embodiment, the bacterial agent is gram Orisratti, Streptococcus parasanguinis, Streptococcus pero positive and selected from the group comprising, but not ris, Streptococcus pneumoniae, Streptococcus porcinus, limited to, Rhodococcus spp. Examples of Rhodococcus spp Streptococcus pseudintermedius, Streptococcus pyogenes, include Rhodococcus equi, Rhodococcus erythropolis, Rho Streptococcus ratti, Streptococcus salivarius, Streptococcus dococcus fasciens, and Rhodococcus rhodochirous. sanguinis, Streptococcus sobrinus, Streptococcus suis, 0027. In another embodiment, the bacterial agent is gram Streptococcus thermophilus, Streptococcus vestibularis, and positive and selected from the group comprising, but not Nutritionally Variant (Deficient) Streptococci (Abiotrophia limited to, Gordonia spp. defectiva, Granulicatella adiacens, Granulicatella elegans, 0028. In another embodiment, the bacterial agent is gram and Granulicatella para-adiacens) and related species Such positive and selected from the group comprising, but not as Rothia mucilaginosa (formerly Stomatococcus mucilagi limited to, Tsukamurella spp. nosus) and Pediococcus. 0029. In another embodiment, the bacterial agent is gram 0022. In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not positive and selected from the group comprising, but not limited to, Acholeplasma spp. limited to, Enterococcus spp. Examples of Enterococcus spp 0030. In another embodiment, the bacterial agent is gram include Enterococcus faecalis, Enterococcus faecium, positive and selected from the group comprising, but not Enterococcus gallinarum, Enterococcus durans, Enterococ limited to, Such as Crossiella equi. cus avium, Enterococcus raffinosus, Enterococcus pallens, 0031. In another embodiment, the bacterial agent is gram Enterococcus gilvus, Enterococcus Cecorum, Enterococcus positive and selected from the group comprising, but not malodoratus, Enterococcus italicus, Enterococcus sanguini limited to, Bacillus spp. Examples of Bacillus spp include cola, Enterococcus mundtii, Enterococcus cassellifavus/fla Bacillus anthracis, Bacillus cereus, Bacillus circulans, vescens, Enterococcus dispar, Enterococcus hirae, Entero Bacillus licheniformis, Bacillus megaterium, Bacillus pumi coccus pseudoavium, and Enterococcus bovis. lus, Bacillus sphaericus, Bacillus subtilis, Brevibacillus bre 0023. In another embodiment, the bacterial agent is gram vis, Brevibacillus laterosporus, and Paenibacillus alvei. positive and selected from the group comprising, but not 0032. In another embodiment, the bacterial agent is gram limited to, Leuconostoc spp. Examples of Leuconostoc spp positive and selected from the group comprising, but not include Leuconostoc mesenteroides, Leuconostoc limited to, Anaerobic Cocci. Examples of Anaerobic Cocci pseudomesenteroides, Leuconostoc paramesenteroides, include Anaerococcus murdochi, Anaerococcus previotii, Leuconostoc citreum, and Leuconostoc lactis. Anaerococcus tetradius, Anaerococcus octavius, Anaero 0024. In another embodiment, the bacterial agent is gram coccus hydrogenalis, Anaerococcus lactolyticus, Anaero positive and selected from the group comprising, but not coccus vaginalis, Atopobium parvulum, Finegoldia magna, limited to, Corynebacterium spp. Examples of Corynebac Gallicola barnesae, Gemella asaccharolytica, Gemella terium spp include nonlipophilic, fermentative Corynebac bergeri, Gemella cuniculi, Gemella haemolysans, Gemella terium spp. Such as Corynebacterium ulcerans, Corynebac morbillorum, Gemella palaticanis, Gemella sanguinis, Par terium pseudotuberculosis, Corynebacterium xerosis, vimonas micra, Peptococcus niger, Peptoniphilus asaccha Corynebacterum striatum, Corynebacterium minutissimum, rolyticus, Peptoniphilus gorbachii, Peptoniphilus indolicus, Corynebacterium amycolatum, Corynebacterium glu Peptoniphilus harei, Peptoniphilus ivorii, Peptoniphilus lac curonolyticum, Corynebacterium argentoratense, Coryne rimalis, Peptoniphilus Olsenii, Peptostreptococcus stomatis, bacterium matriuchotii, Corynebacterium riegelii, Coryne Peptostreptococcus anaerobius, Ruminococcus productus, bacterium confilsum, Corynebacterium cystidis, Slackia heliotrinireducens, and Staphylococcus saccharo Corynebacterium diphtheria, Corynebacterium simulans, lyticus. Corynebacterium sundvallense, Corynebacterium thoms 0033. In another embodiment, the bacterial agent is gram sensii, Corynebacterium frenevi, and Corynebacterium positive and selected from the group comprising, but not aurimucosum, nonlipophilic, nonfermentative Corynebacte limited to, Anaerobic Gram-Positive Nonsporulating Bacilli. rium spp. Such as Corynebacterium afermentans afermen Examples of Anaerobic Gram-Positive Nonsporulating tans, Corynebacterium auris, Corynebacterium pseudodiph Bacilli include Alloscardovia Omnicolens, Atopobium spe theriticum, and Corynebacterium propinquium and lipophilic cies (such as Atopobium minutum, Atopobium rimae, Atopo Corynebacterium spp Such as Corynebacterium jeikeium, bium parvulum, and Atopobium vaginae), Bifidobacteria Corynebacterium urealyticum, Corynebacterium afermen (such as Bifidobacteria adolescentis, Bifidobacteria den tans lipophium, Corynebacterium accolens, Corynebacte tium, Bifidobacteria scardovii), Catabacter hongkongensis, rium macginleyi, Corynebacterium tuberculostearum, Collinsella aerofaciens, Eggerthella (Such as Eggerthella Corynebacterium kroppenstedtii, Corynebacterium lenta, Eggerthella hongkongensis and Eggerthella Sinensis), kutscheri, Corynebacterium pilosum, Corynebacterium Eubacterium and related species (such as Eubacterium bovis, CDC coryneform groups F-1 and G, and Corynebac modatum, Eubacterium tenue, Eubacterium brachy, Eubac terium lipophiloflavum, and other Corynebacterium spp terium infirmum, Eubacterium minutum, Eubacterium noda such as Turicella, Arthrobacter, Brevibacterium, Derm tum, Eubacterium saphenum, Eubacterium sulci, Filifactor abacter; Rothia, Oerskovia, Microbacterium, and Leifsonia alocis, Mogibacterium timidum, Mogibacterium vescum, aquatica. Pseudoramibacter alactolyticus, Bulleidia extructa, and US 2017/007 1884 A1 Mar. 16, 2017

Solobacterium moorei), Lactobacillus species (such as Lac bacterium genavense, Mycobacterium goodii, tobacillus rhamnosus, Lactobacillus casei, Lactobacillus Mycobacterium haemophilum, Mycobacterium heckesho fermentum, Lactobacillus gassen, Lactobacillus plantarum, mense, Mycobacterium heidelbergense, Mycobacterium Lactobacillus acidophilus, Lactobacillus iners and Lacto houstonense, Mycobacterium immunogenium, Mycobacte bacillus ultunensis), Mobiluncus species (such as Mobilum rium interjectum, Mycobacterium intracellulare, Mycobac cus curtisii, Mobiluncus mulieris), Morvella indoligenes, terium Senegalense, Mycobacterium africanum, Mycobac Olsenella oral species (such as Olsenella uli and Olsenella terium avium Subsp paratuberculosis, Mycobacterium profitse), Oribacterium sinus, Propionibacterium (Such as kansasii, Mycobacterium lacus, Mycobacterium lentiflavum, Propionibacterium acnes and Propionibacterium propioni Mycobacterium leprae, Mycobacterium lepraemurium, cum), Slackia exigua, and Turicibacter sanguine. Mycobacterium mageritense, Mycobacterium malmoense, 0034. In another embodiment, the bacterial agent is gram Mycobacterium marinum, Mycobacterium massiliense, positive and selected from the group comprising, but not Mycobacterium microti, Mycobacterium montefiorense limited to, Actinomyces spp. Examples of Actinomyces spp (eels), Mycobacterium moracense, Mycobacterium muco include Actinomyces israelii, Actinomyces naeslundii, genicum, Mycobacterium nebraskense, Mycobacterium neo Actinomyces viscosus, Actinomyces Odontolyticus, Actino aurum, Mycobacterium novocastrense, Mycobacterium myces meyeri, and Actinomyces gerencseriae (formerly palustre, Mycobacterium parmense, Mycobacterium phlei, Actinomyces israeli serotype II), Actinomyces europaeus, Mycobacterium phocaicum, Mycobacterium pinnipedii, Actinomyces neuii, Actinomyces radingae, Actinomyces Mycobacterium porcinum, Mycobacterium pseudoshottsii graevenitzii, Actinomyces hordeovulneris, Actinomyces turi (fish), Mycobacterium pseudotuberculosis, Mycobacterium censis, Actinomyces georgiae, Arcanobacterium (Actinomy Saskatchewanense, Mycobacterium scrofulaceum, Myco ces) pyogenes, Arcanobacterium (Actinomyces) bemardiae, bacterium senuense, Mycobacterium septicum, Mycobacte Actinomyces funkei, Actinomyces lingnae, Actinomyces hou rium simiae, Mycobacterium Smegmatis, Mycobacterium stonensis, and Actinomyces cardiffensis. Szulgai, Mycobacterium terrae/chromogenicum complex, 0035. In another embodiment, the bacterial agent is gram Mycobacterium triplex, Mycobacterium tuberculosis, Myco positive and selected from the group comprising, but not bacterium tusciae, Mycobacterium ulcerans, Mycobacte limited to, Clostridium spp. Examples of Clostridium spp rium wolinskyi, and Mycobacterium xenopi. include Clostridium baratii, Clostridium bifermentans, 0041. In another embodiment, the bacterial agent is gram Clostridium botulinum, Clostridium botulinum (types A, B, positive and selected from the group comprising, but not C, D, E, F, G), Clostridium butyricum, Clostridium difficile, limited to, Trueperella spp. Examples of Trueperella spp Clostridium histolyticum, Clostridium novyi (type A), include Trueperella abortisuis, Trueperella bemardiae, Clostridium novyi (type B), Clostridium perfiringens, Trueperella biallowiezensis, Trueperella bomasi, Trueperella Clostridium perfingens (types A-E), Clostridium raemo pyogenes (Arcanobacterium pyogenes). sum, Clostridium septicum, Clostridium sordelli, 0042. In another embodiment, the bacterial agent is gram Clostridium sphenoides, Clostridium tedium, and positive, gram negative or does not have a cell wall and Clostridium tetani. selected from the group comprising, but not limited to, 0036. In another embodiment, the bacterial agent is gram livestock pathogens. Examples of livestock pathogens positive and selected from the group comprising, but not include Actinobaculum suis, Actinomyces bovis, Arcanobac limited to, Nocardia spp. Examples of Nocardia spp include terium pyogenes, Bacillus anthracis, cereus, licheniformis, Nocardia asteroides, Nocardia brasiliensis, Nocardia farci pumilus, melaninogenicus, subtilis, Clostridium botulinum, nica, Nocardia nova, Nocardia Otitidiscaviarum, and Nocar chauvoei, haemolyticum, novyi, perfingens, septicum, sor dia transvalensis. dellii, tetani, colinum, Corynebacterium pseudotuberculo 0037. In another embodiment, the bacterial agent is gram sis, renale, Dermatophilus congolensis, Enterococcus spp positive and selected from the group comprising, but not (such as E. faecalis, E. faecium, E. durans, E. avium, E. limited to, Erysipelothrix spp., Such as Erysipelothrix rhu hirae), Erysipelothrix rhusiopathiae, Listeria ivanovii, Siopathiae. gravi, innocua, seeligeri, wellshimeri, monocytogenes, 0038. In another embodiment, the bacterial agent is gram Mycobacterium avium, bovis, paratuberculosis (Johne's positive and selected from the group comprising, but not Disease), Mycoplasma (Such as Capricolum Subsp. caprip limited to, Listeria spp., Such as Listeria monocytogenes. neumoniae, Subsp. capricolum, M. mycoides Subsp 0039. In another embodiment, the bacterial agent is gram mycoides, M. agalactiae, M. ovipneumoniae, M. conjuncti positive and selected from the group comprising, but not vae, M. argini, M. bovis, and M. putrefaciens) Mycoplasma limited to, Kytococcus spp., Such as Kytococcus Schroeteri. bovis, dispar; mycoides subsp. mycoides (such as Contagious 0040. In another embodiment, the bacterial agent is gram bovine pleuropneumonia CBPP) Mycoplasma gallisepticum positive and selected from the group comprising, but not (MG), iowae meleagridis (MM), synoviae (MS) Myco limited to, Mycobacterium spp. Examples of Mycobacte plasma haemosuis (formerly Eperythrozoon suis), alkale rium spp include Mycobacterium abscessus, Mycobacterium scens, bovigenitalum, bovirhinis, bovoculi, Californicum, arupense, Mycobacterium asiaticum, Mycobacterium auba Canadense, Cynos, equigenitalium, gateae, haemocanis, hae gnense, Mycobacterium avium complex, Mycobacterium mofelis, hyopneumoniae, hyorhinis, hyosynoviae, iowae, boletii, Mycobacterium bolletii, Mycobacterium branderi, leachii, meleagridis, mycoides subsp capi, wenyoni, Suis, Mycobacterium canettii, Mycobacterium caprae, Mycobac Rhodococcus equi, Staphylococcus epidermidis, Staphyllo terium cellatum, Mycobacterium chelonae, Mycobacterium coccus simulans, Staphylococcus felis, Staphylococcus xylo chimaera, Mycobacterium colombiense, Mycobacterium sus, Staphylococcus chromogenes, Staphylococcus warneri, conceptionense, Mycobacterium conspicuum, Mycobacte Staphylococcus haemolyticus, Staphylococcus Sciuri, rium elephantis, Mycobacterium farcinogenes, Mycobacte Staphylococcus saprophyticus, Staphylococcus hominis, rium florentinum, Mycobacterium fortuitum group, Myco Staphylococcus Caprae, Staphylococcus cohnii Subsp. US 2017/007 1884 A1 Mar. 16, 2017

cohnii, Staphylococcus cohnii Subsp. urealyticus, Staphyllo sia helminthoeca risticii; Neorickettsia sen coccus capitis Subsp. capitis, Staphylococcus capitis Subsp. netsu, Wolbachia pipientis. —Armatimonadaceae:—Arma urealyticus, Staphylococcus hyicus, Staphylococcus aureus, timonas rosea. —Bacteroidaceae:—Bacteroides forsythus, Staphylococcus pseudintermedius, Staphylococcus delphini, Bacteroides fragilis, Bacteroides melaninogenicus, Bacte Staphylococcus Schleiferi Subsp. coagulans, Staphylococcus roides ruber, Bacteroides urealtvicus. —Bartonellaceae:— aureus Subsp. anaerobius, Streptococcus uberis, Streptococ alsatica, Bartonella australis, Bartonella bacil cus Canis, Streptococcus agalactiae, Streptococcus dysga lactiae, Streptococcus pyogenes, Streptococcus bovis, Strep liformis, Bartonella birtlesii, Bartonella bovis, Bartonella tococcus equi Subsp. Zooepidemicus, Streptococcus equinus, capreoi, Bartonella chomelii, Bartonella clarridgeiae, Bar Streptococcus equi (Streptococcus equi Subsp equi), Strep tonella doshiae, , Bartonella graha tococcus equisimilis (Streptococcus dysgalactiae Subsp mii, Bartonella hemselae, Bartonella koehlerae, Bartonella equisimilis), porcinus, suis, ZOOepidemicus, Streptococcus peromysci; Bartonella phoceensis, , ZOOepidemicus (Streptococcus equi Subsp Zooepidemicus), Bartonella rattimassiliensis, Bartonella rochalinae, Barto Streptococcus dysgalactiae Subsp. equisimilis, Propionibac nella Schoenbuchensis, Bartonella telpae, Bartonella terium acnes, Propionibacterium granulosum, Eubacterium, tamiae, Bartonella taylorii, , Barto Peptococcus indolicus, and Peptostreptococcus anaerobius; nella vinsonii subsp. berkhofii, Bartonella vinsonii subsp. and various species of the following Gram negative genera: arupensis, Bartonella vinsonii Subsp. vinsonii. —Bdellovi , Aeromonas, Anaplasma, Arcobacter, Avibac brionaceae:—Bdello spp. —Brachyspiraceae:— terium, Bacteroides, Bartonella, , Borrelia, Brachyspira spp including Brachyspira hampsonii, Brachyspira, Brucella, Campylobacter; Capnocytophaga, Brachyspira hyodysenteriae, Brachyspira murdochi, , Chlamydophila, Chryseobacterium, Coxiella, Brachyspira pilosicoi. —:—Brucala abortus, Cytophaga, Dichelobacter, Edwardsiella, Ehrlichia, Brucalla canis, Brucala Ceti, Brucala melitensis, Brucella Escherichia, Flavobacterium, Francisella, Fusobacterium, Ovis, Brucalla pinnipedialis, Brucalla suis, Ochrobactrum Gallibacterium, , Histophilus, Klebsiella, Law anthropi; Ochrobactrum intermedium. —Burkholderi Sonia, Leptospira, Mannheimia, Megasphaera, , aceae:—Burkholderia aboris, Burkholderia ambifana (ge Neorickettsia, Nicoletella, Ornithobacterium, Pasteurella, nomovar VII); Burkholderia anthina (genomovar VIII); Photobacterium, Piscichlamydia, Piscirickettsia, Poiphy Burkholderia cenocepacia (genomovar III); Burkholderia romonas, PrevOtella, Proteus, Pseudomonas, , cepacia (genomovar I); Burkholderia diffusa, Burkholderia Riemerella, Salmonella, Streptobacillus, Tenacibaculum, dolosa (genomovar VI); Burkholderia latens, . Burkholderia metallica, Burkholderia multivorans Vibrio, and Yersinia. (genomovar II); Burkholderia pseudomallei. Burkholderia 0043. In another embodiment, the bacterial agent is gram pyrrocinia (genomovar IX); Burkholderia seminalis, Burk positive and selected from the group comprising, but not holderia Stabilis (genomovar IV); Burkholderia ubonensis limited to, pathogens of companion animal species such as (genomovar); Burkholderia Vietnamiensis (genomovar IV: cats, dogs and horses. Examples of Such pathogens include Cupriavidus pauculus, Cupriavidus gillardii; Ralstonia pick equine pathogens such as Streptococcus equi, Streptococcus etti, Ralstonia mannitoilytica, Sphaerotilus hippei, Sphaero ZOOepidemicus, Rhodococcus equi, Clostridium difficile, tilus montanus, Sphaerotilus natans. —Campylobacter Clostridium perfingens, Corynebacterium pseudotubercu aceae:—Arcobacter Spp including Arcobacter Skirrowii; losis, Clostridium piliforme, Actinomyces bovis, Staphyllo Campylobacter coli, Campylobacter concisus, Campy coccus aureus, beta haemolytic Streptococcus spp., Der lobacter curvus, Campylobacter fetus, Campylobacter gra matophilus congolense, Clostridium tetani, and Clostridium cilis, Campylobacter helveticus, Campylobacter hominis, botulinum. Further examples include pathogens of dogs and Campylobacter hyointestinalis, Campylobacter insulaemi cats Such as Staphylococcus spp., Streptococcus spp. grae, , Campylobacter lanienae, Clostridium spp., Actinomyces spp., Enterococcus spp. Campylobacter larii Campylobacter laridis, Campy Nocardia spp., Mycoplasma spp., and Mycobacterium spp. lobacter mucosalis, Campylobacter rectus, Campylobacter 0044. In another embodiment, the bacterial agent is gram Showae, Campylobacter sputorum, Campylobacter upsa negative and selected from the group consisting of the liensis. —Candidatus:—Piscichlamydia salmonis. —Car following representative families and species: Acetobacter diobactenaceae:—, Cardiobacte aceae:—Roseomonas cervicalis, Roseomonas fauriae, rium valvarum, Dichelobacter nodosus. Roseomonas gillardii. —Aeromonadaceae:—Aeromonas —Chlamydiaceae:—Chlamydia spp including Chlamydia allosaccharophila, Aeromonas aquariorum, Aeromonas avium, Chlamydia gallinacea, Chlamydia muridarum, Chla caviae, (and Subspecies); Aeromonas mydia suis, , Chlamydophila spp salmonicida, Aeromonas Shubertii Aeromonas veroni bio including Chlamydophila pneumoniae, Chlamydophila var sobria (Aeromonas sobria). —:—Achro pecorum, Chlamydophila psittaci, Chlamydophila abortus, mobacter xylosoxidans, Aicalligenes faecalis, Bordetella Chlamydophila caviae, and Chlamydophila felis. —Chtho ansorpii , Bordetella bronchiseptica, Bor nomonadaceae:—Chthonomonas celidirosea. —Coma detella hinzii; Bordetella holmesii; Bordetella parapertus monadaceae:—Comamonas testosteroni, Verminephro sis, , ; Bordetella bacter spp. —Coxiellaceae:—. trematum, Oligella ureolytica, Oligella urethralis. —Ana —Cytophagaceae:—Cytophaga columnaris, Cytophaga plasmataceae:—Anaplasma phagocytophilum, Anaplasma hutchinsonii, Flexibacter echinicida, Flexibacter elegans, platys, , Anaplasma centrale, Anaplasma Flexibacter flexilis, Flexibacter litorais, Flexibacter poly marginale, Anaplasma Odocoilei, Anaplasma ovis, Ehrli morphus, Flexibacter roseolus, Flexibacter ruber. —Des chia canis, Ehrlichia chafeensis, , Ehrli ulfovibrionaceae:—Bilophila wadsworthia, Lawsonia intra chia muris, Ehrlichia ovina, Ehrlichia ruminantium, cellularis. —Enterobacteraceae:—Cedecea davisae, Neoehrlichia lotoris, Neoehrlichia mikurensis, Neorickett Cedecea lapagei; Cedecea meteri; amalomaticus, Citro US 2017/007 1884 A1 Mar. 16, 2017

