# Patent

## Title
Methods for treating bacterial infections

## Abstract

The invention comprises methods for treating and preventing a bacterial infection in a subject, methods for preparing a medicament for use in treating and preventing a bacterial infection in a subject, and pharmaceutical and veterinary antibacterial compositions when used therein.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 14/888,289 filed 30 Oct. 2015 (allowed), which is a U.S. National Stage Application of PCT/AU2014/000480 filed 1May 2014, which claims priority to Australia Application No. 2013901517 filed 1 May 2013, the contents of all of which are hereby incorporated by reference in their entirety.


TECHNICAL FIELD
This invention relates to methods of treating and preventing a bacterial infection in a subject, methods for preparing a medicament for use in treating and preventing a bacterial infection in a subject, and pharmaceutical and veterinary antibacterial compositions when used therein.
BACKGROUND ART
A marked increase in prevalence of multi-drug resistance in disease-causing Gram-positive (G+ve) (Staphylococcus aureus, Enterococcus spp. and Streptococcus pneumoniae) and Gram negative (G−ve) pathogens (Escherichia coli, Enterobacter spp., Salmonella spp., Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) has coincided with an unprecedented global decline in investment in new anti-infective drugs. There are few currently registered alternatives for multidrug resistant (MDR) bacterial infections, forcing clinicians to consider older generation drugs such as colistin with narrow spectrum and considerable potential for toxic side-effects. In addition, there are fewer novel classes of antiinfective therapeutics moving through the drug development pipeline.
Since the year 2000, a period of almost 15 years, only 5 novel mode of action (MOA) antibacterial agents have been approved by the US FDA—linezolid (an oxazolidinone) in 2000, daptomycin (a lipopeptide) in 2003, retapamulin (a pleuromutilin) in 2007, fidaxomicin (a macrolide tiacumicin) in 2011, and bedaquiline (a diarylquinoline) in 2012. Notably, none of these agents has significant activity against gram negative bacteria. No novel MOA antibacterial agents were approved in 2013 and to date in 2014 only tedizolid and dalbavancin, both analogs of existing classes, have been recommended for approval in the US. While there are more than 300 anti-infective medicines in various stages of development, the large majority of these medicines are previously approved antibacterial compounds or their derivatives that are undergoing studies for new Indications.
Furthermore, the prevalence of multidrug-resistance in animal-specific pathogens together with greater regulation of the registration and usage of antimicrobials in animals, has caused veterinarians to become increasingly reliant on the traditional classes of antimicrobial agents. The risk of transfer of MDR zoonotic organisms from animals to humans has also led to calls for further restrictions on the usage of some recently registered antibacterial drugs such as the fluoroquinolones and the third and fourth generation cephalosporins.
Epidemiology of Antibacterial Resistance Development in Pathogens of Humans and Animals
Much of the evolution in resistance development is driven by changes in the epidemiology of key MDR organisms. Once only restricted to human hospitals and aged care facilities, methicillin resistant Staphylococcus aureus (MRSA) strains are now being isolated from the community in alarming proportions. Furthermore, community-acquired MRSA strains are more likely to carry the Panton-Valentine leukocidin (PVL) toxin, a virulence factor linked to skin and soft tissue lesions as well as a rapid, fulminating, necrotizing pneumonia with significant associated mortality. Recently MRSA strains have become host-adapted in several key animal species including livestock, horses and companion animals and regular cases of human-to-animal and animal-to-human transfer are being documented. This has important consequences for strain transmission and public health. A recent survey of 751 Australian veterinarians for MRSA nasal carriage found that a remarkable 21.4% of equine veterinarians were MRSA-positive compared to 4.9% of small animal veterinarians and 0.9% of veterinarians with little animal contact. These ecological shifts of MRSA together with the emergence of resistance to new drugs developed specifically for MRSA such as linezolid confirm that new MRSA anti-infectives are urgently needed. Furthermore, hospitals that use vancomycin for treating MRSA then have to contend with outbreaks of vancomycin-resistant enterococci (VRE) infections in their patients, once again with limited alternative antimicrobial choices.