bacter diversus, freundii, Citrobacter ko seri; Hebdomadis, Leptospira alexanderi serovar Manhao. 3); Cronobacter condimenti, Cronobacter dublimensis, Crono Leptospira alstoni (including Leptospira alstoni serovar bacter helveticus, Cronobacter malonaticus, Cronobacter Pingchang, Leptospira alstoniserovar Sichuan); Leptospira muytjensii; Cronobacter pulveris, Cronobacter Sakazaki; biflexia (including Leptospira biflexia serovar Ancona, Lep Cronobacter turicensis, Cronobacter universalis, Crono to spira biflexia serovar Canella); Leptospira borg.petersenii bacter Zurichensis, Edwardsiella ictaluri; Edwardsiella (including Leptospira borg.petersenii serovar Hardio, Lep tarda, Enterobacter aerogenes, Enterobacter agglomerans, to spira borgpetersenii serovar Hardio-bovis, Leptospira . Enterobacter Cowanii, Escherichia borg.petersenii serovar Pomona, Leptospira borg.petersenii albertii, Escherichia coli, including AIEC-adherent inva serovar Tarassovi); Leptospira broomii (including Lep sive E. coli, EaggEC enteroaggregative E. coli: to spira broomii serovar Hurstbridge); Leptospira fainei EHEC-enterohemonhagic E. coli: EIEC=enteroinvasive E. (including Leptospira fainei serovar Hurstbridge); Lep coli: EPEC=enteropathogenic E. coli: to spira idonii; Leptospira inadai (including Leptospira ina ETEC=enterotoxigenic E. coli: ExPEC=extraintestinal dai serovar Lyme, Leptospira inadai serovar Malaya); Lep pathogenic E. coli, NMEC=neonatal meningitis E. coli, to spira interrogans (including Leptospira interrogans NTEC-necrotoxigenic E. coli, UPEC=uropathogenic E. serovar Australis, Leptospira interrogans serovar Autumna coli. Escherichiafergusonii, Ewingella americana, Hafinia lis, Leptospira interrogans serovar Bratislava, Leptospira alvei, Hafnia paralvei, , Klebsiella interrogans serovar Canicola, Leptospira interrogans sero Oxytoca, Klebsiella pneumoniae, Kluyvera ascorbata, Kluy var Grippotyphosa, Leptospira interrogans serovar Hardio, vera cryocrescens, Morganella morgani, Pantoea (for Leptospira interrogans serovar Hardio-bovis, Leptospira merly Enterobacter) agglomerans, Photorhabdus asymbi interrogans serovar Icterohaemonrrhagiae, Leptospira otica, Plesiomonas Shigeloides, , Proteus interrogans serovar Pomona, Leptospira interrogans sero penneri; , Providencia alcalifaciens, Provi var Pyrogenes, Leptospira interrogans serovar Tarassovi); dencia rettgeri; , Raoultella electrica, Leptospira kirschneri (including Leptospira kirschneri sero Raoultella Ornithinolytica, Raoultella planticola, Raoultella var Bulgarica, Leptospira kirschneri serovar Cynopten, terrigena, , Subspe Leptospira kirschneri serovar Grippotyphosa); Leptospira cies enterica (many serotypes); Serratia liquifaciens, Ser kinetvi, Leptospira licerasiae, Leptospira meyeri (including ratia marcesans, ; , Shi Leptospira meyeri serovar Sofia); Leptospira noguchi (in gella flexneri; , cluding Leptospira noguchii serovar Panama, Leptospira , Yersinia pseudotuberculosis, Yersinia ruck noguchii serovar Pomona); Leptospira Santarosai; Lep eri. —Fimbriimonadaceae:—Fimbriimonas ginsengisoli. to spira terpstrae, Leptospira vanthielii; Leptospira weilii —Flavobacteriaceae:—Bergeyella zoohelicum, Capnocy (including Leptospira weilii serovar Celledoni, Leptospira tophaga Canimorsus, Capnocytophaga Cynodegni, Capno weilii serovar Sarmin); Leptospira wolbachii; Leptospira cytophaga gingivalis, Capnocytophaga granulosa, Capno Wolffii; Leptospira vanagawae. —Leptotrichiaceae:—Lep cytophaga haemolytica, Capnocytophaga leadbetteri; totrichia buccalis, Streptobacillus moniliformis. —Methyl Capnocytophaga ochracea, Capnocytophaga sputigena, obacteriaceae:—Methylobacterium extorquens group; Meth Chryseobacterium indologenes, Chryseobacterium pisci vlobacterium fiujisawaense, Methylobacterium cola, Elizabethkiingia mening Oseptica, Flavobacterium mesophilicum, Methylobacterium zatmanii. —Moraxel branchiophilum, Flavobacterium columnare, Flavobacte laceae:— (genomic species 2); rium Oncorhynchi, Flavobacterium piscicida, Flavobacte Acinetobacter baylvi; Acinetobacter bouvetii, Acinetobacter rium psychrophilum, Myroides odoratus, Myroides odora calcoaceticus (genomic species 1); Acinetobacter gerneri; timimus, Ornithobacterium rhinotracheale, Riemerella Acinetobacter grimontii, Acinetobacter haemolyticus (ge anatipestifer; Riemerella columbina, Riemerella columbi nomic species 4); Acinetobacter johnsonii (genomic species pharyngis, Tenacibaculum dicentrarchi, Tenacibaculum 7); Acinetobacter junii (genomic species 5), Acinetobacter discolour, Tenacibaculum gallaicum, Tenacibaculum mar lwoffi (genomic species 89); Acinetobacter parvus, Acine itimum, Tenacibaculum soleae; Weeksella virosa. —Franci tobacter radioresistens (genomic species 12); Acinetobacter sellaceae:— Subsp. tularensis, Fran schindleri, Acinetobacter tandoii, Acinetobacter tiernber cisella tularensis Subsp. holarctica, Francisella tularensis giae, Acinetobacter townenri, Acinetobacter ursingii; Subsp. novicida, Francisella philomiragia, Francisella noa Acinetobacter venetianus, Moraxella atlantae, Moraxella tunensis, Francisella noatunensis Subsp. Orientalis (also boevrei. , Moraxella bovoculi. Moraxella termed Francisella asiatica). —Fusobacteriaceae:—Fuso canis, Moraxella caprae, , Moraxella bacterium spp. including Fusobacterium necrophorum, caviae, Moraxella cuniculi, Moraxella equi, Moraxella Fusobacterium nucleatum, Fuso-bacterium polymorphum. lacunata, Moraxella lincolnii Moraxella macacae. Morax —Helicobacteraceae:—. Helicobacter ella nonliquefaciens, Moraxella Oblonga, Moraxella fennelliae, . —Legionelaceae:—Legio Osloensis, Moraxella ovis, Moraxella phenylpyruvica, nella pneumophila and other species including; Moraxella pluranimalium, Moraxella porci. —Moritel anisa, Legionella birminghamensis, Legionella bozen laceae:—Moritella abyssii Moritella dasanensis, Moritella annii; Legionella cincinnatiensis, Legionella dunoffii; japonica, Moritella marina, Moritella pro-funda, Moritella ; ; Legionella hack viscosa, Moritella vavanosi. —Neisseriacae:—Chromobac eliae, , , Legio terium violaceum, , Kingella denitrifi nella longbeachae, Legionella maceachemii; Legionella cans, , Kingella oralis, Kingella potus, micdadei , , Neisseria cinema, Neisseria elongata, Neisseria flavescens, Legionella Sainthelens, Legionella tusconensis, Legionella Neisseria gononrrhoeae, Neisseria lactanica, Neisseria Wadswothii; . —Leptospiraceae:—Lep meningitidis, Neisseria mucosa, Neisseria polysaccharea, to spira alexanderi (including Leptospira alexanderi serovar Neisseria sicca, Neisseria subflava, Neisseria weaver Vit US 2017/007 1884 A1 Mar. 16, 2017

reoscilla spp. —Nitrosomonadaceae:—Nitrosomonas eutro turicatae, Borrelia turicatae, Borrelia valaisiana, pha, Nitrosomonas halophila, Nitrosomonas Oligotropha. Treponema carateum, Treponema pallidum ssp. endemicum, —:—Actinobacillus actinomycetemcomi Treponema pallidum ssp. pallidum, Treponema pallidum tans, Actinobacillus equuli. Actinobacillus lignieresii; Acti ssp. pertenue. —Succinivibrionaceae:—Anaerobiospirillum nobacillus pleuropneumoniae, Actinobacillus seminis, Acti spp. —Sutterellaoeae:—Sutterella spp including Sutterella nobacillus Succinogenes, Actinobacillus ureae, Wadsworthia. —Thermaceae:—Meiothermus spp. —Ther Aggregatibacter actinomycetemcomitans, Aggregatibacter motogaceae:—Thermotoga neapolitana. —Veillonel Segnis, Aggregatibacter aphrophilus, Avibacterium avium, laceae:—Dialister spp., Megamonas spp., Megasphaera spp.; Pectinatus spp., Pelosinus spp., Propionispora spp., Sporo Avibacterium endocarditidis, Avibacterium gallinarum, Avi musa spp., Veillonella spp., Zymophilus spp. —Vibrion bacterium paragallinarum, Avibacterium volantium, Biber aceae:—Photobacterium damselae, Vibrio adaptatus, Steinia , Gallibacterium anatis, Gallibacterium , Vibrio azasii, Vibrio campbellii, Vibrio genomospecies 1; Gallibacterium genomospecies 2: Galli , Vibrio damsel; Vibrio fluvialis, Vibrio filmisii; bacterium genomospecies 3; Gallibacterium group V. Gal Vibrio hollisae, Vibrio metchnikovii Vibrio mimicus, Vibrio libacterium melopsittaci, Gallibacterium salpingitidis, Gal parahaemolyticus, . —Wolbachieae:—Wol libacterium trehalosifermentans, Haemophilus aegyptius, bachia spp. —:—Luteinonas aestuarii; Haemophilus avium, , Haemophilus Luteinonas aquatica, Luteinonas composti, Luteimonas haemolyticus, , Haemophilus para lutimaris, Luteimonas marina, Luteinonas mephitis, Lutei haemolyticus, Haemophilus parainfluenzae, Haemophilus monas vadosa, Pseudoxanthomonas broegbenrnensis, parasuis. Histophilus somni, Mannheimia caviae, Man Pseudoxanthomonas japonensis, Stenotrophomonas malto inheimia glucosida, Mannheimia granulomatis, Man philia, Stenotrophomonas nitritireducens. inheimia haemolytica, Mannheimia ruminalis, Mannheimia varigena, Nicoletella semolina, Pasteurella aerogenes, 0045 Most preferably, the bacterial agent causing the Pasteurella bettvae, Pasteurella caballi, , bacterial infection is gram negative and is selected from the , (Subspecies group comprising: Acinetobacter species, Aeromonas hydro multocida, septicum, gallicida); Pasteurella pneumotropica, phila, Citrobacter species, Enterobacter species, Escheri , Pasteurella trehalosi. —Piscirickett chia coli, Klebsiella pneumoniae, Morganella morganii, siaceae:—Piscirickettsia salmonis. -Plesiomonadaceae:— Pseudomonas aeruginosa, and Stenotrophomonas malto . -Polyangiaceae:- Sorangium philia. cellulosum. —Porphyromonadaceae:—Dysgonomonas cap 0046. In another preferred embodiment, the bacteria nocytophagoides, Dysgonomonas gadei, Dysgonomonas agent causing the bacterial colonisation or infection is hofstadii; Dysgonomonas mossii; Dysgonomonas Oryzarvi, resistant to a conventional antibiotic used to treat the colo Dysgonomonas wimpennyi, Porphyromonas gingivalis. nisation or infection. In one preferred embodiment, the —Prevotellaceae:—Prevotella spp. including Prevotella bacterial agent is resistant to a compound selected from the intermedia, Prevotella melaninogenica. group comprising: one or more of (for —Pseudomonadaceae:—Chryseomonas luteola, example , , , or ); Pseudomonas aeruginosa, Pseudomonas luteola, anti-MRSA cephalosporins (for example ceftaroline); antip Pseudomonas fluorescens, Pseudomonas putida, seudomonal +B-lactamase inhibitors (for example Pseudomonas Stutzeri. Pseudomonas Oryzihabitans. ticarcilin- or -); car —Rhizobiaceae:—Agrobacterium tumefaciens, Rhizobium bapenems (for example , , radiobacter. —:—Orientia chuto, Orientia or ); non-extended spectrum cephalosporins; 1st tsutsugamushi; Rickettsia aeschlimannii; , and 2nd generation cephalosporins (for example or , Rickettsia argasii; , ); extended-spectrum cephalosporins; 3rd and , Rickettsia bellii, Rickettsia Canadensis, 4th generation cephalosporins (for example or Rickettsia Conori, Rickettsia cooleyi, , Rick ); (for example or cefo ettsia heilongjiangensis, Rickettsia helvetica, Rickettsia tetan); fluoroquinolones (for example ); folate homei, Rickettsia hoogstraali, Rickettsia hulinensis, Rick pathway inhibitors (for example -Sulphame ettsia hulinii; , Rickettsia marmionii, thoxazole); (for example ); mono Rickettsia martinet, , Rickettsia bactams (for example ); penicillins (for example monacensis, Rickettsia montanensis, Rickettsia monteiroi; ); penicillins+B-lactamase inhibitors (for example Rickettsia moreli; Rickettsia parked ; -clavulanic acid orampicillin-); pheni Rickettsia philipii; , Rickettsia cols (for example ); phosphonic acids (for raoultii; Rickettsia rhipicephali, ; Rick example ); and (for example colis ettsia Sibirica subgroup, Rickettsia Slovaca, Rickettsia tin); (for example , or tamurae, . —Shewanellaceae:—Shewanella ). Preferably, the bacterial agent resistant to putrefaciens. —Sphingomonadaceae:—Sphingobactenum these compounds is gram negative. multivorum, Sphingobacterium spiritivorum, Sphingomo 0047 Preferably, the bacterial agent is resistant to a nas paucimobilis. —Spirillaceae:—Spirillum minus, Spiril compound selected from the group comprising: penicillins, lum volutans, Spirillum winogradskyi. —Spirochaeta cephalosporins, carbapenems, and other ceae. Borrelia afzeli; Borrelia anserina, Borrelia B-lactam , fusidanes, aminoglycosides, fluoroqui bissettii, Borrelia burgdorferi; Borrelia coriaceae, Borrelia nolones, , tetracyclines, glycylcyclines, duttonii, Borrelia garinii, Borrelia hermsii; Borrelia his chloramphenicol and other phenicols, macrollides and panica, Borrelia japonica, Borrelia lonestari, Borrelia lus , , oxazolidinones, aminocyclitols, itaniae, Borrelia miyamotoi, Borrelia parked Borrelia per polymyxins, glycopeptides, lipopeptides, , mupiri Sica, Borrelia recurrentis, Borrelia spielmanii; Borrelia cin, , , Sulphonamides and US 2017/007 1884 A1 Mar. 16, 2017 trimethoprim. More preferably, the compound is selected folate pathway inhibitors (for example trimethoprim-sul from the group comprising: B-lactams, glycopeptides, lipo phamethoxazole); fucidanes (for example ); gly peptides, macrollides, oxazolidinones and tetracyclines. copeptides (for example Vancomycin, or tella Preferably, the bacterial agent is resistant to the compound vancin); glycylcyclines (for example tigecycline); when the compound is at a concentration range selected lincosamides (for example ); lipopeptides (for from the following: 0.001 ug/mL-10,000 g/mL, 0.01 example daptomycin); macrollides (for example erythromy ug/mL-1000 g/mL, 0.10 g/mL-100 ug/mL, and 1 ug/mL cin); oxazolidinones (for example linezolid or tedizolid); 50 ug/mL phenicols (for example chloramphenicol); phosphonic acids 0048 Most preferably, the bacterial agent causing the (for example fosfomycin); Streptogramins (for example qui bacterial infection is selected from the group comprising, nupristin-dalfopristin); and tetracyclines (for example tetra but not limited to, gram positive bacteria. The bacterial agent cycline, doxycycline or minocycline). Preferably, the bac is most preferably a Gram positive bacterial agent selected terial agent resistant to these compounds is gram positive. from the group comprising Staphylococcus aureus, Staphy 0052. In another most preferred embodiment, the bacte lococcus pseudintermedius, Streptococcus pneumoniae, rial agent is Streptococcus pneumoniae. The Streptococcus Streptococcus pyogenes, Streptococcus agalactiae, Strepto pneumoniae may be a strain that is resistant to one or more coccus uberis, Enterococcus faecium, Enterococcus faeca of B-lactams and macrollides. lis, and Clostridium difficile. 0053. In another most preferred embodiment, the bacte 0049. In one preferred embodiment, the bacterial agent rial agent is Streptococcus pyogenes. has no cell wall. Preferably, the bacterial agent is selected from the group comprising: Mycoplasma spp., Mycoplasma 0054. In another most preferred embodiment, the bacte agalactiae, Mycoplasma alkalescens, Mycoplasma rial agent is Streptococcus agalactiae. amphoriforme, Mycoplasma arginini, Mycoplasma bovi 0055. In another most preferred embodiment, the bacte genitalum, Mycoplasma bovirhinis, Mycoplasma bovis, rial agent is either Enterococcus faecium or Enterococcus Mycoplasma bovOculi, Mycoplasma buccale, Mycoplasma faecalis. The Enterococcus faecium or Enterococcus faeca Californicum, Mycoplasma Canadense, Mycoplasma capri lis may be a strain that is resistant to one or more of colum Subsp. capricolum, Mycoplasma capricolum Subsp. aminoglycosides (for example gentamicin (high level) or capripneumoniae, Mycoplasma conjunctivae, Mycoplasma (for example streptomycin (high level)); car cynos, Mycoplasma dispar, Mycoplasma equigenitalium, bapenems (for example imipenem, meropenem or dorip Mycoplasma faucium, Mycoplasma felis, Mycoplasma fer enem); fluoroquinolones (for example ciprofloxacin, levo mentans (incognitus str.), Mycoplasma gallisepticum (MG), floxacin or ); glycopeptides (for example Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma Vancomycin or teicoplanin); glycylcyclines (for example haemocanis, Mycoplasma haemofelis, Mycoplasma haemo tigecycline); lipopeptides (for example daptomycin); oxazo suis (formerly Eperythrozoon suis), Mycoplasma hominis, lidinones (for example linezolid); penicillins (for example Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, ampicillin); Streptogramins (for example quinupristin-dalfo Mycoplasma hyosynoviae, Mycoplasma iowae meleagridis pristin); tetracycline (for example doxycycline or minocy (MM), Mycoplasma iowae, Mycoplasma leachi, Myco cline). plasma lipophilum, Mycoplasma meleagridis, Mycoplasma 0056. In another most preferred embodiment, the bacte mycoides subsp capri, Mycoplasma mycoides Subsp rial agent is Clostridium difficile. mycoides, Mycoplasma mycoides Subsp. mycoides (such as 0057 The bacterial infection in the subject may cause a Contagious bovine pleuropneumonia CBPP), Mycoplasma disease selected from the group comprising, but not limited orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, to, nosocomial pneumonia caused by Staphylococcus aureus Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma (MDR, XDR, PDR or methicillin-susceptible or -resistant pneumoniae, Mycoplasma primatum, Mycoplasma putrefa strains), or invasive pneumococcal diseases such as pneu ciens, Mycoplasma salivarium, Mycoplasma spermatophi monia, bronchitis, acute sinusitis, otitis media, conjunctivi lum, Mycoplasma suis, Mycoplasma synoviae (MS), Myco tis, meningitis, bacteremia, sepsis, osteomyelitis, septic plasma wenyonii, Mycoplasma, Ureaplasma spp. arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and Ureaplasma parvum, Ureaplasma urealyticum, Urea brain abscess caused by Streptococcus pneumoniae (includ plasma, and Ureoplasma diversum. ing multi-drug resistant strains IMDRSP such as those 0050. In another most preferred embodiment, the bacte resistant to B-lactams and macrollides), complicated skin and rial agent is Staphylococcus aureus. skin structure infections, including diabetic foot infections, 0051. In another preferred embodiment, the bacterial with or without concomitant osteomyelitis, caused by agent is resistant to a compound selected from the group Staphylococcus aureus (methicillin-Susceptible and -resis comprising: one or more of aminoglycosides (for example tant strains), Streptococcus pyogenes, or Streptococcus aga gentamicin); ansamycins (for example ); anti lactiae, uncomplicated skin and skin structure infections MRSA cephalosporins (for example ceftaroline); anti caused by Staphylococcus aureus (methicillin-Susceptible staphylococcal B-lactams (or cephamycins) (for example and -resistant strains) or Streptococcus pyogenes, commu or cefoxitin); carbapenems (for example nity-acquired pneumonia caused by Streptococcus pneumo ertapenem, imipenem, meropenem or doripenem); non-ex niae (including multi-drug resistant strains IMDRSP. tended spectrum cephalosporins; 1st and 2nd generation including cases with concurrent bacteraemia, or Staphyllo cephalosporins (for example cefazolin or cefuroxime); coccus aureus (methicillin-Susceptible and -resistant strains) extended-spectrum cephalosporins; 3rd and 4th generation and Staphylococcus aureus bloodstream infections (bacter cephalosporins (for example cefotaxime or ceftriaxone); aemia), including those with right-sided infective endocardi cephamycins (for example cefoxitin or ); fluoro tis, caused by methicillin-Susceptible and methicillin-resis quinolones (for example ciprofloxacin or moxifloxacin); tant isolates, Vancomycin-resistant Enterococcus infections, US 2017/007 1884 A1 Mar. 16, 2017

including cases with concurrent bacteraemia, and treatment nephritis, recurrent urinary tract infections and atheter of Clostridium difficile-associated diarrhea (CDAD). associated bacteriuria and urinary tract infections). 0058 Gram negative organisms are important causes of 0059. In humans, gram negative bacteria are common causes of intra-abdominal infections (IAIS), urinary tract many infectious diseases in humans and other animal spe infections (UTIs), hospital acquired pneumonia, and bacte cies. Bone and joint infections (Gram-negative organisms or raemia. Escherichia coli (E. coli), Klebsiella pneumoniae mixed bacteria, are an important cause of vertebral osteo (K. pneumoniae), and Pseudomonas aeruginosa (P. aerugi myelitis and septic arthritis), cardiovascular system infec nosa) are important pathogens in the hospital setting, tions (including endocarditis caused by the HACEK accounting for 27% of all pathogens and 70% of all Gram group—Haemophilus parainfluenzae, Haemophilus aphro negative pathogens causing healthcare-associated infections philus, Aggregatibacter actinomycetemcomitans, Cardio Sievert D M. Ricks P. Edwards J R, et al. Antimicrobial bacterium hominis, Eikenella conrrodens, Kingella kingae), resistant pathogens associated with healthcare-associated central nervous system infections (the commonest causes of infections: summary of data reported to the National Health bacterial meningitis are , Streptococ care Safety Network at the Centers for Disease Control and cus pneumoniae and, in nonvaccinated young children, Prevention, 2009-2010. Infect Control Hosp Epidemiol. Haemophilus influenzae type b (Hib), in neonates and 2013: 34:1-14.. infants less than 3 months of age, Streptococcus agalactiae 0060 Gram negative bacteria are showing rising rates of (group B Streptococcus), Escherichia coli and other aerobic resistance to current therapies. The production of extended Gram-negative rods are important pathogens, brain abscess spectrum B-lactamase (ESBL) enzymes is a common or Subdural empyema, the infecting organism(s) vary with mechanism of resistance. Rates of ESBL-producing E. coli the underlying predisposing cause but where the likely site and K. pneumoniae have risen Substantially, with the result of origin is the ear, enteric Gram-negative bacilli are com that these bacteria are increasingly resistant to widely used monly involved), eye infections (common pathogens include antimicrobials. Haemophilus influenza, or Chla 0061 P aeruginosa is the most common Gram-negative mydia trachomatis), gastrointestinal tract infections (a wide cause of nosocomial pneumonia and the second most com range of pathogens are implicated including enterotoxigenic mon cause of catheter-related UTIs in the U.S. Escherichia coli (ETEC), Salmonella, Campylobacter, Shi 0062 E. coli is the most common cause of UTIs. Cases gella, Vibrio cholera and Yersinia enterocolitica), genital of UTI caused by ESBL-producing E. coli and K. pneumo infections (bacterial vaginosis is a polymicrobial clinical nia as well as P. aeruginosa, including MDR strains, are syndrome with high concentrations of anaerobic (eg Mobi increasing. ESBL-producing E. coli and K. pneumoniae are luncus species) and other fastidious bacteria (including also frequently isolated in patients with complicated IAI Gardnerella vaginalis and Atopobium vaginae), and Myco (cIAI). plasma hominis; non-sexually acquired pelvic inflammatory 0063 P aeruginosa is a clinically challenging and viru disease (PID) is usually caused by mixed vaginal flora, lent that can be a cause of common infections in including anaerobes, facultative Gram-negative bacteria and humans such as nosocomial pneumonia, UTI, IAI, and Mycoplasma hominis, while sexually acquired PID is usu bloodstream infections. P. aeruginosa is the most common ally initiated by C. trachomatis or N. gonorrhoeae with Gram-negative organism causing ventilator associated pneu growing evidence that M. genitalium infection is involved in monia and the second most common cause of catheter a significant minority of cases), intra-abdominal infections (peritonitis due to perforated Viscus is usually a polymicro associated UTIs. bial infection with aerobic and anaerobic bowel flora while 0064. The increase in the number of infections caused by spontaneous bacterial peritonitis (SBP) is usually caused by Gram-negative bacteria is being accompanied by rising rates enteric Gram-negative bacilli, Such as Escherichia coli and of resistance. Treatment options to meet this challenge are Klebsiella species, Klebsiella pneumoniae is an increasingly increasingly limited. There is a critical need for new anti identified cause of liver abscess), community-acquired biotics to meet the needs of patients now and in the future. pneumonia (Mycoplasma pneumoniae, Chlamydophila 0065. In another preferred embodiment, robenidine, or a (Chlamydia) pneumoniae, Chlamydophila (Chlamydia) psit therapeutically acceptable salt thereof, is administered taci, Haemophilus influenza, aerobic Gram-negative bacilli together with a compound or agent that removes or Substan including Klebsiella pneumonia, Pseudomonas aeruginosa, tially removes or reduces the integrity of the cell wall of the Acinetobacter baumannii, Burkholderia pseudomallei), oti bacterial agent. As an example, the compound is selected tis externa (including acute diffuse) (bacterial cultures com from the group consisting of B-lactams, fosfomycin, monly yield Pseudomonas aeruginosa, Staphylococcus , polymyxins and chelating agents such as ethyl aureus, and Proteus and Klebsiella species), otitis media enediaminetetraacetic acid (EDTA). As an example, the (including acute) (common bacterial pathogens include agent is an immunological agent (Such as an antibody or Streptococcus pneumoniae, Haemophilus influenzae and vaccine) that reduces the integrity of the cell wall. In one Moraxella catarrhalis), sepsis (including severe) (including preferred embodiment, robenidine, or a therapeutically Acinetobacter baumannii, disseminated gonococcal sepsis, acceptable salt thereof, is administered together with a Gram-negative enteric bacteria, Neisseria meningitidis compound that removes or Substantially removes or weak (meningococcal sepsis) and Pseudomonas aeruginosa), Sys ens the integrity of the outer cell wall of a gram negative or temic infections (Spotted fevers (Rickettsia) and scrub positive bacterial agent. (Orientia), , Cat-scratch disease and other 0066. According to another aspect of the invention, there Bartonella infections, Leptospirosis, Lyme disease, Melioi is provided an antibacterial pharmaceutical composition dosis, , Typhoid and paratyphoid fevers (enteric comprising a therapeutically effective amount of robenidine, fevers), urinary tract infections (acute cystitis, acute pyelo or a therapeutically acceptable salt thereof. US 2017/007 1884 A1 Mar. 16, 2017

0067. According to another aspect of the invention, there dazole, Climbazole, Croconazole (Cloconazole), Clotrima is provided an antibacterial veterinary composition compris Zole, Eberconazole, Econazole, Enilconazole, ing a therapeutically effective amount of robenidine, or a Fenticonazole, Flutrimazole, Fosfluconazole, Isoconazole, therapeutically acceptable salt thereof. Ketoconazole, Lanoconazole, Luliconazole, Miconazole, 0068. The method of treating or preventing a bacterial Neticonazole, Omoconazole, Oxiconazole Nitrate, Parcon infection or colonisation in a Subject, may also comprise the azole, Sertaconazole, Sulconazole, Tioconazole). Thiocar administration of the pharmaceutical or veterinary compo bamates (Liranaftate, Tolciclate, Tolindate, Tolnaftate), Tri sitions of the invention. azoles (Fluconazole, Isavuconazole, Itraconazole, 0069. The pharmaceutical composition may optionally Posaconazole, Ravuconazole, Saperconazole, Terconazole, include a pharmaceutically acceptable excipient or carrier. Voriconazole), and other synthetic agents such as Acrisorcin, The veterinary composition may optionally include a vet Amorolfine, Bromosalicylchloranilide (Bromochlorosalicy erinary acceptable excipient or carrier. lanilide), Buclosamide, Calcium Propionate, Chlorphenesin, 0070 The pharmaceutical or veterinary composition of Ciclopirox, Cloxyquin (Cloxiquine), Coparaflinate. Exal the invention preferably contains robenidine, or a pharma amide, Flucytosine, Haloprogin, Hexetidine, Loflucarban, ceutically acceptable salt, at a concentration of selected from Nifuratel, Nifuroxime, Piroctone, Potassium Iodide, Propi the group consisting of 1 mg/g to 500 mg/g; 5 mg to 400 onic Acid, Pyrithione, Salicylanilide, Sodium Parachlo mg/g; 10 mg/g to 200 mg/g; 20 mg/g to 100 mg/g; 30 mg/g robenzoate, , Sulbentine, Tenonitrozole, to 70 mg/g; and 40 mg/g to 60 mg/g. Triacetin, Trimetrexate, Undecylenic Acid (Undecenoic 0071. In another embodiment, the pharmaceutical or vet Acid), and Zinc Propionate. erinary composition comprises impurities, wherein the 0075. The composition of the invention may comprise an quantity of impurities as a percentage of the total weight of antibiotic adjunct selected from the group comprising, but the composition is selected from the group consisting of not limited to, B-Lactamase Inhibitors (, Clavu less than 20% impurities (by total weight of the composi lanic Acid, Sulbactam, Sultamicilin, Tazobactam), Renal tion); less than 15% impurities; less than 10% impurities: Dipeptidase Inhibitors (Cilastatin), and Renal Protectant less than 8% impurities; less than 5% impurities; less than (Betamipron). 4% impurities; less than 3% impurities; less than 2% impu 0076. In one embodiment, the composition of the inven rities; less than 1% impurities: less than 0.5% impurities: tion comprises a further antibiotic selected from the group less than 0.1% impurities. In one embodiment, the pharma comprising, but not limited to, 2,4-DIAMINOPYRIMI ceutical or veterinary composition comprises microbial DINES, including Baquiloprim, , . impurities or secondary metabolites, wherein the quantity of Ormetoprim, Pyrimethamine, , Trimethoprim; microbial impurities as a percentage of the total weight of , including : AMINOCY the composition is selected from the group consisting of CLITOLS, including , AMINOGLYCO less than 5%; less than 4%; less than 3%; less than 2%; less SIDES, including Amikacin, , , Beka than 1%; less than 0.5%; less than 0.1%; less than 0.01%; namycin, Butirosin, , , less than 0.001%. In one embodiment, the pharmaceutical or Etimicin, Fortimicins (), Framycetin, Gentami veterinary composition is sterile and stored in a sealed and cin, , , Kanamycin, , sterile container. In one embodiment, the pharmaceutical or , Netilmicin, , , veterinary composition contains no detectable level of , , Streptomycin, Tobramycin, Ver microbial contamination. damicin: AMINOMETHYLCYCLINES, including Omada 0072 Preferably, the robenidine is pharmaceutical or cycline: , including , veterinary grade. Methods to synthesise commercial quan Chloramphenicol, , ; ANSAMY tities of robendine are widely available in the art. Commer CINS, including , Rifamide, Rifampin (Rifampi cial quantities of pharmaceutical or veterinary grade robeni cin), , , : ANTISEPTIC dine are available from Zhejiang Esigma Animal Health Co., AGENTS, including Acridine derivatives (including acrifla Ltd, Haining City, Peoples Republic of China. vine, aminoacridine, ethacridine, proflavine), Bispyridines 0073. The pharmaceutical or veterinary composition of (including octenidine dihydrochloride), Brominated salicy the invention may comprise a further antimicrobial agent. lanilides (including bromsalans), Chlorhexidine, Phenol The further antimicrobial agent may be an antifungal agent derivatives (including thymol and triclosan), Quarternary or antibacterial agent. The method of treating or preventing ammonium compounds (including Alkyldimethylethylben a bacterial infection or colonisation in a Subject, may also Zyl Ammonium Chloride, benzalkonium chloride, cetylpyri comprise the administration of robenidine with a further dinium chloride, benzethonium chloride, cetrimonium); antimicrobial agent. ANTITUBERCULAR AGENTS, including , 0074. In one embodiment, the antifungal agent is selected Delamanid, Ethambutol, Ethionamide, (Ftivaz from the group comprising, but not limited to naturally ide), Morinamide, p-Aminosalicylic Acid (PAS), Protiona occurring agents including Echinocandins (Anidulafungin, mide, Pyrazinamide, Terizidone. , Tiocarlide; Caspofungin, Micafungin), Polyenes (Amphotericin B, Can ARSENICALS, including Arsanilic Acid, Roxarsone; BAC dicidin, Filipin, Fungichromin (Pentamycin), Hachimycin, TERIOCINS, including Nisin, (PMX-30063); Hamycin, Lucensomycin, Mepartricin, Natamycin, Nysta |B-LACTAM , including ; tin, Pecilocin, Perimycin), and other naturally occurring B-LACTAM CARBAPENEMS, including , antifungal agents including Griseofulvin, Oligomycins, Pyr Doripenem, Ertapenem, , Imipenem, Mero rolnitrin, Siccanin, and Viridin. The antifungal agent may be , , , , Sulopenem, a synthetic compound selected from the group comprising, , : B-LACTAM CEPHA but not limited to Allylamines (Butenafine, Naftifine, Ter LOSPORINS, including , , , binafine) (Bifonazole, Butoconazole, Chlormi , , , Cefaloridine, Cefa US 2017/007 1884 A1 Mar. 16, 2017

lothin, , , , , Furazolium Chloride, Nifuratel, Nifurfoline, Nifuroxazide, , Cefazolin, , , , Nifurpirinol, , Nifurzide, Nitrofural, , , , Cefnmenoxime, Cefodiz toin, Nitrofurazone; , including ime, , , , , Cefo Dimetridazole, , Omidazole, Ronidazole, taxime, , , , Ce?pimizole, Secnidazole, : OLIGOSACCHARIDES, includ Ce?piramide, Ce?pirome, , , Cefaui ing Avilamycin, Eveminomicin; OTHER ANTIBACTE nome, , , , Ceftaroline, RIAL AGENTS, including Auriciosene, Chloroxine, Chlo , , , , , rquinaldol, , , Halquinol, Lotilibcin, , , Ceftolozane, Ceftradine, Ceftr , Methenamine (hexamine), Nitazole, eZole, Ceftriaxone, CeftroXadine, Cefuroxime, , , PerchloZone, Taurolidine, Thenoic Acid, Pivcefalexin: B-LACTAM CEPHAMYCINS, including Xibomirnol; OXAZOLIDINONES, including , Linezolid, , , , Tedizolid (Tor , Cefnmetazole, Cefninox, Cefotetan, Cefoxi ezolid); PEPTIDE DEFORMYLASE INHIBITORS, includ tin: B-LACTAM MONOBACTAMS, including Aztreonam, ing GSK1322322; PEPTIDES, including Omiganan, Pexi , ; B-LACTAM , ganan; PLEUROMUTILINS, including Retapamulin, including , , Moxalactam; B-LACTAM , ; POLYETHER IONOPHORES, PENICILLINS, including Amdinocillin (), including Laidlomycin, Lasalocid, Maduramicin, Monensin, Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, AZidocil Narasin, Salinomycin, Semduramicin; POLYMYXINS, lin, , , , , including Colistin, B: POLYPEPTIDES, includ , Clemizole , Clometocillin, , ing Amphomycin, Bacitracin, Capreomycin, Enduracidin, Cyclacillin, , , Fenbenicillin, Floxacil Enramycin, Enviomycin, Fusafungine, (s), Ise lin (), , Lenampicillin, Mecillinam, ganan, Magainins, Nosiheptide, Ristocetin, Thiostrepton, , Methicillin Sodium, Meziocillin, , Tuberactinomycin, , , Viomycin; Oxacillin, , Penethamate Hydriodide, Penicillin PSEUDOMONIC ACIDS, including : QUINO G, Penicillin G BenZathine, Penicillin G Procaine, Penicillin LONES, including , , Oxolinic N, Penicillin O, Penicillin V. Phenethicillin Potassium, Pip Acid, Ozenoxacin, , : QUI eracillin, , , , Qui NOXAUNES, including Carbadox, Olaquindox: RIMI nacillin, , , , Temocil NOFENAZINES, including Clofazimine; STATINS, includ lin, ; BICYCLOMYCINS, including ing Atorvastatin, Fluvastatin, Lovastatin, Mevastatin, Bicozamycin; BORON CONTAINING ANTIBACTERIAL Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin; AGENTS, including AN3365 (aminomethylbenzoxa STREPTOGRAMINS, including Dalfopristin, Flopristin, boroles), GSK2251052 (leucyl-tRNA synthetase inhibitors): Linopristin, , Quinupristin, ; CYCLIC ESTERS, including Fosfomycin; FATTY ACID STREPTOTHRICINS, including ; SULFO SYNTHESIS INHIBITORS (FabI), AFN-1252, NAMIDES, including Acetyl Sulfamethoxypyrazine, MUTO56399, FAB-001; FLUOROQUINOLONES, includ ing Avarofloxacin, , , Chinfloxacin, Chloramine-B, Chloramine-T, Dichloramine T. Formosul , Ciprofloxacin, , , fathiazole, , N4-Sulfanilylsulfanilamide, Nopryl , , , , Fina sulfamide, N-Sulfanilyl-3,4-xylamide, Ormaosulfathiazole, floxacin, , , , Gatifloxa Phthalylsulfacetamide, Phthalylsulfathiazole, Salazosul cin, , , , Levofloxa fadimidine. Succinylsulfathiazole, Sulfabenzamide, Sulfac cin, , , Miloxacin, arbamide, , Sulfachlorpyridazine, Sulfach Moxifloxacin, , , , Orbi rysoidine, Sulfaclozine, Sulfacytine, , floxacin, , , , Pruli , , , Sulfa floxacin, , , , , doxine, Sulfaethidole, Sulfaguanidine, Sulfaguanole, Sul , , , , falene, Sulfaloxic Acid, , Sulfameter, Sul Zabofloxacin: FUSIDANES, including Fusidic Acid; GLY famethazine, , Sulfamethomidine, COLIPODEPSIPEPTIDE, including ; GLYCO , , Sulfamethyl PEPTIDES, including Avoparcin, Dalbavancin, Norvanco thiazole, Sulfametopyrazine, , Sulfami mycin, , Teicoplanin, , Vancomycin; dochrysoidine, Sulfamonomethoxine, , Sulfa GLYCOPHOSPHOLIPIDS, including Bambermycins nilamide, Sulfanily lurea, Sulfaperine, , (bambermycin, moenomycins, flavophospholipol): GLY Sulfaproxyline, Sulfapyrazine, , Sulfaquinoxa CYLCYCLINES, including Tigecycline: HYBRIDS, Cada line, , , Sulfatroxazole, Sulfiso Zolid (Oxazolidinone-quinolone), TD-1792 (glycopeptide midine, Sulfisoxazole (); SULFONES, includ ); LINCOSAMIDES, including Clindamycin, ing Acediasulfone, , Glucosulfone Sodium, , ; LIPOPEPTIDES, including Dap p-Sulfanilylbenzylamine. Succisulfone, Sulanilic Acid, Sul tomycin, Surotomycin; , including Azithro foxone Sodium, Thiazolsulfone; TETRACYCLINES, mycin, Carbomycin, , , including , , Demeclocy , , Fidaxomicin, , cline, Doxycycline, , Guamecycline, Lymecy Gamithromycin, , , Leucomycin, cine, Meciocycline, Methacycline, Minocycline, Oxytetra Meleumycin, Midecamycins, Miokamycin, Mirosamycin, cycline, , Pipacycline, , , Primycin, , Rosaramicin, Rox , Tetracycline. ithromycin, Sedecamycin, , , 0077. The composition of the invention may further , Terdecamycin, Tildipirosin, Tilmicosin, Tro comprise an excipient selected from the group comprising, leandomycin, Tulathromycin, , Tylvalosin: NITRO but not limited to, binders and compression aids, coatings FURANS, including Furaltadone. Furazidin, , and films, colouring agents diluents and vehicles disinte US 2017/007 1884 A1 Mar. 16, 2017

grants, emulsifying and Solubilising agents, flavours and (0093 FIG. 9 shows a graph of the effect of NCL812 on Sweeteners, repellents, glidants and lubricants, plasticisers, cell wall macromolecular synthesis in Staphylococcus preservatives, propellants, solvents, stabilisers, Suspending aureus (ATCC29213) according to example 2: agents and viscosity enhancers. (0094 FIG. 10 shows a graph of the effect of NCL812 on 0078. According to a further aspect of the invention, lipid macromolecular synthesis in Staphylococcus aureus there is provided a medical device when used in a method of (ATCC29213) according to example 2: treating or preventing a bacterial infection in the Subject. 0.095 FIG. 11 shows a graph summarising the effect of 0079 According to further aspect of the invention, there NCL812 on macromolecular synthesis in Staphylococcus is provided a medical device comprising the composition of aureus (ATCC29213) according to example 2: the invention. The composition of the invention may be any (0096 FIG. 12 shows a graph of the effect of NCL812 on slow release form, and/or in the form of a coating of the ATP release from Staphylococcus aureus (ATCC29213) medical device. according to example 3: 0080. The medical device may be in a form selected from the group comprising: an implant, a plaster, a bandage, and 0097 FIG. 13 shows a table of Staphylococcus aureus other dressing applied to a bacterial infection in a Subject. clone/isolate name, type, Source, antibiogram, clindamycin 0081. According to further aspect of the invention, there resistance status, multi-locus sequence type (MLST), is provided a method of killing bacteria, the method includ staphylococcal cassette chromosome (SCCmec) type, clonal ing the step of contacting the bacteria with robenidine, or a complex, Panton-Valentine leukocidin status (PVL), and spa therapeutically acceptable salt thereof. type for isolates used according to example 4. MSSA: 0082. According to further aspect of the invention, there methicillin-susceptible S. aureus. HA-MRSA; hospital-ac is provided the use of robenidine, or a therapeutically quired methicillin-resistant S. aureus. CA-MRSA; commu acceptable salt thereof, to kill bacteria, said use comprising nity-associated methicillin-resistant S. aureus. M. B; M. the step of contacting the bacteria with robenidine, or a Barton (University of South Australia). G: Gribbles pathol therapeutically acceptable salt thereof. ogy (South Australia). J. P.; J. Perry (University of Adelaide). VIMP, Nares of students from Veterinary Immu 0083) Terms used herein will have their customary mean nology, Microbiology, & Public Health (University of ings in the art unless specified. Adelaide). S. P. S. Polyak (University of Adelaide). G. C.; Geoff Coombs (PathWest Laboratory Medicine, Western BRIEF DESCRIPTION OF THE DRAWINGS Australia). Em; Erythromycin. Ci: Ciprofloxacin. Gn; Gen 0084. Further features of the present invention are more tamicin. Tm; Trimethoprim. Te; Tetracycline. FA; Fusidic fully described in the following description of several non Acid. Rf; Rifampicin. Mp; Mupirocin: limiting embodiments thereof. This description is included 0.098 FIG. 14 shows a table of the percentage of pre solely for the purposes of exemplifying the present inven Sumptively identified S. aureus isolates reporting positive to tion. It should not be understood as a restriction on the broad selected phenotypic and genotypic tests according to Summary, disclosure or description of the invention as set Example 4. HA-MRSA; hospital-acquired S. aureus. CA out above. The description will be made with reference to MRSA; community-associated S. aureus. S. aureus isolates the accompanying drawings in which: were identified as testing positive to protein. A latex agglu I0085 FIG. 1 shows a table of the Minimum Inhibitory tination (Protein A), slide , Voges-Proskauer and Concentrations for the individual Staphylococcus aureus resistance tests, as well as testing positive for isolates according to example 1: polymerase chain reaction (PCR) and real-time PCR ampli I0086 FIG. 2 shows a table of the Minimum Inhibitory fication of the Spa gene. Methicillin-resistant S. aureus Concentrations for the individual Enterococcus isolates isolates were identified as isolates testing positive to the according to example 1: criteria described above, as well as positive for PCR and I0087 FIG. 3 shows a table of the Minimum Inhibitory real-time PCR of the mecA gene: Concentrations for the individual Streptococcus pneumoniae 0099 FIG. 15 shows a table of the resistance of S. aureus isolates according to example 1: isolates to antibacterial agents using the Kirby-Bauer disc 0088 FIG. 4 shows a table of the NCL812 MICs, diffusion method according to Example 4. HA-MRSA: MIC, MIC mode and MIC range for Australian isolates of hospital-acquired methicillin-resistant S. CaS MRSA, VRE and Str. pneumoniae according to example 1. CA-MRSA; community-associated methicillin-resistant S. Comparative MIC values for ampicillin are shown in paren attrett.S, thesis; 0100 FIG. 16 shows a table of the number and percent I0089 FIG. 5 shows a table of the Minimum Inhibitory age of identified mec gene complexes in 20 S. aureus strains Concentrations values for NCL812 (robenidine) and lin classified as methicillin-resistant according to Example 4. eZolid against Staphylococcus aureus ATCC29213 accord Respective staphylococcal cassette chromosome (SCCmec) ing to example 2: complexes and types expressing phenotypic resistance to 0090 FIG. 6 shows a graph of the effect of NCL812 on oxacillin and cefotetan are indicated as well as real-time DNA macromolecular synthesis in Staphylococcus aureus mecA status, and the average negative dF/dT peak obtained according to example 2: from melting point analysis from real-time PCR of the mecA 0091 FIG. 7 shows a graph of the effect of NCL812 on gene. Figures in parentheses indicate percentages; RNA macromolecular synthesis in Staphylococcus aureus 0101 FIG. 17 shows a graph showing the average melt according to example 2: ing point peaks for the negative derivative plot-dF/dT after 0092 FIG. 8 shows a graph of the effect of NCL812 on real-time polymerase chain reaction of the mecA gene in protein macromolecular synthesis in Staphylococcus aureus methicillin-resistant S. aureus isolates grouped by mec gene (ATCC29213) according to example 2: complexes, A (n=4), B (n=10), C2 (n=4) and unclassified US 2017/007 1884 A1 Mar. 16, 2017

(n2). Groups indicated with different SuperScripts are sig 0111 FIG. 27 shows a graph illustrating the 12-hour nificantly different (P<0.05), according to Example 4; time-kill of S. pneumoniae strain D39 treated with NCL812, 01.02 FIG. 18 shows a table of the characteristics of adopted from FIG. 40 according to example 5: antibacterial NCL812 and the B-lactam antibacterial ampi 0112 FIG. 28 shows a graph illustrating the 48-hour cillin according to Example 4, detailing antibacterial Solu time-kill of S. pneumoniae strain D39 treated with amplicil bility in dimethyl sulfoxide (DMSO), solubility in cation lin according to example 5: adjusted Mueller-Hinton II broth (CAMHB), and average 0113 FIG. 29 shows a graph illustrating the 48-hour minimum inhibitory concentrations (MIC) (ug/ml at 24-h) time-kill of S. pneumoniae strain D39 treated with erythro against methicillin-resistant S. aureus (MRSA) determined mycin according to example 5: from preliminary studies and those determined during this 0114 FIG. 30 shows a graph illustrating the 48-hour present study. ATCC 49775; methicillin-susceptible S. time-kill of S. pneumoniae strain D39 treated with NCL812 aureus isolate and ATCC control strain. MRSA580; methi and 5% choline chloride according to example 5: cillin-resistant S. aureus isolate #580. MRSA698; methicil 0115 FIG. 31 shows a graph illustrating the 12-hour lin-resistant S. aureus isolate #698; time-kill of S. pneumoniae strain D39 treated with NCL812 and 5% choline chloride according to example 5: (0103 FIG. 19 shows a table of in vitro activities of the 0116 FIG. 32 shows a graph illustrating the relative novel antibacterial NCL812 and the 1-lactam antibacterial minimum bactericidal concentration (MBC) of S. pneumo ampicillin against S. aureus clinical isolates according to niae strain D39 treated with amplicillin over a 48-hour time example 4. HA-MRSA; hospital-acquired methicillin-resis period according to example 5: tant S. aureus. CA-MRSA; community-associated methicil 0117 FIG. 33 shows a graph illustrating the relative lin-resistant S. aureus. MIC: minimum inhibitory concen MBC for S. pneumoniae strain D39 treated with erythromy tration (ug/ml). MBC, minimum bactericidal concentration cin over a 48-hour time period according to example 5: (ug/ml). MIC/MBCrange; minimum and maximum MIC/ 0118 FIG. 34 shows a graph illustrating the viable count MBC for all isolates. MIC/MBC50; MIC/MBC at which (logo CFU/ml) of S. pneumoniae strain D39 treated with 50% of isolates are inhibited. MIC/MBC90; MIC/MBC at NCL812 from a macro-broth dilution of time-kill over 24 which 90% of isolates are inhibited; hours according to example 5: 0104 FIG. 20 shows a graph of the optical densities of 0119 FIG. 35 shows a graph illustrating the viable count the unsupplemented growth control, ampicillin and different (logo CFU/ml) of S. pneumoniae strain D39 treated with concentrations of antibacterial agent NCL812 against methi ampicillin from a macro-broth dilution of time-kill over 24 cillin-susceptible S. aureus ATCC 49775 using broth hours according to example 5: microdilution methodology according to example 4. The I0120 FIG. 36 is a bar graph illustrating the mean cell concentrations of NCL812 tested were at the MIC and four membrane thickness of treated and untreated D39 according times the MIC determined under test conditions, up to 24-h to example 5: incubation. Ampicillin was tested at the MIC. Bactericidal I0121 FIG. 37 is a bar graph illustrating the mean width activity was tested at 0-, 1-, 2-, 4-, 8-, 12-, and 24-h for of periplasmic space of treated (16 ug/ml NCL812) and antibacterials; untreated D39 samples according to example 5: 0105 FIG. 21 shows a graph of kill kinetic curves for 0.122 FIG. 38 is a table showing the Staphylococcus methicillin-susceptible S. aureus ATCC 49775 demonstrat pseudintermedius isolates tested according to example 6: ing bactericidal activity of NCL812 using the Clinical and I0123 FIG. 39 is a table showing the antibiotic resistance Laboratory Standards Institute macrodilution methodology profile of the Staphylococcus pseudintermedius isolates in a 10-mi vial according to example 4. The concentrations tested according to example 6. of antibacterials tested were at 1.x and 4x the MIC deter 0.124 FIG. 40 is a graph illustrating the effectiveness of mined under test conditions. Bactericidal activity was deter NCL812 against gram-negative E. coli spheroplasts accord mined at 0-, 1-, 2-, 4-, 8-, 12- and 24-h after antibacterial ing to example 7: addition. Bactericidal activity was defined as a 3-log 10 0.125 FIG. 41 is a graph illustrating the cumulative (99.9%) decrease in the number viable bacteria from the release of NCL812 from Formulation B according to initial inoculum size; example 9: 0106 FIG. 22 shows a table of the antibacterial suscep 0.126 FIG. 42 shows the kill kinetics assay of Staphylo tibility of 20 S. pneumoniae isolates for six different anti coccus aureus KCO1 at different concentrations of NCL812, bacterials according to example 5: up to 24 h incubation according to example 11; and 0107 FIG. 23 shows a graph indicating the change of pH I0127 FIG. 43 shows the kill kinetics assay of Entero during macro-broth dilution assay for S. pneumoniae strain coccus faecalis USA01 at different concentrations of D39 exposed to 4 lug/ml in NCL812 and 0.0023 ug/ml NCL812, up to 24 h incubation according to example 11. ampicillin according to example 5: DESCRIPTION OF EMBODIMENTS 0108 FIG. 24 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with NCL812 General according to example 5: I0128. Before describing the present invention in detail, it 0109 FIG. 25 shows a graph illustrating in the 14-hour is to be understood that the invention is not limited to time-kill of S. pneumoniae strain D39 treated with NCL812 particular exemplified methods or compositions disclosed according to example 5: herein. It is also to be understood that the terminology used 0110 FIG. 26 shows a graph illustrating the 14-hour herein is for the purpose of describing particular embodi time-kill of S. pneumoniae strain D39 treated with amplicil ments of the invention only, and is not intended to be lin according to example 5: limiting. US 2017/007 1884 A1 Mar. 16, 2017

0129. All publications referred to herein, including pat effective amounts can be dosages that are recommended in ents or patent applications, are incorporated by reference in the modulation of a diseased state or signs or symptoms their entirety. However, applications that are mentioned thereof. Effective amounts differ depending on the compo herein are referred to simply for the purpose of describing sition used and the route of administration employed. Effec and disclosing the procedures, protocols, and reagents tive amounts are routinely optimized taking into consider referred to in the publication which may have been used in ation pharmacokinetic and pharmacodynamic connection with the invention. The citation of any publica characteristics as well as various factors of a particular tions referred to herein is not to be construed as an admission patient, such as age, weight, gender, etc and the area affected that the invention is not entitled to antedate such disclosure by disease or disease causing microbes. by virtue of prior invention. 0.136. As referred to herein, the terms “treatment” or 0130. In addition, the carrying out of the present inven “treating refers to the full or partial removal of the symp tion makes use of unless otherwise indicated, conventional toms and signs of the condition. For example, in the treat microbiological techniques within the skill of the art. Such ment of a bacterial infection or colonisation, the treatment conventional techniques are known to the skilled worker. completely or partially removes the signs of the infection. 0131. As used herein, and in the appended claims, the Preferably in the treatment of infection, the treatment singular forms “a”, “an', and “the include the plural unless reduces or eliminates the infecting bacterial pathogen lead the context clearly indicates otherwise. ing to microbial cure. 0132 Unless otherwise indicated, all technical and sci 0.137 As referred to herein, the term “bacteria' refers to entific terms used herein have the same meanings as com members of a large of prokaryotic microorganisms. monly understood by one of ordinary skill in the art to which Typically a few micrometres in length, bacteria have a this invention belongs. Although any materials and methods number of shapes, ranging from spheres to rods and spirals similar to, or equivalent to, those described herein may be and can be present as individual cells or present in linear used to carry out the present invention, the preferred mate chains or dusters of variable numbers and shape. Preferably rials and methods are herein described. the terms “bacteria” and its adjective “bacterial' refer to 0133. The invention described herein may include one or bacteria Such as the Gram positive Staphylococcus spp. more ranges of values (e.g. size, concentration, dose etc). A Streptococcus spp., Bacillus spp., Enterococcus spp., Listeria range of values will be understood to include all values spp., Mycoplasma spp., and anaerobic bacteria; Gram nega within the range, including the values defining the range, tive Escherichia coli, Enterobacter spp., Klebsiella spp and and values adjacent to the range that lead to the same or Pseudomonas spp.; and the cell wall free bacteria such as Substantially the same outcome as the values immediately Mycoplasma spp and Ureaplasma spp. The terms may refer adjacent to that value which define the boundary of the to an antibiotic-sensitive strain or an antibiotic-resistant range. strain. In a preferred embodiment, the terms refer to MRSA 0134. The pharmaceutical for veterinary compositions of or MRSP. In another preferred embodiment, the terms refer the invention may be administered in a variety of unit to MDR Staphylococcus spp., Streptococcus spp., Enterococ dosages depending on the method of administration, target cus spp., Clostridium difficile, Escherichia coli, Enterobacter site, physiological state of the patient, and other medica spp., Klebsiella spp and Pseudomonas spp. ments administered. For example, unit dosage form Suitable 0.138 Referred to herein, the term “methicillin-resistant for oral administration include Solid dosage forms such as bacteria' (such as methicillin-resistant Staphylococcus) powder, tablets, pills, and capsules, and liquid dosage forms, refers a bacteria isolate that demonstrates resistance at any Such as elixirs, syrups, solutions and Suspensions. The active dose to all 3 lactams including penicillins, carbapenems and ingredients may also be administered parenterally in sterile first to fourth generation cephalosporins, but not to the fifth liquid dosage forms. Gelatin capsules may contain the active generation anti-MRSA cephalosporins (for example ceftaro ingredient and inactive ingredients such as powder carriers, line). Multidrug-resistant (MDR) is defined as acquired , , , , , cellulose or cel non-Susceptibility to at least one agent in three or more lulose derivatives, magnesium Stearate, Stearic acid, sodium antimicrobial categories, extensively drug-resistant (XDR) saccharin, talcum, magnesium carbonate, and the like. is defined as non-susceptibility to at least one agent in all but 0135 The phrase “therapeutically effective amount’ as two or fewer antimicrobial categories (i.e. bacterial isolates used herein refers to an amount sufficient to inhibit bacterial remain Susceptible to only one or two categories) and growth associated with a bacterial infection or colonisation. pandrug-resistant (PDR) is defined as non-susceptibility to That is, reference to the administration of the therapeutically all agents in all antimicrobial categories currently available. effective amount of robenidine according to the methods or (0.139. An example of susceptible, MDR, XDR and PDR compositions of the invention refers to a therapeutic effect in bacteria includes the following. Wild type, antibacterial which substantial bacteriocidal or bacteriostatic activity unexposed isolates of Staphylococcus aureus that are likely causes a substantial inhibition of bacterial infection. The to be susceptible to all of the following antibacterial cat term “therapeutically effective amount’ as used herein, egories (and agents): aminoglycosides (for example gen refers to a sufficient amount of the composition to provide tamicin); ansamycins (for example rifampicin); anti-MRSA the desired biological, therapeutic, and/or prophylactic cephalosporins (for example ceftaroline); anti-staphylococ result. The desired results include elimination of bacterial cal B-lactams (or cephamycins) (for example oxacillin or infection or colonisation or reduction and/or alleviation of cefoxitin); carbapenems (for example ertapenem, imipenem, the signs, symptoms, or causes of a disease, or any other meropenem or doripenem); non-extended spectrum cepha desired alteration of a biological system. An effective losporins; 1st and 2nd generation cephalosporins (for amount in any individual case may be determined by one of example cefazolin or cefuroxime); extended-spectrum ordinary skill in the art using routine experimentation. In cephalosporins; 3rd and 4th generation cephalosporins (for relation to a pharmaceutical or veterinary composition, example cefotaxime or ceftriaxone); cephamycins (for US 2017/007 1884 A1 Mar. 16, 2017

example cefoxitin or cefotetan); fluoroquinolones (for cinnamic acid, mandelic acid, methanesulfonic acid, ethane example ciprofloxacin or moxifloxacin); folate pathway Sulfonic acid, p-toluenesulfonic acid, salicylic acid, and the inhibitors (for example trimethoprim-sulphamethoxazole); like. fucidanes (for example fusidic acid); glycopeptides (for 0142. The pharmaceutically or veterinary acceptable salts example Vancomycin, teicoplanin or telavancin); glycylcy of the compounds useful in the present disclosure can be clines (for example tigecycline); lincosamides (for example synthesized from the parent compound, which contains a clindamycin); lipopeptides (for example daptomycin); basic or acidic moiety, by conventional chemical methods. macrollides (for example erythromycin); oxazolidinones (for Generally, Such salts can be prepared by reacting the free example linezolid or tedizolid); phenicols (for example acid or base forms of these compounds with a stoichiometric chloramphenicol); phosphonic acids (for example fosfomy amount of the appropriate base or acid in water or in an cin); streptogramins (for example quinupristin-dalfopristin; organic solvent, or in a mixture of the two; generally, and tetracyclines (for example tetracycline, doxycycline or nonaqueous media like ether, ethyl acetate, ethanol, isopro minocycline). Isolates that are non-Susceptible to more than panol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences. 17th ed., one agent in more than three antimicrobial categories are Mack Publishing Company, Easton, Pa. (1985), p. 1418, the classified as MDR (all MRSA, for example, meet the disclosure of which is hereby incorporated by reference. definition of MDR). Isolates that are non-susceptible to Examples of Such acceptable salts are the iodide, acetate, more than one agent in all but one or two antimicrobial phenyl acetate, trifluoroacetate, acrylate, ascorbate, benzo categories are classified as XDR. Isolates that are non ate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, susceptible to all listed antibacterial agents are PDR. methoxybenzoate, methylbenzoate, o-acetoxybenzoate, 0140) Pharmaceutically and veterinary acceptable salts naphthalene-2-benzoate, bromide, isobutyrate, phenylbu include salts which retain the biological effectiveness and tyrate, Y-hydroxybutyrate, 3-hydroxybutyrate, butyne-1,4- properties of the compounds of the present disclosure and dioate, hexyne-1,4-dioate, hexyne-1,6-dioate, caproate, which are not biologically or otherwise undesirable. In many caprylate, chloride, cinnamate, citrate, decanoate, formate, cases, the compounds disclosed herein are capable of form fumarate, glycollate, heptanoate, hippurate, lactate, malate, ing acid and/or base salts by virtue of the presence of amino maleate, hydroxymaleate, malonate, mandelate, mesylate, and/or carboxyl groups or groups similar thereto. Acceptable nicotinate, isonicotinate, nitrate, oxalate, phthalate, base addition salts can be prepared from inorganic and terephthalate, phosphate, monohydrogenphosphate, dihy organic bases. Salts derived from inorganic bases, include by drogenphosphate, metaphosphate, pyrophosphate, propio way of example only, sodium, potassium, lithium, ammo late, propionate, phenylpropionate, salicylate, sebacate. Suc nium, calcium and magnesium salts. Salts derived from cinate, Suberate, Sulfate, bisulfate, pyrosulfate, Sulfite, organic bases include, but are not limited to, salts of primary, bisulfite, Sulfonate, benzenesulfonate, p-bromophenylsul secondary and tertiary amines, such as by way of example fonate, chlorobenzenesulfonate, propanesulfonate, ethane only, alkyl amines, dialkyl amines, trialkyl amines, Substi Sulfonate, 2-hydroxyethanesulfonate, methanesulfonate, tuted alkyl amines, di(Substituted alkyl)amines, tri(Substi naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-tolu tuted alkyl)amines, alkenyl amines, dialkenyl amines, tri enesulfonate, Xylenesulfonate, tartarate, and the like. alkenyl amines, Substituted alkenyl amines, di(Substituted 0143. The pharmaceutical or veterinary compositions of alkenyl)amines, tri(Substituted alkenyl)amines, cycloalkyl the invention may be formulated in conventional manner, amines, di(cycloalkyl)amines, tri(cycloalkyl)amines, Sub together with other pharmaceutically acceptable excipients stituted cycloalkyl amines, disubstituted cycloalkyl amines, if desired, into forms suitable for oral, parenteral, or topical trisubstituted cycloalkyl amines, cycloalkenyl amines, administration. The modes of administration may include di(cycloalkenyl) amines, tri(cycloalkenyl) amines, Substi parenteral, for example, intramuscular, Subcutaneous and tuted cycloalkenyl amines, disubstituted cycloalkenyl intravenous administration, oral administration, topical amines, trisubstituted cycloalkenyl amines, aryl amines, administration and direct administration to sites of infection diaryl amines, triaryl amines, heteroaryl amines, dihet Such as intraocular, intraaural, intrauterine, intranasal, intra eroaryl amines, triheteroaryl amines, heterocyclic amines, mammary, intraperitoneal and intralesional. diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine 0144. The pharmaceutical or veterinary compositions of are different and are selected from the group consisting of the invention may be formulated for oral administration. alkyl, substituted alkyl, alkenyl, substituted alkenyl, Traditional inactive ingredients may be added to provide cycloalkyl, Substituted cycloalkyl, cycloalkenyl, Substituted desirable colour, taste, stability, buffering capacity, disper cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. sion, or other known desirable features. Examples include Also included are amines where the two or three substitu red iron oxide, silica gel, sodium laurel Sulphate, titanium ents, together with the amino nitrogen, form a heterocyclic dioxide, edible white ink, and the like. Conventional or heteroaryl group. diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as Sustained 0141 Pharmaceutically and veterinary acceptable acid release compositions for the continual release of medication addition salts may be prepared from inorganic and organic over a period of time. Compressed tablets may be in the form acids. The inorganic acids that can be used include, by way of Sugar coated or film coated tablets, or enteric-coated of example only, hydrochloric acid, hydrobromic acid, Sul tablets for selective disintegration in the gastrointestinal furic acid, nitric acid, phosphoric acid, and the like. The tract. Liquid dosage forms for oral administration may organic acids that can be used include, by way of example contain colouring and/or flavouring to increase patient com only, acetic acid, propionic acid, glycolic acid, pyruvic acid, pliance. As an example, the oral formulation comprising oxalic acid, malic acid, malonic acid, Succinic acid, maleic NCL812 may be a tablet comprising anyone, or a combi acid, fumaric acid, tartaric acid, citric acid, benzoic acid, nation of the following excipients: calcium hydrogen phos US 2017/007 1884 A1 Mar. 16, 2017 phate dehydrate, microcrystalline cellulose, lactose, techniques such as those known in the art. Nanotechnology hydroxypropyl methyl cellulose, and talc. based drug delivery systems have the advantage of improv 0145 The compositions described herein may be in the ing bioavailability, patient compliance and reducing side form of a liquid formulation. Examples of preferred liquid effects. compositions include Solutions, emulsions, injection solu 0152 The formulation of the composition of the inven tions, Solutions contained in capsules. The liquid formula tion includes the preparation of nanoparticles in the form of tion may comprise a solution that includes a therapeutic nanosuspensions or nanoemulsions, based on compound agent dissolved in a solvent. Generally, any solvent that has solubility. NanoSuspensions are dispersions of nanosized the desired effect may be used in which the therapeutic agent drug particles prepared by bottom-up or top-down technol dissolves and which can be administered to a Subject. ogy and stabilised with Suitable excipients. This approach Generally, any concentration of therapeutic agent that has may be applied to robenidene which has poor aqueous and the desired effect can be used. The formulation in some lipid solubility in order to enhance saturation solubility and variations is a solution which is unsaturated, a saturated or improve dissolution characteristics. An example of this a Supersaturated Solution. The solvent may be a pure solvent technique is set out in Sharma and Garg (2010) (Pure drug or may be a mixture of liquid solvent components. In some and polymer-based nanotechnologies for the improved solu variations the solution formed is an in situ gelling formu bility, stability, bioavailability, and targeting of anti-HIV lation. Solvents and types of Solutions that may be used are drugs. Advanced Drug Delivery Reviews, 62: p. 491-502). well known to those versed in such drug delivery technolo Saturation solubility will be understood to be a compound gies. specific constant that depends on temperature, properties of 0146 The composition described herein may be in the the dissolution medium, and particle size (<1-2 um). form of a liquid Suspension. The liquid Suspensions may be 0153. The composition of the invention may be provided prepared according to standard procedures known in the art. in the form of a nanosuspension. For nanosuspensions, the Examples of liquid Suspensions include micro-emulsions, increase in the Surface area may lead to an increase in the formation of complexing compounds, and stabilising saturation solubility. NanoSuspensions are colloidal drug Suspensions. The liquid Suspension may be in undiluted or delivery systems, consisting of particles below 1 um. Com concentrated form. Liquid Suspensions for oral use may positions of the invention may be in the form of nanosus contain Suitable preservatives, antioxidants, and other pensions including nanocrystalline Suspensions, Solid lipid excipients known in the art functioning as one or more of nanoparticles (SLNs), polymeric nanoparticles, nanocap dispersion agents, Suspending agents, thickening agents, Sules, polymeric micelles and dendrimers. NanoSuspensions emulsifying agents, wetting agents, solubilising agents, sta may be prepared using a top-down approach where larger bilising agents, flavouring and Sweetening agents, colouring particles may be reduced to nanometre dimensions by a agents, and the like. The liquid Suspension may contain variety of techniques known in the art including wet-milling and water. and high-pressure homogenisation. Alternatively, nanosus 0147 The composition described herein may be in the pensions may be prepared using a bottom-up technique form of an oral paste. The oral paste may be prepared where controlled precipitation of particles may be carried according to standard procedures known in the art. out from Solution. 0148. The composition is described herein may be in the 0154 The composition of the invention may be provided form of a liquid formulation for injection, such as intra in the form of a nanoemulsion. Nanoemulsions are typically muscular injection, and prepared using methods known in clear oil-in-water or water-in-oil biphasic systems, with a the art. For example, the liquid formulation may contain droplet size in the range of 100-500 nm, and with com polyvinylpyrrolidone K30 and water. pounds of interest present in the hydrophobic phase. The 014.9 The composition is described herein may be in the preparation of nanoemulsions may improve the solubility of form of topical preparations. The topical preparation may be robenidine described herein, leading to better bioavailabil in the form of a lotion or a cream, prepared using methods ity. Nanosized suspensions may include agents for electro known in the art. For example, a lotion may be formulated static or steric stabilisation Such as polymers and Surfactants. with an aqueous or oily base and may include one or more Compositions in the form of SLNs may comprise biode excipients known in the art, functioning as Viscosity enhanc gradable lipids such as triglycerides, steroids, waxes and ers, emulsifying agents, fragrances or perfumes, preserva emulsifiers such as soybean lecithin, egg lecithin, and poloX tive agents, chelating agents, pH modifiers, antioxidants, and amers. The preparation of a SLN preparation may involve the like. For example, the topical formulation comprising dissolving/dispersing drug in melted lipid followed by hot or NCL812 may be a gel comprising anyone, or a combination cold homogenisation. If hot homogenisation is used, the of the following excipients: PEG 4000, PEG 200, glycerol, melted lipidic phase may be dispersed in an aqueous phase propylene glycol. The NCL812 compound may further be and an emulsion prepared. This may be solidified by cooling formulated into a solid dispersion using SoluPlus (BASF, to achieve SLNs. If cold homogenisation is used, the lipidic www.soluplyS.com) and formulated with anyone, or a com phase may be solidified in liquid nitrogen and ground to bination of the following excipients: PEG 4000, PEG 200, micron size. The resulting powder may be subjected to glycerol, propylene glycol. high-pressure homogenisation in an aqueous Surfactant solu 0150. For aerosol administration, the composition is of tion. the invention they be provided in finely divided form 0155 Robenidine as described herein may be dissolved together with a non-toxic Surfactant and a propellant. The in oils/liquid lipids and stabilised into an emulsion formu surfactant is preferably soluble in the propellant. Such lation. Nanoemulsions may be prepared using high- and Surfactants may include esters or partial esters of fatty acids. low-energy droplet reduction techniques. High-energy 0151. The compositions of the invention may alterna methods may include high-pressure homogenisation, ultra tively be formulated using nanotechnology drug delivery Sonication and microfluidisation. If the low-energy method US 2017/007 1884 A1 Mar. 16, 2017

is used, solvent diffusion and phase inversion will generate the basis of their colony appearance on this agar and a spontaneous nanoemulsion. Lipids used in nanoemulsions identification as Staphylococcus aureus was determined may be selected from the group comprising triglycerides, using colony characteristics on non-selective Sheep Blood Soybean oil, safflower oil, and sesame oil. Other components Agar and phenotypic characteristics such as , Such as emulsifiers, antioxidants, pH modifiers and preser positive test, positive coagulase test (tube coagulase Vatives may also be added. test using rabbit plasma) and clumping factor (agglutination 0156 The composition may be in the form of a con with the Oxoid Staphytect latex test), positive Voges trolled-release formulation may include a degradable or Proskauer test, and the ability to produce acid from treha non-degradable polymer, hydrogel, organogel, or other lose. A positive cefoxitin resistance screen confirmed the physical construct that modifies the release of the polyether isolates as MRSA. All Enterococcus isolates underwent a ionophore. It is understood that such formulations may standard biochemical identification. Biochemical profiling include additional inactive ingredients that are added to provisionally identified four of the VRE isolates as Entero provide desirable colour, stability, buffering capacity, dis coccus faecalis and the remainder as Enterococcus faecium, persion, or other known desirable features. Such formula however this is not 100% reliable for human Enterococcus tions may further include liposomes, such as emulsions, strains and full biochemical profiling using API-ZYM pro foams, micelles, insoluble monolayers, liquid crystals, phos filing will be undertaken to 100% confirm identity. All Str. pholipid dispersions, lamellar layers and the like. Liposomes pneumoniae isolates were identified on the basis of standard for use in the invention may be formed from standard biochemical profiling. vesicle-forming lipids, generally including neutral and nega tively charged phospholipids and a sterol. Such as choles Preparation of Antimicrobials terol. 0157. The formulations of the invention may have the (0162 Analytical grade NCL812 (batch 20081214) with a advantage of increased solubility and/or stability of defined potency of 1000 mg/g (ie 100%) was obtained The NCL812, particularly for those formulations prepared using powder was stored at a temperature of -20° C. at the study nanotechnology techniques. Such increased stability and/or site, in a locked freezer. Aliquots (1 ml) of Stock Solution stability of NCL812 may improve bioavailability and (25.6 mg/ml) were prepared in DMSO and stored at -80°C. enhance drug exposure for oral and/or parenteral dosage and defrosted immediately before use. forms. 0158. Throughout this specification, unless the context Minimum Inhibitory Concentration Assay requires otherwise, the word “comprise' or variations such 0163 Minimum inhibitory concentration tests were per as “comprises” or “comprising, will be understood to imply formed according to CLSI Standards (CLSI 2008). 90 uL of the inclusion of a stated integer or group of integers but not one of the test compound solutions, or amplicillin, was added the exclusion of any other integer or group of integers. to the end column of a 96 well plate that contained 90 uL of CAMHB in each well. The solutions were then serially EXAMPLES diluted across the row, leaving 2 columns for positive and Example 1 negative controls. A bacterial Suspension was prepared by adding fresh colonies obtained from an overnight culture on The Minimum Inhibitory Concentrations (MIC) for Sheep Blood Agar (SBA) to a 9.1 g/L saline solution. This NCL812 in Methicillin-Resistant Staphylococcus Suspension was adjusted to a concentration of between aureus (MRSA), Vancomycin-Resistant 4x10' and 5x10 CFU/mL. Concentration of the suspension Enterococcus Spp. (VRE) and Streptococcus was determined by measuring optical density (OD) using a pneumoniae spectrophotometer at a wavelength of 600 nm where the correct concentration was determined to have an optical 0159. In this example and other examples in the specifi density of between 1.00 and 1.20. One millilitre of this cation, the term NCL812 is used to indicate robenidine. suspension was added to 9 mL of saline before being added 0160 This study was undertaken to determine minimum to all wells, excluding the negative control wells, in 10 LI inhibitory concentrations (MIC) for a new antibacterial volumes giving a final concentration of between 4x10 and agent, NCL812. The antibacterial agent represents a poten 5x10 CFU/mL in each well. The tests were then incubated tially new class of drug with a perceived narrow spectrum of for 24 hours at 37° C. and then assessed both visually and activity against bacteria and a novel mechanism of action. using OD readings from a microplate reader at a wavelength This study focused on recent isolates of three major oppor of 600 nm. These tests were performed in duplicate but tunistic pathogens of humans where the development of repeated if discrepancies were observed. antibacterial resistance to existing antibacterial classes is 0164. The minimum inhibitory concentration (MIC) was problematic: methicillin-resistant Staphylococcus aureus determined to be the lowest concentration of antibiotic that (MRSA), Vancomycin-resistant Enterococcus spp. (VRE) prevented growth of bacteria both visually and using OD and Streptococcus pneumoniae. readings. Direct statistical comparisons between the test compounds and amplicillin could not be performed in light of Materials and Methods confidential information restrictions, such as restrictions on disclosure of information relating to the compound struc Bacterial Isolate Collection and Identification ture, such as molecular weight. Instead, MIC values were 0161 Sixty one test isolates were sourced from clinical collated and used to determine the lowest concentration of diagnostic microbiology laboratories. The MRSA isolates each compound that was effective against 50% and 90% of were originally cultured on selective Brilliance MRSA Chro isolates, referred to as the MICs and MICso respectively. mogenic Agar (Oxoid). Suspect colonies were selected on These values, as well as the range of MIC values, were then US 2017/007 1884 A1 Mar. 16, 2017 used for direct comparisons between test compounds and for and the culture was grown to early exponential growth phase general comparisons with amplicillin. (OD600–0.2 to 0.3) while incubating in a shaker at 35° C. and 200 rpm. Results (0165 Ampicillin MIC values obtained for the ATCC DNA, RNA, and Protein Synthesis control strains were within the normal range expected on the 0171 When cells reached early exponential phase, 100 ul basis of CLSI recommendations. The NCL812 and ampi of culture was added to triplicate wells containing various cillin MIC values for each isolate are indicated in FIG. 1 concentrations of test compound or control antibiotics (5ul) (MRSA isolates), FIG. 2 (VRE isolates) and FIG. 3 (Str. at 20x the final concentration in 100% DMSO. A 5% DMSO pneumoniae isolates). treated culture served as the “no drug control for all (0166 The pooled MIC50, MIC90, MIC mode and MIC experiments. Cells were added in MHBII at 105% to account range for NCL812 for each of the species of bacteria tested for the volume of drug added to each reaction or in M9 are shown in FIG. 4. minimal medium for protein synthesis reactions. Following (0167 NCL812 MIC values were remarkably consistent 15 min incubation at room temperature, either 3H thymi within and between each of the three species. MIC50 and dine (DNA synthesis), 3H uridine (RNA synthesis) or 3H MIC90 values were both equal (4 g/ml) for MRSA, VRE leucine (protein synthesis) was added at 0.5-1.0 uCi per and Str. pneumoniae isolates, with less than 10% of isolates reaction, depending on the experiment. Reactions were showing MIC values either 1-2 dilutions below or only one allowed to proceed at room temperature for 15-30 min and dilution above this figure. then stopped by adding 12 ul of cold 5% trichloroacetic acid (TCA) or 5% TCA/2% casamino acids (protein synthesis Example 2 only). Reactions were incubated on ice for 30 min and the TCA precipitated material was collected on a 25 mm GF/A Effect of NCL812 on Staphylococcus aureus filter. After washing three times with 5 ml of cold 5% TCA, Macromolecular Synthesis the filters were rinsed two times with 5 ml 100% ethanol, allowed to dry, and then counted using a Beckman LS3801 Materials and Methods liquid Scintillation counter. Test Compounds Cell Wall Synthesis 0168 Test compound NCL812 was transported to the 0172 Bacterial cells in early exponential growth phase experimental facility under conditions of ambient tempera were transferred to M9 minimal medium and added to 1.5 ml ture and then stored at 2-8°C. until assayed. Stock solutions eppendorf tubes (100 ul/tube) containing various concentra were made by dissolving NCL812 dry powder in 100% tions of test compound or control antibiotics (5ul) at 20x the DMSO to a concentration of 6,400 ug/ml. final concentration in 100% DMSO as described above. Following a 5 min incubation at 37° C., 14CN-acetylglu Minimal Inhibitory Concentration Testing cosamine (0.4 uCi/reaction) was added to each tube and incubated for 45 min in a 37° C. heating block. Reactions (0169. The MIC assay method followed the procedure were stopped through the addition of 100 ul of 8% SDS to described by the Clinical and Laboratory Standards Institute, each tube. Reactions were then heated at 95°C. for 30 min or CLSI (Clinical and Laboratory Standards Institute). Meth in a heating block, cooled, briefly centrifuged, and spotted ods for Dilution Antimicrobial Susceptibility Tests for Bac onto pre-wet HA filters (0.45uM). After washing three times teria That Grow Aerobically, Approved Standard Eighth with 5 ml of 0.1% SDS, the filters were rinsed two times Edition. CLSI document M07-A8 ISBN 1-56238-689-1. with 5 ml of deionized water, allowed to dry, and then Clinical and Laboratory Standards Institute, 940 West Valley counted using a Beckman LS3801 liquid scintillation coun Road, Suite 1400, Wayne, Pa. 19087-19898 USA, 2009), ter. and employed automated liquid handlers to conduct serial dilutions and liquid transfers. The medium employed for the Lipid Synthesis MIC assay was Mueller Hinton II Broth (MHB II Becton Dickinson, Sparks, MD; Cat No 212322; Lot 9044411). S. 0173 Bacterial cells were grown to early exponential aureus ATCC 29213 served as the quality control strain, and growth phase in MHBII broth and added to 1.5 mileppendorf linezolid was utilized as the quality control antibiotic to tubes (in triplicate) containing various concentrations of test validate the assay. NCL812 and linezolid were both dis compound or control antibiotics as described above. Fol solved in 100% DMSO before addition to the growth lowing a 5 min incubation at room temperature, 3H medium. glycerol was added at 0.5 uCi per reaction. 0.174 Reactions were allowed to proceed at room tem Macromolecular Synthesis Assays perature for 15 min and then stopped through the addition of 375ul chloroform/methanol (1:2) followed by vortexing for Bacteria and Growth Conditions 20 seconds after each addition. Chloroform (125 ul) was then added to each reaction, vortexed, followed by the (0170 The effect of NCL812 on whole cell DNA, RNA, addition of 125 ul dH2O and vortexing. Reactions were cell wall, protein and lipid synthesis was investigated using centrifuged at 13,000 rpm for 10 min, and then 150 ul of the S. aureus ATCC 29213. Cells were grown at 35° C. over organic phase was transferred to a Scintillation vial and night on . A colony from the plate was allowed to dry in a fume hood for at least 1 hr. Samples were used to inoculate 10 ml of Mueller Hinton broth II (MHBII), then counted via liquid Scintillation counting. US 2017/007 1884 A1 Mar. 16, 2017

Results of pathways was observed, all five macromolecular synthe (0175 Susceptibility testing was conducted with NCL812 sis reactions were similarly sensitive to NCL812. and S. aureus ATCC 29213 to determine the concentrations of drug needed in the macromolecular synthesis assays. Example 3 (0176 FIG. 5 shows that the MIC for NCL812 was 4 ug/mL, while the quality control agent lineZolid was within Effect of NCL812 on ATP Release from the CLSI-established quality control range (Clinical and Staphylococcus aureus Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing, Nineteenth Informa Materials and Methods tional Supplement. CLSI document M100-S20 ISBN 1-56238-716-2. Clinical and Laboratory Standards Insti Test Compounds tute, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087 0.184 The test compound NCL812 was shipped under 1898 USA, 2010). Precipitation of NCL812 was observed at conditions of ambient temperature and then stored at 2-8°C. 28 ug/mL in plates that were prepared in an identical until assayed. Stock Solutions were made by dissolving fashion, but did not receive an inoculum of S. aureus. NCL812 dry powder in 100% DMSO to a concentration of Macromolecular synthesis inhibition studies were per 1,600 ug/ml. The comparator agent was polymyxin B formed using concentrations of NCL812 that were equiva (Sigma, P-4932 (Lot 044K11905)). lent to 0, 0.25, 0.5, 1, 2, 4 or 8-fold the MIC value (4 ug/ml) for S. aureus ATCC 29213 (FIGS. 6-11). Test Organism 0177 FIG. 6 shows the effect of NCL812 on DNA synthesis. NCL812 demonstrated no inhibition at 0.25-fold 0185. S. aureus ATCC 29213 was originally acquired the MIC, 40% inhibition at 0.5-fold, and approximately 95% from the American Type Culture Collection (Manassas, Va.). inhibition at the MIC. This is compared to the control ciprofloxacin which showed approximately 51% at 8-fold ATP Release Assay the MIC (0.5 g/ml). (0178. The results for NCL812 inhibition of RNA synthe 0186. The CellTiter-Glo Luminescent Cell Viability sis were very similar to the DNA synthesis study, with Assay (Promega) was utilized to measure the leakage of ATP rifampicin serving as the positive control (FIG. 7). It should from bacteria. Cultures were grown to early exponential be noted that precipitation was observed at 4 to 8-fold the phase (0.2-0.3 optical density units at 600 nm) in Mueller MIC in the Mueller Hinton broth II utilized in the DNA and Hinton Broth II and then treated with seven different con RNA synthesis assays. centrations of either NCL812 or polymyxin B (positive 0179 Protein synthesis was inhibited in a dose-depen control) utilizing the MIC for each compound as a guide (0. dent manner at 0.25, 0.5, and 1-fold the MIC value of 0.25, 0.5, 1, 2, 3, 4, or 8-fold the MIC). The negative control NCL812 showing up to 97% inhibition at the MIC (FIG. 8). received 2% DMSO, which represented the final DMSO Linezolid demonstrated approximately 61% inhibition of concentration in each assay. After a 30 min exposure to drug, protein synthesis at 8-fold the MIC (2 ug/ml). Precipitation cells were sedimented by centrifugation and the Supernatant of NCL812 occurred at 4 and 8-fold the MIC in the protein was analyzed for the presence of ATP. Results were synthesis assay. expressed as ATP concentration released to the medium 0180. In FIG. 9, NCL812 also showed a somewhat dose (LM). dependent inhibition of cell wall synthesis, though there was a large increase in inhibition from 1 to 2-fold the MIC. Results However, inhibition dropped to approximately 68% and 52% at 4-fold and 8-fold the MIC, respectively. Precipitation 0187. The MIC for NCL812 has been previously deter of NCL812 occurred at 2, 4, and 8-fold the MIC in the M9 mined to be 4 ug/ml. The ATP release assay is conducted by minimal medium used for the cell wall synthesis assay, and growing S. aureus to exponential phase and then adding the that is the likely cause of the decline in inhibition. In antibiotic at multiples of the MIC in an effort to detect a comparison, the positive control Vancomycin showed 96% dose-dependent response. inhibition at 8-fold the MIC (2 ug/ml). 0188 As shown in FIG. 12, the positive control poly 0181 NCL812 demonstrated a similar inhibition profile myxin B released ATP from S. aureus cells in a dose against lipid synthesis as that shown for DNA and RNA dependent fashion with maximal release of approximately synthesis, reaching approximately 90% inhibition at the 0.34 uM ATP at 8-fold the MIC (256 ug/ml). ATP release in MIC (FIG. 10). The positive control inhibitor cerulenin the presence of NCL812 was dose-dependent at 0.5-1 fold demonstrated 72% inhibition at 8-fold the MIC (32 lug/ml). the MIC, resulting in maximal release (0.33 uM) observed at 0182 FIG. 11 represents a composite of all five macro the MIC (4 ug/ml). ATP release actually decreased thereafter molecular synthesis reactions. It can be observed that the at 2 to 8-fold the MIC. It should be noted that in previous inhibition curves were similar for each pathway, Suggesting studies, precipitation of NCL812 was observed at 4 to 8-fold a global inhibition of several pathways simultaneously by the MIC in Mueller Hinton broth II. NCL812. It is possible that NCL812 targets the cell mem 0189 In summary, NCL812 demonstrated dose-depen brane, causing leakage of essential ions and/or metabolites, dent release of ATP from actively growing S. aureus cells. thereby leading to a global shutdown of the cell synthesis ATP release from the cells into the growth medium reached pathways. maximum levels at the MIC value, and this was followed by 0183. In summary, NCL812 inhibited DNA, RNA, pro a decrease in ATP release at higher doses. The data indicated tein, cell wall, and lipid pathways in a growing culture of S. that NCL812 may interact with the cell membrane of S. aureus. Though some instances of dose-dependent inhibition aureus, causing leakage of vital metabolites such as ATP. US 2017/007 1884 A1 Mar. 16, 2017 20

Example 4 primers (Invitrogen, Australia). Conventional PCR amplifi cation was performed in a 20-ul Volume containing 10-ul In Vitro Antibacterial Activity of NCL812 Against HotStarTaq Plus Master Mix (Qiagen, Australia), 0.5-uM of Methicillin-Resistant and Methicillin-Susceptible each spa primer, 0.2-1 uM of each mecA primer, and 3-li of Staphylococcus aureus extracted DNA. An automated thermal cycler (T100 Ther mal Cycler, Bio-Rad) was used for PCR amplification of the Materials and Methods Spa and mecA genes.

Antimicrobial Agents TABLE 1.

0.190 Aliquots of stock solution of NCL812 (25.6-mg/ PCR and RT PCR reaction conditions ml) was prepared in dimethyl sulfoxide, stored at -80° C. and defrosted immediately before use. Ampicillin stock was Temperature Time obtained from Sigma-Aldrich (Australia). Antimicrobial (° C.) (seconds) Number of cycles discs were obtained from Thermo Fisher Scientific (Austra PCR stage lia). Enzyme activation 95 3OO 1 Amplification: 94 30 Microorganisms Denaturation Annealing 50 30 38 (0191 Twenty nine clinical isolates of MRSA were Extension 72 38 obtained (FIG. 13), along with S. aureus control organism Cooling 2O ce 1 ATCC 49775. Isolate identification was confirmed by con Real-time PCR stage ventional phenotypic methodologies, including the slide Enzyme activation 95 3OO 1 coagulase test, Vogues-Proskauer test, polymyxin B sensi 95 15 tivity (300-units), and Staphytect Plus Protein A latex slide Amplification: 50 2O 40 agglutination (Thermo Fisher Scientific Australia). Bacteria Denaturation were stored at -80° C. in 40% glycerol broth and routinely Annealing 70 40 Single acquisition 95 5 grown from Stock on sheep blood agar (SBA) incubated at Melting curve 55 2O 1 37°C. In subsequent experiments, only fresh cultures<24-h 95 O Continuous acquisition were used. Cooling 40 30 1 Isolate Resistotyping 0194 The mecA and spa amplified products of 325- and 0.192 Antibiotic-susceptibility profiling of the isolate 120-bp, respectively, were detected by GelRed staining collection was undertaken using Kirby-Bauer disc diffusion, followed by electrophoresis in 2% agarose gels. as recommended by the Clinical and Laboratory Standards Institute (CLSI) on Mueller-Hinton agar. Isolates were grown overnight on SBA at 37°C. Colonies were suspended Minimum Inhibitory Concentration Testing in physiological saline. Turbidity was adjusted to a 0.5 (0195 The in vitro activities of NCL812, and ampicillin McFarland standard and Suspensions were spread over the as a positive control, were determined by broth microdilu medium. Antibiotic discs were transferred onto the inocu tion as recommended by the CLSI in cation-adjusted Muel lated medium and analysed after 24-h incubation at 37° C. ler-Hinton II broth. Microtiter plates containing two-fold Isolates labelled as MRSA that were not f-lactam-resistant dilutions of each antimicrobial agent were inoculated with on the basis of the Kirby-Bauer test were grown from stock ~10-CFU/ml of each isolate in a 100-ul final volume. Plates on plate count agar Supplemented with 5-lug/ml amplicillin were incubated for 24-h at 37° C. Turbidity (absorbance at and subject to repeat testing, as Penicillin Binding Protein2a ODoo) was measured using a Bio-Rad Benchmark Plus expression can be induced by exposure to B-lactam antimi microplate spectrophotometer in Microplate Manager) ver crobials. sion 5.2.1 (Bio-Rad). Minimum inhibitory concentration Molecular Detection of the Protein A and mecA Genes to (MIC) endpoints were defined as the lowest antimicrobial Confirm MRSA Status concentration assessed by the spectrophotometer that inhib 0193 Isolate identities were confirmed genotypically ited bacterial growth. ATCC 49775 was included in the using a novel, duplex conventional polymerase chain reac isolate collection as a control organism using breakpoints tion (PCR) test targeting the spa (protein A) and mecA defined by the CLSI. The MICs, MIC (concentrations that (methicillin resistance) genes. In addition, the isolates were inhibited growth of the lower 50% and 90% of total organ tested in a mecA and spa Sybr green real-time PCR. isms, respectively), and MIC range (minimum and maxi Approximately ten colonies of each overnight bacterial mum) were calculated to profile the antimicrobial Suscepti subculture was suspended in 1x phosphate buffered saline bility of the isolate collection. (pH 7.4) and vortexed. Isolates were subject to DNA extrac tion using the QIAampO DNA Mini Kit (Qiagen, Australia) Bactericidal Activity following the manufacturers protocols. Template DNA was eluted in 50-ul of elution buffer and either used directly in (0196. The bactericidal activity of NCL812 was estab PCR, or stored at -20° C. prior to DNA amplification using lished by determination of the minimum bactericidal con the spa forward (5'-TGATACAGTAAATGACATTG-3") and centration (MBC) and time-kill analyses using CLSI guide reverse (5'-TTCTTATCAACAACAAGTTC-3) primers and lines. The MBC was defined as the lowest drug mecA forward (5'-TTCGTGTCTTTTAATAAGTGAGG-3') concentration at which 99.95% of the original inoculum was and reverse (5'-ATGAAGTGGTAAATGGTAATATCG-3') eliminated. US 2017/007 1884 A1 Mar. 16, 2017

(0197) Time-kill assays for ATCC 49775 were performed clavulanic acid, cefotetan, cephalexin, clindamycin, eryth in cation-adjusted Mueller-Hinton II broth in Microtiter romycin, oxacillin, and penicillin-G, whereas the majority of plates and again in 10-ml Volumes for macrodilution assays CA-MRSA isolates were resistant to only clindamycin, at antimicrobial concentrations equivalent to 1x and 4x the erythromycin, and penicillin-G. None of the isolates tested MIC. Bactericidal activity in macrodilution assays was were Vancomycin resistant. Overall, the most prevalent identified as a 3-logo decrease from the initial inoculum resistance phenotypes were penicillin-G (83.33%), erythro size. Bacteria were cultured overnight at 37° C. on SBA. mycin (73.33%), and clindamycin (43.33%), whilst only Colonies were suspended in broth and the turbidity was single isolates (3.33%) were resistant to trimethoprim-sul adjusted to a 0.5 McFarland standard to obtain a bacterial famethoxazole and rifampicin. suspension of ~10-CFU/ml. Bacterial suspensions were incubated at 37°C. with shaking. Aliquots were removed at mec Gene Complex Interactions 0-, 1-, 2-, 4-, 8-, 12-, and 24-h after antimicrobial addition, 0202 All MRSA isolates belonging to mec gene complex diluted, plated onto SBA and incubated for 48-hat 37°C. for A expressed resistance to both oxacillin and cefotetan (FIG. viable count determination. Turbidimetric growth curves for 16). However, only 20% of mec gene complex B MRSA S. aureus were obtained for Microtiter plate assays by isolates were phenotypically resistant to these antimicrobi monitoring optical density changes using a Bio-Rad Bench als. Of the MRSA isolates belonging to mec gene complex mark Plus microplate spectrophotometer at 600 nm. Optical C2, only a single isolate expressed methicillin resistance to densities were measured at 0-, 1-, 2-, 4-, 8-, 12, and 24-h oxacillin and only two isolates expressed resistance to after antimicrobial addition. cefotetan. Unclassified MRSA isolates expressed full resis tance to oxacillin and cefotetan. Statistical Methodology 0203 Melting point peaks for the mecA real-time PCR 0198 Microbiological data was interpreted using CLSI negative derivative plot -dF/dT differed between mec gene guidelines. Data was examined using the student's t-test, complex (P<0.003) (FIG. 17). On average, mec gene com Fisher's exact test, analysis of variance, and a generalized plex B and unclassified isolates demonstrated higher melting linear model for tests of between-subjects effects where point peaks than other SCCmec types (P<0.012). appropriate. Differences were considered significant at the 0.05 level in IBM SPSS(R) version 19.0 (University of Physical Properties and MIC of NCL812 Adelaide). (0204 Initial tests of NCL812 showed that it was soluble in DMSO, but produced a cloudy solution when dissolved in Results cation-adjusted Mueller-Hinton II broth (CAMHB) (FIG. 0199 Confirmation of Staphylococcus aureus Identity 18). In initial testing, NCL812 was found to have consistent and mecA Status MIC values (FIG. 18). 0200 Latex agglutination tests confirmed that all 30 isolates were protein A positive. The isolates tested positive In Vitro Antibacterial Activities: Minimum Inhibitory for coagulase activity using slide agglutination. Voges Concentrations Proskauer and polymyxin B resistance tests confirmed that 0205 MICs and MIC values for compound NCL812 all isolates were S. aureus except for a single methicillin (4- and 4-8-g/ml) are shown in FIG. 19. MIC values susceptible isolate; MSSA DE-25 (FIG. 14). Based on spa differed by S. aureus classification (susceptible, HA- or gene PCR amplification, this isolate was not identified as a CA-MRSA) (P<0.005). In many cases, NCL812 had sig S. aureus isolate despite testing positive in the protein A nificantly increased activity against CA-MRSA and methi latex agglutination and slide coagulase tests. This canine cillin-Susceptible staphylococci by one dilution when com origin Staphylococcus spp. was identified as Staphylococcus pared to HA-MRSA (P<0.002 and P-0.020, respectively), pseudintermedius based on biochemical characteristics. The however there were no significant differences between MIC mecA conventional and real-time PCR results confirmed that values for methicillin-susceptible staphylococci and CA 66.66% of the isolates were classified as methicillin-resis MRSA (P)-0.05). Ampicillin MIC values were within the tant on the basis of possession of the mecA gene. There were normal range expected on the basis of CLSI guidelines. no significant differences between the ability of conven tional and real-time PCR to detect the mecA gene (P-0.05). In Vitro Antibacterial Activities: Minimum Bactericidal Staphylococcus aureus Antimicrobial Susceptibility Profiles Concentrations 0201 Antimicrobial susceptibility assays revealed that (0206. The MBCs determined from NCL812 were equiva HA-MRSA isolates had the highest mean prevalence of lent to the MIC for 93.33% and 83.33% of S. aureus isolates, resistance to multiple antimicrobial classes (P<0.000). CA respectively (FIG. 19). In all remaining cases, MBCs were MRSA isolates were next most resistant (P<0.007), followed one dilution higher. For NCL812, MBCs ranged from 2-8- by methicillin-susceptible staphylococci (P<0.037) (FIG. ug/ml and 4-16-ug/ml, respectively. 15). Oxacillin resistance was expressed in only 80.00% and 10.00% of HA-MRSA and CA-MRSA isolates, respectively. Time-Kill Studies Cefotetan resistance was expressed in 80.00% and 20.00% of HA-MRSA and CA-MRSA isolates, respectively. 0207. In comparison to the turbidimetric growth curve of Although oxacillin and cefotetan did not significantly differ ATCC 49775, no visible bacterial growth was observed in their ability to detect MRSA (P>0.05), detection was when ATCC 49775 was inoculated into cation-adjusted significantly improved when using the mecA PCR when Mueller Hinton II broth supplemented with NCL812 at 1x compared to disc diffusion (P<0.013). The majority of and 4x the MIC in microdilution assays (P<0.033 and HA-MRSA isolates expressed resistance to amoxicillin P<0.038, respectively) (FIG. 20). US 2017/007 1884 A1 Mar. 16, 2017 22

0208. When analysed in 10-mi macrodilution assays, need to be administered every 8-h in future in vivo safety broth supplemented with antimicrobials at 1x and 4x the and efficacy experiments to maintain adequate systemic MIC and inoculated with ATCC 49775 displayed signifi concentrations, though it would appear from the time-kill cantly reduced viable counts for both NCL812 when com profile that the NCL compound series are concentration pared to the growth control (0.000

0215. The National Collection of Type Cultures (NCTC) TABLE 3-continued control strain D39 was used as a growth control for all MIC and MBC assays. D39 was later designated for kill kinetics, Interpretive Standards for Zone Diameters point of resistance assays and transmission electron micros Interpretive Standards for copy (TEM) studies as it is a well documented laboratory Zone Diameters (mm strain with a defined in vivo pathogenesis that displayed consistent NCL812 MICs and MBCs. Resis- Inter 0216 For all in vitro assays, fresh pneumococcal isolates Antibiotic tant mediate Sensi were grown overnight (O/N) on horse blood agar (HBA) Class Antimicrobial (Ig) (R) (I) tive Fluoroquin- Ciprofloxacin (5 g)* s22 s22 22 plates (39 g/L Columbia blood agar base Oxoid 5% V/v olone defribinated horse blood (Oxoid at 37° C. with 5% supple Folate pathway Trimethoprim- s15 16-18 e19 mented CO. Mueller-Hinton blood agar with 5% defibri inhibitor Sulphamethoxazole nated sheep blood (MHSBA Roseworthy Media and Blood (1.25/23.75 g) Glycopeptide Vancomycin (30 g) s17 Service) was used for disk diffusion analysis as directed by Lincosamide Clindamycin (2 g) s15 16-18 e19 Clinical Laboratory Standards Institute (CLSI) standards. Macrollide Erythromycin (15 g) s15 16-20 21 Pneumococci were routinely grown in broth consisting of Clarithromycin (15 g) s16 17-20 21 4% lysed horse blood (LHB) with Cation Adjusted Mueller Phenicol Chloramphenicol (30 g) s20 21 Hinton Broth (CAHMB, Difco) at 37° C. with 5% supple Rifamycin Rifampin (5 g) s16 17-18 e19 mented CO. Horse serum broth (HSB, 10% (v/v) donor Tetracycline Tetracycline (30 g) s18 19-22 e23 horse serum in nutrient broth 10 g/L peptone, 10 g/L Lab Lemco (Oxiod) and 5 g/L NaCl) was also used in some MIC 0219 Standardised bacterial suspensions were spread assays. Isolates were stored in HSB at -80° C. onto MHSBA using a sterile cotton Swab. Bacterial sus pensions from of Streptococcus pneumoniae were stan Antibiotic Stocks and Reagents dardised to an ODoo, between 0.08 and 0.1 using a spectrophotometer and then diluted 1:20. Bacterial colonies 0217 NCL812 was provided in dry powder form by were taken from an O/N horse blood agar plate. To ensure Neoculi. A total of 256 mg was dispensed into 10 ml of the purity of the 1:20 bacterial suspension, 50 uL was spread 100% dimethylsulphoxide (DMSO) to make a stock of 25.6 plated onto horse blood agar and incubated O/N at 37° C. mg/ml, which was then diluted 1:100 in CAHMB to make with 5% CO. The CFU was calculated and compared to the a final working stock of 256 ug/ml. Ampicillin dry powder initial plate counts. Antibiotic disks (Sigma) were placed was from Sigma A0166. The original 25.6 mg/ml stock was using a disk dispenser (Oxoid) according to CLSI standards. diluted in saline 1:100, 1:4, 1:20 and finally 1:16 in CAMHB MHSBA plates were incubated for 16 hrs-24 hrs at 37° C. in to make a final working Stock of 0.18 g/ml. Erythromycin 5% CO. Zones of complete inhibition were measured in was from Sigma E077 and choline chloride was from Roche triplicate to the nearest millimetre using a ruler on natural Diagnostics. Twenty micro litres of 0.05 g/ml erythromycin light-reflected growth, and the mode was represented as the was diluted 1:25 in 4.980 mls of CAMHB to give a final diameter for each isolate. Pneumococcal isolates were cat working stock of 0.2 g/ml. Choline chloride (0.5%) was egorised as sensitive, intermediate (I) or resistant (R) by added to 4% LHB:CAMHB for specific kill kinetic assays. CLSI standards and quality control (QC) ranges (FIG. 22). Defining Antimicrobial Susceptibility of Pneumococcal Determination of NCL812 MIC50, MIC90, MIC Range and Isolates MBC50, MBC90, MBC Range 0218 Isolate susceptibility to 12 different antimicrobials 0220 MICs for NCL812 for all isolates listed in Table 2 (FIG. 22) were determined by CLSI and European Com were determined by measuring optical density at 600 nm mittee on Antimicrobial Susceptibility Testing (EUCAST) (OD600 nm) (Spectramax spectrophotometer, Molecular methods. Antimicrobials were selected based upon the CLSI Devices Corporation) as an indicator of bacterial growth and EUCAST guidelines. Zone diameters for antimicrobials using 96-well microtitre trays after incubation for 24 hrs at other than Ciprofloxacin for S. pneumoniae were determined 37° C. in 5% CO. Micro-broth dilutions and 96-well trays by CLSI standards; whilst Zone diameters for Ciprofloxacin are prepared by the following method: 90 uL of 4% LHB: antimicrobial Susceptibility to S. pneumoniae were deter CAMHB is aliquotted into all wells using a multichannel mined by EUCAST. pipette. 90 ul of working antimicrobial stocks were no serial diluted down the tray by a 1:2 dilution. Negative broth TABLE 3 controls and dilution control were taken into account when planning the set up of a 96-well tray. 10 uL of bacterial Interpretive Standards for Zone Diameters suspension was then added to the appropriate wells in the 96 Interpretive Standards for well tray. Appropriate positive (no antimicrobial), negative Zone Diameters (mm (no antimicrobial or bacteria) and negative dilution (a serial Resis- Inter dilution control of antimicrobial and broth) controls were Antibiotic tant mediate Sensi included in each assay. MBC and plate counts for kill kinetic Class Antimicrobial (Ig) (R) (I) tive assays were determined by aliquotting 20 uL from each well of the 96-well microtitre tray onto HBA, and incubating at B-lactam Oxacillin (1 g) s20 s20 e20 Ampicillin (10 Ig) s20 s20 e20 37°C. with 5% CO2. The MBC was determined by a 99.95% Amoxicillin-clavulanate s20 s20 e20 inhibition of S. pneumoniae, taking into account the dilution (20/10 ug) factor. MICs and MBCs were determined in quadruplicate and the mode was taken as the representative value. The US 2017/007 1884 A1 Mar. 16, 2017 24

MIC50, MIC90 and MIC range and MBC50, MBC90 and points using pH indicator strips. Confluent growth was MBC range were determined according to CLSI standards. defined when more than 1000 colonies were counted per The MIC50 and MIC90, or MBC50 and MBC90, are defined plate. A bactericidal effect was defined as a 3-log 10-unit by the lowest concentrations which, when all the MICs and reduction (99.9%) of the original cell suspension determined MBCs of the isolates are arranged from lowest to highest, at 24 hrs for each concentration. inhibited the 50th and 90th percentile of the total amount of isolates, respectively. Point of Resistance Assay for NCL812 Micro-Broth Dilution Time Kill Studies with NCL812 Using Strain D39 0225 Macro-broth dilutions were prepared as above. 0221 Bacterial suspensions were added in triplicate to a Broth cultures of strain D39 (10 ml) were incubated in the 96-well microtitre tray containing NCL812 with a starting presence of 2 ug/ml and 4 ug/ml of NCL812, and 0.022 concentration of 128 Jug/ml and serially diluted 1:2 sequen ug/ml of Ampicillin for 6 hrs at 37°C. in 5% CO. Samples tially to a concentration of 0.25 ug/ml. Negative dilution were centrifuged at a relative centrifugal force (RCF) of controls were subtracted from the median growth value to 101.45xg for 10 mins and washed in 50 mls of phosphate obtain a suitable indicator of overall bacterial production. buffered saline (PBS) twice to remove any residual antimi The 96-well tray was incubated at 37° C. in 5% CO, and crobial, and/or bacterial end products and media. Washed read every 2 hrs for the first 12 hrs followed by final reads bacteria were resuspended and MICs were performed. at 24 and 48 hrs at 600 nm. To further supplement this data, a separate experiment in which a 96-well tray was read Effect of NCL812 on D39 Cell Membrane Ultra-Structure automatically at half hourly intervals using a spectropho tometer (Spectramax spectrophotometer, Molecular Devices Transmission Electron Microscopy Corporation) for 14 hrs was performed to confirm the trends 0226. Morphological appearance and morphometric in growth curves observed from original micro-broth dilu analysis of the cell membrane was determined using trans tion studies. mission electron microscopy (TEM). Bacterial Suspensions MBC Time Kill Studies with NCL812. Using Strain D39 and 10 ml cultures of D39 were prepared as before. Samples 0222 MBC kill kinetics assays involved the preparation were incubated at 37° C. in 5% CO, with gentle manual of three 96-well microtitre trays. At specific time points, tilting of the cultures every 10 mins. Cultures were exposed aliquots obtained from these trays provided viable counts to either 1 lug/ml. 4 ug/ml or 16 g/ml of NCL812 and following incubation at 37°C. in 5% CO on HBA, and the harvested at 6 or 12 hrs by centrifugation at 101.45xg for 20 MBC was determined after 24 hrs of growth. mins and washed twice in 50 mls of PBS. Critical time Macro-Broth Dilution Time Kill Studies of D39 with points for TEM work were determined by analysing trends NCL812 in the growth curves produced from the kill kinetics studies. 0223 Bacterial suspensions and working antibiotic Samples were resuspended in PBS containing 20% glycerol stocks were prepared as described above. For preparing and stored at -80° C. until required. Before fixation, 20% macro-broth dilutions, 20 ml tubes were filled each with 9 glycerol was removed by centrifugation and washing on ice mls of 4% LHB:CAMHB. 9 mls of a working antimicrobial three times in 50 mls of PBS. stock was diluted 1:2 when added to one of the tubes, and 0227 Samples were fixed using modified protocols then serial diluted down from a high to low concentration of defined by a previous study examining cell wall ultrastruc antimicrobial. 1 ml of S. pneumoniae bacterial Suspension ture of S. pneumoniae (Hammerschmidt, S. et al. 2005. was added to the appropriate tubes, including the positive Infect Immun 73:4653-4667). A lysine-acetate-based form control. Tubes were incubated at 37° C. with 5% CO, with aldehyde-glutaraldehyde ruthenium red-osmium fixation gentle manual tilting of the tubes treated with NCL812 every procedure involved fixing the bacterial pellets with a 10 mins for the first 12 hrs. At every 2-3 hrs during the first cacodylate buffer solution containing 2% formaldehyde, 12 hrs of growth and then at 24 hrs and 48 hrs, 50 u, of each 2.5% glutaraldehyde, 0.075% ruthenium red and 0.075 M of bacterial Suspension was spread plated onto HBA and incu lysine acetate for 1 hr. After washing with cacodylate buffer bated at 37° C. with 5% CO, for 16-24 hrs. containing 0.075% ruthenium red three times, a second fixation in cacodylate buffer solution containing 2% form TABLE 4 aldehyde, 2.5% glutaraldehyde and 0.075% ruthenium red was undertaken for 1.5 hrs. Cells were subsequently washed Concentration of antimicrobials three times with cacodylate buffer containing 0.075% ruthe Serial dilution NCL-812 (g/ml) Ampicillin (g/ml) nium red and underwent a final fixation in 1% osmium tetroxide in cacodylate containing 0.075% ruthenium red for 1 128 O.09 1 hr. The samples were then washed three times in cacody 2 64 O.O45 3 32 O.O23 late buffer containing 0.075% ruthenium red only. 4 16 O.O11 0228 Samples were washed and dehydrated using a 5 8 O.OO6S graded series of ethanol (70, 90, 95 and 100%) for 10-20 6 4 min, two times for each step. Samples were infiltrated using 7 2 50:50 LR White resin in 100% ethanol for 1 hr, and subsequently washed with 100% LRWhite resin for 1 hr and 0224 Cultures were incubated at 37°C. in 5% CO, with left O/N in a third change of 100% LR white to ensure gentle manual tilting every 10 mins for the first 12 hrs. adequate infiltration of resin. The samples were then embed Viable counts from 50 uL aliquots of each concentration ded in fresh LR White resin and incubated at 50° C. for 48 were read following incubation at 37°C. in 5% CO for 24 hrs. Sections were cut to 1 Lum using a glass knife, stained hrs. The pH of each sample was measured at specific time with Toluidene Blue and viewed under a light microcrope at US 2017/007 1884 A1 Mar. 16, 2017

400x to identify the presence of stained pneumococci. At resulted in a threefold increase in the MIC for D39 treated least four ultra-thin sections were then cut to 90 nm using a with NCL812 with a twofold increase for the positive diamond knife and placed on matrix grids, one section per ampicillin control. grid. Ultra-thin sections were then stained with uranyl acetate and lead citrate alternatively at 5 min intervals, TABLE 6 followed by three washes with distilled water in-between each exposure. Stained sections were then placed on grids Difference in activity of NCL812 in different media. and viewed between 25000x and 130000x on a Philips CM100 Transmission Electron Microscope. Images were obtained at 130000x magnification and analysed using Relative MIC with media type for D39 analysIS Olympus Soft Imaging Systems. 10% horse serum- Fold Statistical Analysis Antimicrobial 4% LHB:CAMHB Supplemented broth increase 0229 Statistical analyses were conducted using statistics NCL812 4 32 3 program GraphPad Prism (5th ed, GraphPad Software Inc.) Ampicillin O.O23 O.09 2 for Windows. For growth curves, data presented were the mean and standard error of mean (SEM) (represented as error bars) for each data point except for macro-broth 0235. There was no change in MIC for D39 with differing dilution studies where multiple replicates could not be storage conditions of pre-prepared 96-well microtitre trays. obtained due to the high costs involved in this assay. Two tailed, unpaired t-tests were performed. TABLE 7 Results Storage conditions of prepared micro titre trays for micro broth dilution. 0230 Pharmacodynamics of NCL812 in S. pneumoniae Quality Control Disk Diffusion Analysis for 20 S. pneumo Storage condition niae Isolates Antimicrobial -2° C. 4° C. 0231. Although nine out of the 12 antimicrobials used for NCL812 8 8 disk diffusion analysis had established QC ranges by Ampicillin O.O23 O.O23 EUCAST, QC ranges were not defined for amoxicillin clavulanate, clarithromycin and clindamycin (Table 3). 0232 WCH16 and WCH184 were both resistant to at 0236. During macro-broth dilutions, the pH of the media least two antimicrobials whereas EF3030 and WCH137 did not change compared to appropriate controls (FIG. 23). were intermediate and resistant to trimethoprim-Sulphame thoxazole respectively (FIG. 22). The other remaining 16 Determination of S. pneumoniae In Vitro Susceptibility to isolates were sensitive to all 12 antimicrobials. Sensitivity to NCL812 ampicillin was confirmed for each isolate, enabling the use Determination of NCL812 MIC50, MIC90, MIC range of amplicillin as a positive control in later micro-broth 0237 NCL812 exhibited a MIC50 and MIC90 of 8 g/ml dilution assays (FIG. 22). and MIC range of 4-8 ug/ml when tested against all 20 strains. The MIC for ampicillin was comparable to recent Solubility and Activity of NCL812 in Different Media published findings using micro-broth dilution as an endpoint 0233 NCL812 was brought in 100% DMSO but devel for antimicrobial resistance in pneumococcal isolates, thus oped turbidity when it was further diluted into CAMHB or confirming the accuracy of MICs obtained for NCL812. PBS. TABLE 8 TABLE 5 MIC and MBC valuesfor isolates treated Visual analysis of NCL812 and anpicillin solubility with NCL812 and anpicillin NCL812 Ampicillin NCL812 (ig/ml) Ampicillin (g/ml) Diluent MICso 8 O.O23 MICoo 8 O.O23 CAMHB Turbid Transparent MIC Range 4-8 O.O11-0.09 DMSO Transparent Transparent MBCso 8 O.O23 PBS Precipitate Transparent MBCoo 8 O.O23 Media MBC Range 4-8 O.O11-0.09

4% LEHB:CAMHB Turbid Transparent 10% horse serum- Precipitate Transparent supplemented broth Determination of NCL812 MBC50, MBC90, MBC Range 0234 Growth of S. pneumoniae strain D39 in an MIC 0238 Minimum bactericidal concentrations (MBC50, assay for NCL812 using 10% HSB (220 mls of horse serum MBC90 and MBC range respectively) were determined for is filtered to 10% in 180 mls of Lemco nutrient broth) NCL812 and ampicillin for all twenty isolates. US 2017/007 1884 A1 Mar. 16, 2017 26

TABLE 9 TABLE 10 MIC and MBC values for NCL812 and amplicillin MIC and MBC values of D39 exposed to 2 for each pneumococcal isolate ug/ml or 4 ug/ml of NCL812 for 6 hours NCL812 Ampicillin MIC of D39 MBC of D39 following following MIC MBC MIC MBC Original exposure to Original exposure to MIC of NCL812 for MBC of NCL812 for D39 4 8 O.O23 O.O23 D39 6 hrs. D39 6 hrs EF3030 8 8 O.O23 O.O23 A66.1 8 8 O.045 O.045 D39 exposed to 2 4 8 8 8 TIGR4 4 8 O.O23 O.O23 g/ml NCL812 WU2 4 8 O.O23 O.O23 D39 exposed to 4 4 8 8 8 L82O16 8 8 O.O23 O.O23 g/ml NCL812 P9 8 8 O.O23 O.O23 D39 exposed O.O23 O.O45 O.O23 O.O23 P21 4 8 O.O23 O.O23 0.023 g/ml WCH158 4 8 O.O23 O.O23 Ampicillin WCH89 4 4 O.O23 O.O23 D39 growth * 8 8 8 8 WCH57 8 8 O.O23 O.O23 D39 growth2 ** 8 8 8 8 WCH77 4 8 O.O23 O.O23 WCH46 4 4 O.O23 O.045 * D39 growth control: S. pneumoniae strain D39 grown for 6 hrs in 4% LHB:CAMHB. WCH86 4 8 O.O23 O.O23 ** D39 growth2 control: S. pneumoniae strain D39 on HBAON, resuspended in saline WCH137 4 8 O.O23 O.O23 (0.1 OD600) and diluted "/20 in sterile saline. WCH184 4 4 O.045 O.045 WCH16 8 autolysis O.O23 Autolysis WCH43 4 8 O.O23 O.O23 Micro-Broth Dilutions by Measuring Relative MBC at WCH92 8 8 O.09 O.09 Specific Time Points WCH211 4 8 O.O23 O.O23 0242 Relative MBCs were determined at specific time intervals from using broth dilution assays incubated for 48 hrs for NCL812 and control antimicrobials amplicillin and Micro-Broth Dilution Time Kill Studies of D39 Treated with erythromycin (FIGS. 32 and 33). MICs of ampicillin (0.023 NCL812 ug/ml) and erythromycin (0.00275 g/ml) for D39 were 0239 D39 exposed to sub-inhibitory concentrations (s2 similar in range to published findings for other pneumococ ug/ml) of NCL812 grew similar to unexposed controls over cal isolates. A comparative difference in growth between a 48 hour period (FIG. 24). Higher concentrations of NCL812, amplicillin, and erythromycin was observed (FIGS. NCL812 (>16 ug/ml) resulted in no bacterial growth for 48 32 and 33). Ampicillin and erythromycin demonstrated a hrs (FIG. 24). These growth characteristics were validated time-dependent reduction in bacteria. NCL812 exhibited by a micro-broth kill kinetic study using a Spectramax fast bactericidal action, evidenced by an approximate 3-fold spectrophotometer, which measured growth (represented as decrease in MBC within 5 hrs. A consistent bactericidal concentration (8 ug/ml) was maintained for the full 48 hrs OD600) at half-hourly intervals for 14 hrs for NCL812 and for NCL812. ampicillin (FIGS. 25 and 26). The commencement of expo Macro-Broth Dilution Time Kill Studies of D39 with nential growth for D39 treated with NCL812 is shown in NCL812 FIG. 27. 0243 Viable counts for each time point were represented 0240. The growth of D39 treated with NCL812 was as a log 10 CFU/ml reduction for NCL812 (FIG. 34) and compared to D39 treated with ampicillin or erythromycin ampicillin (FIG. 35). Consistent confluent growth (deter over 48 hrs (FIGS. 28 and 29). D39 treated with ampicillin mined by a limit of 2x10 CFU) was observed for unexposed exhibited similar growth to D39 exposed to NCL812 over 48 controls and 2 ug/ml NCL812. Complete bactericidal activ hrs (FIG. 28). Erythromycin-treated D39 produced very ity (defined by a 3-log reduction in CFU) for 128 ug/ml of different growth curves from NCL812 where a larger dif NCL812 was observed by a 4-log reduction of colony forming units (CFU) in 3 hrs and concentrations between 16 ference in growth between concentrations was observed ug/ml and 64 ug/ml NCL812 were effective at eliminating (FIG. 29). The addition of 5% choline chloride to the media bacterial growth within 8 hrs (FIG. 34). NCL812 at 4 g/ml over a 48 hour period resulted in no significant difference in and 8 ug/ml appeared to be inactivated at 11 hrs post growth for NCL812 compared to positive and growth con exposure, as increased growth of strain D39 after this time trols (FIGS. 30 and 31). point was observed (FIG. 34). Point of Resistance Testing Transmission Electron Microscopy 0244 Morphometric analysis revealed significant 0241 D39 treated with s4ug/ml NCL812 entered a log changes to the cell membrane in strain D39 exposed to 16 phase of growth at 6 hrs (FIG. 24), as shown in four ug/mL NCL812 for 6 hrs compared to growth controls. independent experiments. The possibility of antimicrobial Samples treated with 4 ug/ml as well as 12 hr cultures were resistance to NLC812 between 5 and 6 hrs was investigated not considered for morphemetric analysis due to the lack of by determining further MICs on D39 exposed to 2 ug/ml bacterial cells available in each section. Treated samples NCL812, 4 ug/ml NCL812 and 0.0225 ug/ml amplicillin for possessed significantly thicker cell membranes (6.43+0.29 6 hrs. Results showed no significant increase in MIC for all nm) compared to untreated samples (4.35+0.24 nm) (p<0. samples of D39 exposed to NCL812 compared to growth 0001) (FIG. 36). The periplasmic space (intracellular space controls, and amplicillin between the cell membrane and the cell wall) of D39 treated US 2017/007 1884 A1 Mar. 16, 2017 27 with 16 g/ml NCL812 was significantly wider (4.54+0.096 0250 In conclusion, this in vitro study has demonstrated nm) compared to untreated samples (3.91+0.14 nm) (p<0. that NCL812 has many desirable characteristics as a fast 001) (FIG. 37). acting concentration-dependent bactericidal antimicrobial that appears to target the cell membrane of S. pneumoniae. TABLE 11 These characteristics are desirable to treat acute pneumo coccal infections. As NCL812 may possess a mechanism of Mean cell membrane thickness and periplasmic Space action that targets the cell membrane, it will act much more Treatment (16 g/ml quickly than time-dependent antimicrobials such as Growth control NCL812 for 6 hrs) B-lactams and macrollides and potentially could be more Statistical test effective than other bactericidal concentration-dependent Unpaired t-test antimicrobials such as fluoroquinolones which have intrac Mean SEM Mean SEM (P value) ellular targets. Cell membrane 4.35 + 0.24 nm, 6.43 + 0.29 mm, P< O.OOO1 Example 6 n = 12 n = 13 Periplasmic 3.91 + 0.14 nm, 4.54 + 0.096 nm, P<0.001 Characterization of Methicillin-Susceptible and Space n = 11 n = 11 Methicillin-Resistant Isolates of Staphylococcus Pseudintermedius from Australia and Preliminary 0245. In summary, NCL812 produced highly consistent In Vitro Efficacy of a New Anti-Staphylococcal MICs and equivalent MBCs for the S. pneumoniae strain Compound collection, confirming that it is bactericidal against this organism. In kill kinetics experiments, which measured the Materials and Methods relative MBC over a 48 hr period, a consistent bactericidal effect was elicited in D39 after 6 hrs from initial exposure to Sample Collection and Identification of Methicillin NCL812. Susceptible Staphylococcus Pseudintermedius (MSSP) and 0246 This demonstration of bactericidal activity is the Methicillin Resistant Staphylococcus Pseudintermedius first to be observed in S. pneumoniae. This demonstrates that (MRSP) NCL812 is effective against pneumococcal in vitro. 0251 A total of 23 Staphylococcus pseudintermedius 0247 Competitive binding between components in isolates were obtained from dogs (FIG. 38). blood, serum or broth decreased the antimicrobial activity of 0252 Ten methicillin susceptible and 13 methicillin NCL812. This was reflected in the increase of MIC observed resistant Staphylococcus pseudintermedius were collected between different broth types and diluents. Following the for the study. Isolates were phenotypically classified as completion of these studies, recent independent research methicillin resistant on the basis of in vitro resistance to confirmed precipitation of NCL812 in PBS and reported oxacillin and genetically for the presence of mecA gene complete solubility in water containing 4% DMSO, follow according to standard procedures. ing initial dilution in 100% DMSO. A water-soluble 0253) Oxacillin and cefoxitin susceptibility testing using NCL812 will greatly improve in vivo bioavailability and disk diffusion technique and Epsilometer testing were per negative interaction between blood or serum proteins. formed. Identification of mecA gene was performed using 0248 Based on the findings of this study, NCL812 exhib polymerase chain reaction (PCR) its a mechanism of action against S. pneumoniae that is 0254 CLSI disk diffusion susceptibility testing was per different from lactam or macrollide classes, as it appears to formed on the 23 Staphylococcus pseudintermedius isolates exhibit concentration-dependent bactericidal activity as for the following antimicrobials: penicillin, amoxicillin, opposed to time-dependant qualities. Identifying the maxi erythromycin, gentamicin, clindamycin, ciprofloxacin, mum pharmacokinetic serum concentration of NCL812 in cephalexin, chloramphenicol, tetracycline, , Vivo will assist confirmation of its concentration-dependant Vancomycin, cefotetan, moxifloxacin and rifampin (FIG. pharmacodynamic activity. Furthermore, the addition of 39). choline chloride to the media confirmed that the mechanism 0255 Minimum inhibitory concentration (MIC) and of action for NCL812 is not associated with the affinity to minimum bactericidal concentration (MBC) testing was cell wall choline binding proteins, and therefore may not be undertaken using CLSI methodology for NCL812 and cell wall associated. included amplicillin as a control. Anti-staphylococcal com pounds were then tested againstall 23 isolates and minimum 0249 Morphometric analysis of the cell membrane and inhibitory concentrations (MIC) were determined according periplasmic space of D39 treated with 16 ug/ml NCL812 for to standard protocols. After the MICs were determined, the 6 hrs showed that the cell membrane and periplasmic space minimum bactericidal concentrations were performed to was larger in treated Samples, compared to control samples. determine if these compounds are bacteriostatic or bacterio The apparent increase in membrane size could be due to an cidal. accumulation of electron dense intracellular material beneath the cell membrane. The increase in the size of the periplasmic space may be have been due to disruption of the Results cell membrane, potentially by depolarisation or ATP inhibi 0256 The mecA gene was present in 13 isolates of MRSP tion. The mechanism of action of NCL812 may not be and negative in 10 MSSP All MRSP isolates were resistant calcium-dependant as it appears that no competitive binding to oxacillin based on disc diffusion (<=17 mm) and E-test between NCL812 and ruthenium red, a calcium channel MIC (>=0.5 mg/L). inhibitor of lipid bilayers, was observed in electron micro 0257. When cefoxitin resistance breakpoint was set at graphs. <=24 mm, 3/13 (23%) and 5/13 (38%) of MRSP tested were US 2017/007 1884 A1 Mar. 16, 2017 28 susceptible to cefoxitin. When cefoxitin resistance break 0263 Methicillin resistant Staphylococcus pseudinterme point was set at <30 mm, only 1/13 (7.7%) of MRSP tested dius (MRSP) is an emerging problem in dogs, cats and at Veterinary Diagnostic Laboratory was susceptible. horses. Two major clonal MRSP lineages have been reported 0258. The MRSP isolates were resistant to multiple anti from dogs in Europe (ST 71) and North America (ST 68). biotic classes. Of the 13 MRSP isolates, all 13 were sus There have also been reports of MRSP affecting dogs in ceptible to rifampin. 3/13 (23%) were susceptible to Japan and a single case of MRSP in a veterinary worker in chloramphenicol; 10/13 (77%) were susceptible to Vanco Hong Kong. mycin. 0264. In this study, MRSP isolates were determined using 0259 Interestingly, 3/13 (23%) of the MRSP isolates a combination of presence of mecA gene and in vitro were susceptible to amoxicillin: 8/13 (62%) were suscep resistance to oxacillin. Cefoxitin susceptibility has been tible to cephalothin; 12/13 (92%) susceptible to cefotetan used as a substitute for oxacillin for methicillin resistant Staphylococcus aureus. However, cefoxitin disk diffusion and 12/13 (92%) susceptible to moxifloxacin, tests using interpretive guidelines recommended for human 0260 All 23 isolates were susceptible to NCL812 based isolates of methicillin resistant Staphylococcus aureus and on MICs. In addition, NCL812 has been shown to be coagulase negative staphylococci are unreliable in identify bactericidal based on minimal bactericidal concentrations ing MRSP. A cefoxitin breakpoint resistance of <=30 (MBC). mm resistant and >=31=Susceptible has been proposed by 0261 The MIC range of NCL812 against the Staphylo Bemis et a, 2012. This study is in agreement that this coccus pseudintermedius isolates was found to be between breakpoint may be more reliable in predicting methicillin 1 Lig/mL and 4 g/mL resistant Staphylococcus pseudintermedius. MRSP isolates are generally resistant to multiple antibiotic classes. Bacte TABLE 12 rial culture and antibiotic susceptibilities are therefore rec ommended for all suspect MRSP infections to allow appro MIC range of NCL812 against Staphylococcus pseudintermedius priate selection of antibiotics. A limitation noted in this study isolates is the apparent in vitro susceptibility of MRSP isolates to Isolate AMP NCL812 amoxicillin and cephalosporins (cephalothin and cefotetan). S1P1 128 4 NCL812 was effective against all 23 isolates of both MSSP S2P2 128 2 and MRSP. A larger scale study is warranted to confirm the S3P3 128 2 effectiveness of NCL812 against Staphylococcus pseudin S4P4 128 1 termedius as it may provide a safe alternative antibiotic SSP5 16 2 S6P6 64 2 option for emerging MRSP infections in domestic animals. S7B7 128 2 S8P8 128 2 Example 7 S9P9 32 2 S1OP10 64 2 Activity of NCL812 Against Gram-Negative S11P11 128 4 S12P12 32 2 Organisms S13P13 O.25 2 S14P14 1 2 0265. The aim of this study was to determine if a target S15P15 4 4 of the antibacterial activity of NCL812 is present within S16P16 O.25 2 gram-negative cells. To determine if a target of NCL812 is S17P17 1 2 within the gram-negative cell, the outer membrane, and most S18P18 4 4 S1919 O.S 4 of the cell wall, was removed using amplicillin, then the S2OP20 4 4 modified cells (known as spheroplasts) were treated with S21P21 O.1 2 NCL812 at various concentrations. S22P22 8 4 S23P23 32 2 Induction of Spheroplast State 0266 E. coli ATCC 25922 was grown overnight on agar 0262 The MIC 50 and MIC 90 of NCL812 against the at 37°C. Two colonies from the overnight incubation were Staphylococcus pseudintermedius isolates was found to be 2 used to inoculate ~20 ml of cation adjusted mueller hinton ug/mL and 4 ug/mL respectively. The MIC mode and MIC broth. Inoculated broth was incubated for 18 hours at 37° C. range of NCL812 against the Staphylococcus pseudinterme Six ml of overnight broth culture was added to 20 ml of dius isolates was found to be 2 ug/mL and 1-4 ug/mL Supplemented cation adjusted mueller hinton broth (Supple respectively. mented with 50 mg/ml amplicillin, 0.4 M sucrose, 8 mM MgSO) and incubated overnight at 37° C. Formation of TABLE 13 spheroplasts was checked using a phase contrast micro Scope. Combined MIC values of NCL812 against Staphylococcus pseudintermedius isolates Activity of NCL812 Column1 AMP NCL812 0267 Three ml of spheroplast culture was added to each MIC50 (ig/ml) 32 2 test tube and 50 ul of DMSO containing the appropriate MIC90 (ig/ml) 128 4 MIC mode (ug/ml) 128 2 concentration of NCL812 was added to each test tube. Fifty MIC range (ig/ml) O.1-128 1-4 microlitres of DMSO only was added to the control test tube. Spheroplasts were incubated for 24 hours with 20 ul samples taken at 0, 2, 4, 6, 8 and 24 hours. Twenty microliter samples US 2017/007 1884 A1 Mar. 16, 2017 29 were serially diluted 1:10 and 10 ul samples of appropriate Formulation C Topical Formulation PEG-Based Gel dilutions were spotted in triplicate onto brain heart infusion with NCL812-Soluplus agar. Brain heart infusion agar was incubated at 37° C. for 0284 2.5 g PEG 4000: 48 hours and colonies were counted at 24 and 48 hours to 0285 4.0 g PEG 200; determine the number of colony forming units. 0286 2.5 g propylene glycol; (0287 1.0 g water; and Imaging of NCL812 Treated Spheroplasts (0288 1.8 g. solid dispersion of NCL812-SoluPlus. 0268 Samples were taken after 24 hours of exposure to 0289 Soluplus was purchased from BASF (www.solu the NCL812 compound, stained with Trypan blue and plus.com). NCL812-SoluPlus was prepared using standard imaged. methods in the art. 0290 PEG 4000, PEG 200, NCL812-SoluPlus and pro pylene glycol were mixed and heated to 150° C. and until all Results solid crystals were dissolve. Water was added and then the 0269. A spheroplast induction rate above 99% was con Solution was Sonicated. The solution was allowed to cool sistently observed. A target of NCL812 was found to be and solidify. Formulation C demonstrated acceptable vis present within E. coli cells with a significant decrease in the cosity, ease of skin application, uniform suspension and number of colony forming units observed over time as the consistent and fine texture. concentration of NCL812 increased 232 ug/ml (FIG. 40). The experiment was repeated in triplicate with one repre Formulation D. Tablet Formulation sentative shown in FIG. 40. Images of the spheroplasts taken 0291 30 mg Calcium hydrogen phosphate dehydrate; after 24 hours of exposure to the NCL812 compound 0292 80 mg Microcrystalline cellulose; showed the development of pleomorphic cells increasing in 0293 50 mg Lactose; frequency as the concentration of NCL812 increased (data 0294 8 mg Hydroxypropyl methyl cellulose not presented). These results show the effectiveness of 0295) 1.5 mg Talc NCL812 as an antibacterial agent against gram-negative 0296 10 mg of NCL812 bacteria. 0297. The excipients were weighed and mixed for 5 minutes. The mixture was fed into a feed hopper of a tablet Example 8 press machine and the machine was operated according to standard procedures in the art. Formulations of NCL812 0298 Formulation D demonstrated acceptable tablet 0270. The following formulations were prepared using hardness, disintegration and frability. standard methods in the art. Formulation a Topical Formulation PEG-Based Gel with Formulation E. Oral Suspension NCL812 0299 2.0 ml Glycerol; 0271 4.0 g PEG 4000; 0300 1.5 ml Absolute ethanol: (0272. 3.5g PEG 200; (0301 600 mg NCL812; and 0273 0.6 propylene glycol; (0302) To 60 ml Vehicle (Ora Sweet and Ora Plus, 1:1). 0274 1.9 g water; and (0303 NCL 812 powder was sieved through a 75 um 0275 0.204 g of NCL812. sieve. 600 mg of sieved NCL 812 was mixed with 2.0 ml (0276 PEG 4000, PEG 200 and propylene glycol were glycerol and 1.5 ml absolute ethanol. The mixture was mixed and heated to 150° C. and until all solid crystals were placed in a mortar and manually milled until all NCL 812 dissolved. NCL812 was added to water and sonicated for 30 was suspended uniformly. The Suspension was Sonicated for minutes until fully suspended. The NCL812 solution and gel 30 minutes. Vehicle (55 ml of Ora Sweet and Ora Plus solutions were mixed and allowed to cool and solidify. mixture) was then added to the suspension and milled for Formulation. A demonstrated acceptable viscosity, ease of another 10 minutes. Volume was made up with the Ora plus skin application, uniform Suspension and consistent and fine and Ora Sweet mixture to 60 ml by transferring to a texture. measuring cylinder Formulation B Topical Formulation PEG-Based Gel 0304 Formulation E demonstrated acceptable suspen with NCL812 sion and demonstrated acceptable short term stability. (0277 3.0 g PEG 4000; 0278 1.0 g PEG 8000; Formulation F Intramuscular Injection (0279 3.0 g PEG 200; (0305 20 mg/ml Polyvinylpyrrolidone K30 (PVPK30): 0280) 1.0 g propylene glycol; (0306 0.09 mg/ml NCL812; and 0281 1.9 g water and 0307 50 ml water. 0282 0.202 g of NCL812. (0308 Two percent of w/v PVP K30 solution was pre 0283 PEG 4000, PEG 8000, PEG 200 and propylene pared by the addition of 1.0 g of PVPK30 to 50 ml of MilliO glycol were mixed and heated to 150° C. and until all solid water. The solution was then placed in a sonicator for 30 crystals were dissolved. NCL812 was added to water and minutes to equilibrate and 4.5 mg of NCL 812 was added to sonicated for 30 minutes until fully suspended. The NCL812 the PVP solution and placed on an shaker at a Solution and gel solutions were mixed and allowed to cool maximum speed of 10 rpm over a period of 24 hours, with and solidify. Formulation B demonstrated acceptable vis controlled temperature of 25+1° C. Solution was transferred cosity, ease of skin application, uniform Suspension and to 5 ml vials and checked for clarity, appearance, pH and consistent and fine texture. short-term stability. The pH of solution was 7.25. US 2017/007 1884 A1 Mar. 16, 2017 30

0309 Formulation F demonstrated acceptable transpar tions (synergy, antagonism, no effect) of NCL812 in com ency and short term stability. bination with tetracycline, chloramphenicol, erythromycin (macrollide), amplicillin (B-lactam broad-spectrum), gen Example 9 tamicin (), ciprofloxacin (fluoroquinolone), Sulfamethoxazole (Sulphonamide), or penicillin G (B-lactam Release of NCL812 from Formulation B narrow-spectrum). For initial experiments, a laboratory 0310. The objective of this study was to measure the strain of Staphylococcus aureus T3-129 was used, however release of NCL812 from the Formulation B prepared in this strain gave inconsistent results for Some of the antimi example crobials and a new strain of Staphylococcus spp. designated 0311 Franz diffusion cells were utilized to quantify the MK1 (definitive species identification currently in progress) release rate of NCL 812 from its topical formulations. Five that was sensitive to all tested antimicrobials was used in millilitres of absolute ethanol, which was chosen as the Subsequent tests. desired release medium, was loaded into the receptor cham ber. Temperature of the receptor fluid was kept constant, at 0314 Firstly, the MIC of each antibiotic alone was deter 32t1° C. using a water jacket. Acetyl cellulose membranes, mined in accordance to CLSI standard guidelines. Secondly, with pore size of 0.45 um (Pall Corporation) was selected the combination of NCL812 with each of above antibiotics and placed between donor and receptor chamber. Followed was tested in duplicate. To evaluate the effect of the com by that, a number of test samples (Formulation B) were bination the fractional inhibitory concentration (FIC) was loaded into the donor chamber. One millilitre of receptor calculated for each antibiotic as follows: FIC of tested fluid was collected at regular time intervals of 0.25, 0.50, antibiotic=MIC of tested antibiotic in combination/MIC of 0.75, 1, 2, 3, 4, 5, 6, 7, 8 and 24 hours through the sampling antibiotic alone: FIC of NCL812=MIC of NCL812 in com port. One millilitre of fresh absolute ethanol was immedi bination/MIC of NCL812 alone; and FIC=FIC index=FIC ately returned to the receptor chamber. UV-HPLC was of NCL812+FIC of each tested antibiotic. utilized to analyse the content of the receptor fluids attained. 0315 According to the checkerboard guidelines, Synergy 0312 FIG. 41 presents the cumulative release of NCL812 (S) was defined as an FIC-0.5. No effect (NE) was defined over time. This study demonstrates that Formulation B as 0.5

MIC (Lig/ml MIC ml

With With Antibiotic Name Experiment Repeat NCL812 Alone FIC Antibiotic Alone FIC, FIC, Result Tetracycline' O.25 O.S O.S 1 4 0.25 0.75 NE O.25 O.S O.S 1 8 O.125 0.62 NE O.O31 O.25 O.12S 4 8 O.S O.625 NE O.O31 O.25 O.12S 4 8 O.S O.625 NE Chloramphenicol' 4 8 O.S 1 4 0.25 0.75 NE 2 4 O.S 2 8 0.25 0.75 NE 4 8 O.S 2 8 0.25 0.75 NE O.S 8 O.0625 4 8 O.S O.S62 NE Erythromycin O.O31 O.125 0.25 2 4 O.S 0.75 NE O.OO7 O.125 0.063 2 4 O.S O.S62 NE O.OO7 O.25 O.25 2 8 O.25 O.S NE O.OO7 O.25 O.O31 4 8 O.S O.S31 NE Ampicillin' s O.125 O.25 O.S 1 4 0.25 0.75 NE O.25 O.S O.S O.12S 4 O.O31 O.S3 NE 0.062 O.125 O.S 2 8 0.25 0.75 NE O.125 O.25 O.S 2 8 0.25 0.75 NE Gentamicin 0.062 O.125 O.S O.S 4 O.125 0.625 NE 0.062 O.125 O.S 1 4 0.25 0.75 NE O.S 1 O.S 1 4 0.25 0.75 NE O.OO7 O.S O.O156 2 4 O.S 0.515 NE US 2017/007 1884 A1 Mar. 16, 2017

TABLE 14-continued MICs. FICS, FIC, and the interaction between NCL812 and eight antibiotics. Antibiotic NCL812

MIC ml MIC (Lig/ml

With With Antibiotic Name Experiment Repeat NCL812 Alone FIC Antibiotic Alone FIC, FIC Result Ciprofloxacin’ 1 1st O.062 O.12S O.S 2 4 O.S 0.75 NE 2nd O.OO3 O.12S O.O2S 4 2 O.S 0.525 NE 2 1st O.12S O.25 O.S O.S 4 O.125 0.625 NE 2nd O.12S O.25 O.S O.25 4 O.0625 O.S62 NE Sulfamethoxazole' 1 1st 4 8 O.S 1 4 O.25 0.75 NE 2nd 4 8 O.S 2 4 O.S 1 NE 2 1st 4 8 O.S 1 4 O.25 0.75 NE 2nd 4 8 O.S 2 4 O.S 1 NE Penicillin G’ 1 1st O.062 O.12S O.S 2 4 O.S 1 NE 2nd O.062 O.125 O.S 2 4 O.S 1 NE 2 1st O.062 O.12S O.S 2 4 O.S 1 NE 2nd O.O31 O.25 O.12S 2 4 O.S O.625 NE S. aureus strain T3-29 'Staphylococcus spp. Strain MK1 FIC = MIC ofanitbiotic in combination with NCL812/MIC of antibiotic alone FIC = MIC of NCL812 in combination with antibiotic/MIC of NCL812 alone FIC = FIC index

Example 11 the bacteria were added to 9 mL of CAMHB containing various concentrations (up to 4xMIC) of NCL, to achieve a The Effects of NCL812 on Antimicrobial Sensitive final bacterial concentration of 1 to 3x106 CFU/mL. The Isolates of Staphylococcus aureus and Enterococcus tubes were incubated at 37° C., with constant shaking. In faecalis order to determine the number of viable bacteria present at various time points, a 100 uL aliquot was removed from Materials and Methods each tube and diluted. Then, 100 uL of each dilution were spread onto colony count agar, in duplicate, and incubated Strain Information for 48 h at 37° C. After 24 h the numbers of colonies present 0317. Two Staphylococcus aureus isolates were used in on each plate were counted and therefore the number of the following experiments; S. aureus MK01 a human skin viable bacteria present in the original Suspension enumer strain, and S. aureus KCO1 an equine skin strain. These ated. Plates were re-checked after 48 hours. isolates were identified by Gram stain and biochemical methods, including the Remel Staphaurex commercial kit. Results One Enterococcus faecalis isolate (USA01), was not iden tified as a VRE strain. As this isolate has previously been Minimum Inhibitory Concentration (MIC) speciated, it was not subjected to further testing, except for 0320. The NCL812 MIC for isolates S. aureus MKO1 and observation of pure, characteristic growth on blood agar. KC01, and E. faecalis USA01 was investigated. The results were: S. aureus MK01=4-8 ug/mL, S. aureus KCO1=2 Investigation of Minimum Bactericidal Concentration ug/mL, E. faecalis USA 01-4 ug/mL. (MBC) 0321 S. aureus isolates MK01 and KCO1 were investi CLSI Methodology gated and no growth, or growth only at low concentrations of NCL812 (2 g/ml), was observed, indicating that 0318. As in previous experiments, 10 uL of the contents NCL812 is bactericidal against S. aureus. For the E. faecalis of each well starting at the MIC was inoculated on to a isolate tested (USA01) however, growth of bacteria was Columbia SBA plate and incubated at 37° C. for 48 h. Plates observed at all concentrations of NCL812 tested. There was were examined at 24 and 48 h and the MBC was recorded an obvious reduction in the number of bacteria with increas as the lowest concentration of NCL812 at which no colonies ing concentration, but growth was present compared with no of bacteria were observed on the plate (or significant inhi growth for S. aureus. A Summary of these results can be seen bition of growth was observed compared to the control) in Table 15. Table 15 shows the results for NCL812 MBC (CLSI 2005). tests on two non-MRSA S. aureus isolates and one non-VRE Kill Kinetics Assays for S. aureus KCO1 & E. faecalis E. faecalis isolate. Each MBC test was performed in dupli USA01 Method cate. No change in the results was observed at 48 h. Table 0319 S. aureus KCO1 and E. faecalis USA01, not deter 16 shows NCL812 MBC values (ug/mL) for 20 MRSA mined to be MRSA or VRE, respectively, were grown isolates. Each MBC test was performed in duplicate starting overnight on Columbia SBA at 37° C. A few colonies of from NCL812 MIC concentration to 16 times of MIC. Table bacteria were then suspended in CAMHB (cation-adjusted 17 shows NCL812 MBC values (ug/ml) for 10 VRE isolates. Mueller Hinton broth) and adjusted to ODoo of 0.08 to 0.10. Each MBC test was performed in duplicate starting from The bacterial suspension was diluted 1:10. One millilitre of NCL812 MIC concentration to 32 times the MIC. US 2017/007 1884 A1 Mar. 16, 2017 32

TABLE 1.5 TABLE 17

NCL812 MBC tests on two non-MRSA Staphylococcus aureus NCL812 MBC values (Ig/ml) for 10 VRE isolates. isolates and one non-VRE Enterococcus faecalis isolate. NCL812 MBC

NCL812 MBC Organism 2 4 8 16 32 64 128 Sample No. |g/ml g/ml Lig/ml g/ml Ig/ml Ig/ml Ig/ml Organism 2 4 8 16 32 64 128 VRE 1st 90: 2O 4OOO M M M M Sample No. Ig/ml g/ml Ig/ml g/ml Ig/ml Ig/ml g/ml 26c(dc) - 2 O 70 3500 M M M M VRE 1st 500 1OO 2O 2SO M M M 37c - 2nd M 50 100 1100 14OO M M S. atiretts 1st O O O O O O VRE 1st O O O 720 O O O (KCO1) 2nd -- -- O O O O 35t - 2nd O O O O 10 2O 10 S. atiretts 1st O (5) O 0 0 (N) () (N) () (N) () (N) VRE 1st 90 330 O M M M M (MKO1) 2nd O O O O O O O 16c(dc) - 2nd 2OO O 2O M M M M VRE 1st O 120 2O 10 M M M E. faecalis 1 N + (488) + + + (7) + (1) + 23c. - 2nd O O O O 570 M M ( USAO1 2nd N ------VRE 1st O O M M M M M 2Sc - 2nd 20 20 M M M M M + = Growth on Sheep Blood Agar; VRE 1st 10 82O 98O M M M M 16c - 2nd M 790 890 M M M M 0 = No Growth on Sheep Blood Agar; VRE 1st O O O 18O 10 110 M N = Not Cultured; 19t - 2nd 30 O O 70 40 M M Numbers in Parenthesis are the Number of Bacteria Growing after 24 hours per ml of VRE 1st 10 O 10 O 18O 970 M sample (CFU/ml) 14t - 2nd O O O 40 780 M M VRE 1st O O O M M M M 12c - 2nd O M 3OO M M M M TABLE 16 *Number of bacteria growing after 24 hours per ml of sample (CFU/ml); NCL812 MBC values (Ig/ml) for 20 MRSA isolates. M = many bacteria growing on the plate (too many to count) NCL812 MBC Kill Kinetics Assays for S. aureus KCO1 & E. faecalis USA01 Method Organism? 4 8 16 32 64 0322 Colony counts were performed at t=0, 120, 240, Sample No. Ig/ml g/ml Ig/ml Ig/ml g/ml and 360 min, then again at 24 h. At the 2 h time point S. MRSA 1 1st O O O O N* * aureus KCO1 showed a minimum of a 2.5 logo reduction in - 2nd O O O O N bacterial numbers from initial numbers, and greater than a 3 MRSA 2 1st O O O O N logo reduction in comparison to the control at the same time - 2nd O O O O N point. A minimum of a 2 logo reduction was still evident at MRSA 3 1st O GB* O O N - 2nd O O O O N 6 h incubation, however after 24 h the numbers of bacteria MRSA 4 1st O O O O N present had increased and this was not significantly different - 2nd O O O O N to the control. MRSAS 1st O O O O N 0323. Similar results were obtained with E. faecalis - 2nd O O O O N USA01, however the reduction in bacterial numbers MRSA 6 1st O O O O N - 2nd O O O O N observed was less than for S. aureus KCO1. A 2 logo MRSA 7 1st O O O O N reduction in CFU/mL was observed at 2 h, compared to the - 2nd O GB O O N growth control. However, the reduction in CFU/mL com MRSA 8 1st GB O O O N pared to the original bacterial numbers was only just greater - 2nd O O O O N than 1 logo. At concentrations of 4-16 ug/mL of NCL812 MRSA9 1st O O O O N this reduction in bacterial numbers remained consistent until - 2nd O O O O N MRSA 10 1st O O O O N the 6 h time point. At concentrations of 32 and 64 Lug/mL - 2nd O O O O N however, there was approximately a 1 logo rise in bacterial MRSA 11 1st O O O O N numbers over the same time period. At 24 h bacterial - 2nd GB O O O N numbers at all concentrations had increased to almost the MRSA 12 1st O O O O N - 2nd O GB O O N same level as the growth control. MRSA 13 1st O O O O N 0324. The results observed with these strains of S. aureus - 2nd O O O O N and E. faecalis are consistent with the results observed for MRSA 14 1st O O O O N the kill kinetics assay for all MRSA and VRE isolates tested. - 2nd O O O O N MRSA S16 1st O O O O O The kill kinetics assay of Staphylococcus aureus KCO1 at - 2nd O O O O O different concentrations of NCL812, up to 24 h incubation MRSA S70 1st O O O O O are shown in FIG. 42. The kill kinetics assay of Enterococ - 2nd O O O O O cus faecalis USA01 at different concentrations of NCL812, MRSA S8O 1st O O O O O - 2nd O O O O O up to 24 h incubation are shown in FIG. 43. MRSA 606 1st O O O O GB Example 12 - 2nd O O O O O MRSA 610 1st O O O O O A Method of Treating Bacterial Infection. In Vivo - 2nd O GB O O O by the Administration of NCL812 GB = Bacterial Growth on Sheep Blood Agar 0325 The objective of this study was to determine the N** = Not cultured on Sheep Blood Agar efficacy of an Investigational Veterinary Product containing NCL812 in the treatment of a skin infection in mice US 2017/007 1884 A1 Mar. 16, 2017

Summary of the Model were pelleted through centrifugation at 7,500 rpm for 10 mins. Broth Supernatant was removed and bacteria Sus 0326. A useful animal model system should be clinically pended in 10 ml Phosphate Buffered Saline (PBS). These relevant, experimentally robust, ethically acceptable, con steps were repeated a further two times. The density of the venient to perform and should provide reliable and repro Suspension was checked by measuring absorbance at 600 ducible results. There are many animal models of topical nm, using a spectrophotometer with Saline as a blank, to skin infection that have been described including the croton confirm the target density at a reading of approximately oil-inflamed skin model (Akiyama, H., H. Kanzaki, Y. Abe, 0.100, consistent with a bacterial density of 2.5x107 CFU/ J. Tada and J. Arata (1994). “Staphylococcus aureus infec ml. The Suspension was placed into a rack placed into a tion on experimental croton oil-inflamed skin in mice.” lockable transport box with an ice brick to maintain refrig Journal of Dermatological Science 8(1): 1-10), the burnt eration during transport, followed by storage in cool room skin model (Stieritz, D. D., A. Bondi, D. McDermott and E. upon arrival at the mouse skin infection laboratory. Final B. Michaels (1982). “A burned mouse model to evaluate Suspension was mixed thoroughly before inoculating the anti-pseudomonas activity of topical agents.” Journal of skin wounds created in mice. Antimicrobial Chemotherapy 9(2): 133-140), the skin 0329. In order to ensure the purity and accuracy of the suture-wound model (McRipley, R. J. and R. R. Whitney Suspension, the following steps were performed prior to (1976). “Characterization and Quantitation of Experimental placement into lock box. Surgical-Wound Infections Used to Evaluate Topical Anti 0330 Purity of bacterial suspension ensured by spreading bacterial Agents.' Antimicrobial Agents and Chemotherapy 100 ul of the final suspension onto a SBA (sheep blood agar) 10(1): 38-44), the skin tape-stripping model (Kugelberg, E., plate which was incubated at 37° C. for 18 hours and T. NorstrOm, T. K. Petersen, T. Duvoid, D. I. Andersson and examined to confirm uniform growth of one colony type. D. Hughes (2005). “Establishment of a Superficial Skin Viable counts were performed on final suspension by prep Infection Model in Mice by Using Staphylococcus aureus ping saline in Eppendorf tubes (approximately 900 ul per and Streptococcus pyogenes.' Antimicrobial Agents and tube), removing 100 ul sample and adding to first Eppendorf Chemotherapy 49(8): 3435-3441) and the linear full thick tube, Vortexing the mixture and repeating using 2" Eppen ness scalpel cut method (Guo, Y. R. I. Ramos, J. S. Cho, N. dorf tube containing saline. This process was continued for P. Donegan, A. L. Cheung and L. S. Miller (2013). “In Vivo 5-6 tubes. Finally, 100 ul of 5" and 6" dilutions were plated Bioluminescence Imaging To Evaluate Systemic and Topi out on plate count agar, incubated at 37°C. for 18 hours and cal Antibiotics against Community-Acquired Methicillin colony counts performed to confirm that the CFU/ml was Resistant Staphylococcus aureus-Infected Skin Wounds in approximately 2.5x107. Following inoculation of the Mice.’ Antimicrobial Agents and Chemotherapy 57(2): 855 wounds, this process was repeated to ensure that no con 863). tamination or decrease in viable counts had occurred during 0327 Preliminary studies prior to the conduct of the the time of the Surgery. current study established a new method of skin infection arising from a detailed study of the models mentioned Skin Wound Surgical Procedure above. Briefly, study mice are anaesthetised, a patch of 0331 Each mouse was placed into induction chamber dorsal skin is clipped to reveal the skin and a circular area and anaesthesia induced using 2% isoflurane. Eyes of each of skin is removed with a hand held punch, leaving a wound anaesthetised mouse were covered with veterinary eye lubri on the dorsum with a central cavity. The wound is infected cant in order to prevent corneal dehydration. Each mouse with a known number of the challenge organism. Approxi removed from induction chamber and placed onto Surgical mately four to six hours after infection, the wound is either area, in front of individual aesthetic nose cone. While under treated topically with a vehicle formulation or an active anaesthesia each mouse was monitored for assessment of formulation. The infected skin wound is retreated every 12 depth of anaesthesia (response to pain, blink reflex, skeletal hours for a total of 14 treatments. Mice are humanely muscle tone) and respiratory and cardiac function. Back skin euthanased, the area of the original infected wound is hair was shaved from Surgical area with mechanical clippers. dissected and removed and its bacterial content quantified by Shaved area was cleaned using 70% ethanol applied to paper standard microbiologic tests. In this way, the change in towel followed by 10% w/v povidone-iodine solution. Once bacterial concentration due to treatment with the active the iodine Solution was dry, a Subcutaneous injection of the formulation can be readily determined by examining the nonsteroidal anti-inflammatory agent meloxicam was reduction in bacterial burden compared with the vehicle administered. Dorsal skin was pinched gently to allow control. creation of a circular full-thickness wound using ear punch/ biopsy punch. Vehicle control and NCL812 treated mice had Materials and Methods wounds inoculated with 10 ul of bacterial Suspension using a micropipette (2.5x10 CFU/10 ul). Once the bacterial Preparation of Infection Inoculum Suspension was dry, mice were placed into individual recov ery boxes labelled with the mouse number. The time of 0328 Fresh cultures of bacteria (Staphylococcus aureus) inoculation was recorded. Initial body weights of each were grown on Sheep Blood Agar at 37° C. for 16-18 hours. mouse were recorded on the appropriate score sheet. Mice A few typical colonies were selected and suspended in 10 ml recovered to full consciousness within 5 minutes. Recovered of Tryptic Soy Broth and incubated overnight in a shaking mice were returned to individual housing and monitored incubator (240 rpm) at 37°C. The overnight suspension was hourly for post-Surgical or anaesthetic complications. vortexed and diluted (1:100) in fresh Tryptic soy broth (100 ul 0.1 ml) in 9.9 ml broth). The fresh suspension was Post-Surgical Care (4 Hours Post-Surgery) incubated for 3 hours in a shaking incubator (as above) in 0332 Mice were assessed for post-surgical complications order to obtain mid-logarithmic phase bacteria. Bacteria and observations were recorded on clinic record sheet. Each US 2017/007 1884 A1 Mar. 16, 2017 34 mouse was carefully removed from IVC and placed into an lowing day, viable counts were performed using incubated assessment container, avoiding excessive handling or touch plate count agar plates and the identity of Staphylococcus ing of the Surgical site. Once the mouse was inside assess aureus (the challenge organisms) as the harvested Strain was ment container, it was assessed and the observations confirmed. recorded on the post-surgical clinical record sheet. When ever the Suggested wellness breakpoints were reached, post Results operative analgesia was administered and recorded on the 0336. The mean colony count per gram of tissue observed clinical record sheet. in vehicle treated group was 5,888.436 (6.77 log 10). The mean colony count per g of tissue observed in NCL812 Animal Monitoring and Daily Care group was 141,254 (5.15 log 10). The log 10 colony forming 0333 Antibiotic Administration (7 am and 6 pm). The units per gram of tissue and % reduction are summarised in first administration of vehicle or NCL812 ointment occurred the following table. 4 hours post-Surgically. Each ointment container was weighted prior to administration and the weight recorded. TABLE 1.5 Each mouse was carefully restrained. Ointment (vehicle or Log10 colony forming units per gram of tissue and percentage reduction NCL812) was applied to the lesion area and the treated following topical administration of vehicle and treatment mouse was returned to IVC where each mouse was observed to ensure ointment was not immediately removed by groom Treatment Logio (CFU/g) % reduction ing. The weight of the ointment container post-administra Vehicle 6.77 tion was recorded. The vehicle and active NCL products NCL812 5.15 97.6 were applied to the skin wound each 12 hours following the first administration for a total of 14 consecutive treatments. The NCL812 ointment (Formulation B, as presented in It is clear from this table that treatment with NCL812 leads Example 8) contained robenidine at a concentration of 20 to high level reduction in the number of infecting Staphy mg/g. Approximately 0.1-0.2 g of ointment was applied on lococcus aureus. These results demonstrate effective treat each occasion, delivering a total topical dose of NCL812 ment of a bacterial colonisation or infection in vivo. between 28 and 56 mg to mice weighing between 18 g and 1-38. (canceled) 25 g. 39. A method of treating or preventing a bacterial colo 0334 Daily Monitoring. Monitoring of each mouse took nization or infection by a bacterial agent in a subject, the place once daily at around 12 pm. Each mouse carefully method comprising the step of removed from IVC and placed into observation container, administering to the subject a therapeutically effective avoiding excessive handling or touching Surgical site. The amount of robenidine or a therapeutically acceptable coat, posture, eyes, behaviour, Vocalisation and activity salt thereof, wherein the bacterial agent is selected from whilst in the container were carefully assessed and obser the group consisting of Mycoplasma spp. Ureaplasma Vations recorded on assessment sheet. Mouse faeces (either spp., and Mycobacterium spp. on floor of cage or in container) were checked for consis 40. The method according to claim 39, wherein the tency and observations recorded. The weight of each mouse Subject is selected from the group consisting of human, was determined whilst it was in the container and change in canine, feline, bovine, ovine, caprine, porcine, avian, piscine body weight calculated and recorded. The observation con and equine species. tainer was disinfected with ethanol and set aside to dry while 41. The method according to claim 39, wherein the a fresh container was used for the next mouse. For every robenidine is administered to the subject at a dose in the second day, mice were again anaesthetised using 2% iso range of 0.1 mg/kg to 250 mg/kg bodyweight. flurane and photographed using a ruler for size referencing. 42. The method according to claim 39, wherein the These photos were used to assess lesion size and infection bacterial agent is selected from the group consisting of progression during the trial period. Mycoplasma spp., including Mycoplasma agalactiae, Myco plasma alkalescens, Mycoplasma amphoriforme, Myco Tissue Analysis and Assessment of Antibacterial Efficacy plasma arginini, Mycoplasma bovigenitalum, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, 0335. At the end of the 7 day skin wound assessment Mycoplasma buccale, Mycoplasma Californicum, Myco period, all test mice were euthanased prior to wound col plasma Canadense, Mycoplasma capricolum Subsp. capri lection for post mortem examination. The skin wound was colum, Mycoplasma capricolum Subsp. Capripneumoniae, dissected from the dorsum of each mouse. The sample was Mycoplasma conjunctivae, Mycoplasma cynos, Myco placed in a sample tube and weighed before 1 ml PBS and plasma dispar, Mycoplasma equigenitalium, Mycoplasma sterile tissue homogenisation beads were added. Tissue faucium, Mycoplasma felis, Mycoplasma fermentans (incog samples were homogenised for 10 mins using a tissue nitus str.), Mycoplasma gallisepticum (MG), Mycoplasma homogeniser (Next Advance Bulet Blender) and then Vor gateae, Mycoplasma genitalium, Mycoplasma haemocanis, texed for approximately 30 seconds. 100 ul of supernatant Mycoplasma haemofelis, Mycoplasma haemosuis (formerly was removed and placed into an Eppendorf tube containing Eperythrozoon suis), Mycoplasma hominis, Mycoplasma 900 ul of PBS. This procedure was repeated using serial hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyo dilutions for a total of 8 dilutions. Finally, 100 ul of each synoviae, Mycoplasma iowae meleagridis (MM), Myco dilution was pipetted onto a plate count agar in duplicate and plasma iowae, Mycoplasma leachii, Mycoplasma lipophi incubated overnight at 37° C. Ten microlitres of original lum, Mycoplasma meleagridis, Mycoplasma mycoides Subsp Suspension was placed onto sheep blood agar to assess capri, Mycoplasma mycoides Subsp mycoides, Mycoplasma culture purity and incubated overnight at 37° C. The fol mycoides subsp. mycoides (such as Contagious bovine pleu US 2017/0071884 A1 Mar. 16, 2017 ropneumonia CBPP), Mycoplasma orale, Mycoplasma ovip terium intracellulare, Mycobacterium Senegalense, MyCO neumoniae, Mycoplasma ovis, Mycoplasma penetrans, bacterium africanum, Mycobacterium avium Subsp Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma paratuberculosis, Mycobacterium kansasii, Mycobacterium primatum, Mycoplasma putrefaciens, Mycoplasma sali lacus, Mycobacterium lentiflavum, Mycobacterium leprae, varium, Mycoplasma spermatophilum, Mycoplasma suis, Mycobacterium lepraemurium, Mycobacterium mager Mycoplasma synoviae (MS), Mycoplasma wenyonii. itense, Mycobacterium malmoense, Mycobacterium mari 43. The method according to claim 39, wherein the num, Mycobacterium massiliense, Mycobacterium microti, bacterial agent is selected from the group consisting of Mycobacterium montefiorense (eels), Mycobacterium mora Ureaplasma spp., including Ureaplasma parvum, Urea cense, Mycobacterium mucogenicum, Mycobacterium plasma urealyticum, Ureaplasma, and Ureoplasma diver nebraskense, Mycobacterium neoaurum, Mycobacterium S2. novocastrense, Mycobacterium palustre, Mycobacterium 44. The method according to claim 39, wherein the parmense, Mycobacterium phlei, Mycobacterium phocai bacterial agent is selected from the group consisting of cum, Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacterium spp., including Mycobacterium abscessus, Mycobacterium pseudoshottsii (fish), Mycobacterium Mycobacterium arupense, Mycobacterium asiaticum, MyCO pseudotuberculosis, Mycobacterium Saskatchewanense, bacterium aubagnense, Mycobacterium avium complex, Mycobacterium scrofitlaceum, Mycobacterium senuense, Mycobacterium bolletii, Mycobacterium bolletii, Mycobac Mycobacterium septicum, Mycobacterium simiae, MyCO terium bovis, Mycobacterium branderi, Mycobacterium bacterium smegmatis, Mycobacterium Szulgai, Mycobacte canettii, Mycobacterium caprae, Mycobacterium cellatum, rium terrae/chromogenicum complex, Mycobacterium tri Mycobacterium chelonae, Mycobacterium chimaera, MyCO plex, Mycobacterium tuberculosis, Mycobacterium tusciae, bacterium colombiense, Mycobacterium conceptionense, Mycobacterium ulcerans, Mycobacterium wolinskyi, and Mycobacterium conspicuum, Mycobacterium elephantis, Mycobacterium xenopi. Mycobacterium farcinogenes, Mycobacterium florentinum, Mycobacterium fortuitum group, Mycobacterium 45. The method according to claim 39, wherein the genavense, Mycobacterium goodii, Mycobacterium haemo therapeutically effective amount of robenidine or a thera philum, Mycobacterium heckeshornense, Mycobacterium peutically acceptable salt thereof is administered to the heidelbergense, Mycobacterium houstonense, Mycobacte subject by oral, parenteral or topical administration. rium immunogenium, Mycobacterium interjectum, Mycobac *k ck ck ck ck