The global emergence and spread within the community of highly virulent MDR Gram-negative (G−ve) bacteria such as E. coli O25b:ST131 confirms that bacterial pathogens can simultaneously evolve both virulence and resistance determinants. Echoing recent MRSA epidemiology, E. coli O25b:ST131, a major cause of urinary tract and bloodstream infections in humans, has now been isolated from extraintestinal infections in companion animals, and poultry. The increasing significance of E. coli O25b:ST131 and other MDR Enterobacteriaceae with combined resistance to fluoroquinolones and extended spectrum beta-lactams and carbapenems is another worrying trend, especially considering there have been few recent breakthroughs in the development of G−ve spectrum anti-infectives apart from incremental advances in the carbapenem family.
The World Health Organisation has identified antibiotic resistance as one of the three major future threats to global health. A recent report from the US Centers for Disease Control and Prevention (CDC) estimated that “in the United States, more than two million people are sickened every year with antibiotic-resistant infections, with at least 23,000 dying as a result.” The extra medical costs, in the USA alone, associated with treating and managing a single case of antibiotic-resistant infection are estimated to be between US$18,588 and US$29,069 per year resulting in an overall direct cost to the US health system of over US$20 billion annually. In addition, the cost to US households in terms of lost productivity is estimated at over US$35 billion per annum. Twenty five thousand patients in the European Union (EU) still die annually from infection with MDR bacteria despite many EU countries having world's best practice hospital surveillance and infection control strategies. The EU costs from health care expenses and lost productivity associated with MDR infections are estimated to be at least 1.5 billion per year.
There is an unmet clinical need for antibacterial agents with novel mechanisms of action to supplement and replace currently available antibacterial agents, the efficacy of which is increasingly undermined by antibacterial resistance mechanisms. There remains a need for alternative antibacterials in the treatment of infection by multi-resistant bacteria. However, as reported by the Infectious Diseases Society of America and the European Centre for Disease Control and Prevention, few new drugs are being developed that offer promising results over existing treatments (Infectious Diseases Society of America 2010, Clinical Infectious Diseases, 50(8):1081-1083).
It is an object of the present invention to overcome at least one of the failings of the prior art.
The discussion of the background art set out above is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.
SUMMARY OF INVENTION
According to one aspect of the invention, there is provided a method of treating or preventing a bacterial colonisation or infection in a subject, the method comprising the step of: administering a therapeutically effective amount of robenidine, or a therapeutically acceptable salt thereof, to the subject. In this aspect, the bacterial colonisation or infection is caused by a bacterial agent.
According to another aspect of the invention, there is provided the use of robenidine, or a therapeutically acceptable salt thereof, in the manufacture of medicament for the treatment of a bacterial colonisation or infection in a subject. In this aspect, the bacterial colonisation or infection is caused by a bacterial agent.
The subject may be any subject capable of colonisation or infection by bacteria. The subject may be mammalian, or may be piscine or avian. Preferably, the subject is selected from the group comprising, but not limited to, human, canine, feline, bovine, ovine, caprine, other ruminant species, porcine, equine, avian, or piscine.
As used herein, the term robenidine, (also known as 1,2-bis[(E)-(4-chlorophenyl)methylideneamino]guanidine, or, as described by this specification, NCL812) refers to a compound having the following chemical structure:




The robenidine may be administered to the subject in a dose selected from the group comprising 0.1 mg/kg to 250 mg/kg body weight, preferably 1 mg/kg to 100 mg/kg body weight, and more preferably 5 mg/kg to 50 mg/kg body weight. The robenidine may be administered to the subject using a dosing schedule selected from the group consisting of: hourly, 3 times daily; twice daily; daily; every second day; twice weekly; once weekly; once fortnightly; once monthly; once every two months or by constant rate or variable rate infusion. Preferably, the robenidine is administered until colonisation or the signs and symptoms of infection have at least been partially treated or alleviated.
In one embodiment, the concentration of robenidine (or a robenidine metabolite) in the subject's blood after treatment is within a range selected from the group comprising, but not limited to: between 0.1 and 10 ug/mL at 2 hours, 1 and 200 ug/mL after 12 hours; between 0.1 and 5 ug/mL after 24 hrs; between 0.01 and 2 ug/mL after 48 hours; between 0.0001 and 1 ug/mL after 72 hrs. Preferably, the concentration is selected from the group comprising, but not limited to: less than 200 ug/mL after 12 hours; less than 5 ug/mL after 24 hours; less than 1 ug/L after 48 hours and less than 0.5 ug/mL after 72 hours.
The agent causing the bacterial infection is a bacterial agent. In one preferred embodiment, the agent is not a protozoan species. In one preferred embodiment, the agent is not a coccidian protozoan. More preferably, the agent is not Clostridium perfringens nor a heterotrophic bacterial species present in soil samples collected by Hansen et al from Jyndevad Denmark as discussed in the following papers: Hansen et al. 2012, Chemosphere, 86:212-215; and Hansen et al. 2009, Environmental Pollution 157:474-480.
In another embodiment, the bacterial agent is gram negative. In another embodiment, the bacterial agent is gram positive. In another embodiment, the bacterial agent has no cell wall. In another embodiment, the bacterial infection is caused by a mixture of at least two agents selected from the group consisting of: gram negative, gram positive and bacterial agents with no cell wall.
The bacterial agent causing the bacterial infection may be a gram positive bacterial agent selected from the group comprising, but not limited to, Staphylococcus spp, Streptococci, Enterococcus spp, Leuconostoc spp, Corynebacterium spp, Arcanobacteria spp, Trueperella spp, Rhodococcus spp, Bacillus spp, Anaerobic Cocci, Anaerobic Gram-Positive Nonsporulating Bacilli, Actinomyces spp, Clostridium spp, Nocardia spp, Erysipelothrix spp, Listeria spp, Kytococcus spp, Mycoplasma spp, Ureaplasma spp, and Mycobacterium spp.
In one embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to Staphylococcus spp. Examples of Staphylococcus spp include Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus auricularis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus carnosus, Staphylococcus cohnii, Staphylococcus hominis, Staphylococcus pasteuri, Staphylococcus pettenkoferi, Staphylococcus pulvereri, Staphylococcus saccharolyticus, Staphylococcus simulans, Staphylococcus schleiferi, Staphylococcus warneri, Staphylococcus xylosus, Staphylococcus arlettae, Staphylococcus caseolyticus, Staphylococcus chromogenes, Staphylococcus condimenti, Staphylococcus delphini, Staphylococcus equorum, Staphylococcus felis, Staphylococcus fleurettii, Staphylococcus gallinarum, Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus kloosii, Staphylococcus lentus, Staphylococcus lutrae, Staphylococcus muscae, Staphylococcus nepalensis, Staphylococcus piscifermentans, Staphylococcus pseudintermedius, Staphylococcus sciuri, Staphylococcus simiae, Staphylococcus succinus, and Staphylococcus vitulinus. 
In another embodiment, the bacterial agent is gram positive and is selected from the group comprising, but not limited to, Streptococcus spp. Examples of Streptococcus spp include Streptococcus agalactiae, Streptococcus alactolyticus, Streptococcus anginosus, Streptococcus canis, Streptococcus constellatus, Streptococcus cricetus, Streptococcus cristatus, Streptococcus downed, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus dysgalactiae subsp. equisimilis, Streptococcus equi subsp. equi, Streptococcus equi subsp. zooepidemicus, Streptococcus ferus, Streptococcus gallolyticus subsp. gallolyticus (formerly Streptococcus bovis biotype i), Streptococcus gallolyticus subsp. pasteurianus (formerly Streptococcus bovis biotype ii/2), Streptococcus gordonii, Streptococcus hyointestinalis, Streptococcus hyovaginalis, Streptococcus infantarius, Streptococcus infantarius subsp infantarius, Streptococcus infantis, Streptococcus iniae, Streptococcus intermedius, Streptococcus lutetiensis (formerly Streptococcus bovis biotype ii.1), Streptococcus macaccae, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus orisratti, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae, Streptococcus porcinus, Streptococcus pseudintermedius, Streptococcus pyogenes, Streptococcus ratti, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Streptococcus suis, Streptococcus thermophilus, Streptococcus vestibularis, and Nutritionally Variant (Deficient) Streptococci (Abiotrophia defectiva, Granulicatella adiacens, Granulicatella elegans, and Granulicatella para-adiacens) and related species such as Rothia mucilaginosa (formerly Stomatococcus mucilaginosus) and Pediococcus. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Enterococcus spp. Examples of Enterococcus spp include Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinarum, Enterococcus durans, Enterococcus avium, Enterococcus raffinosus, Enterococcus pallens, Enterococcus gilvus, Enterococcus cecorum, Enterococcus malodoratus, Enterococcus italicus, Enterococcus sanguinicola, Enterococcus mundtii, Enterococcus casselifavus/flavescens, Enterococcus dispar, Enterococcus hirae, Enterococcus pseudoavium, and Enterococcus bovis. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Leuconostoc spp. Examples of Leuconostoc spp include Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc paramesenteroides, Leuconostoc citreum, and Leuconostoc lactis. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Corynebacterium spp. Examples of Corynebacterium spp include nonlipophilic, fermentative Corynebacterium spp such as Corynebacterium ulcerans, Corynebacterium pseudotuberculosis, Corynebacterium xerosis, Corynebacterum striatum, Corynebacterium minutissimum, Corynebacterium amycolatum, Corynebacterium glucuronolyticum, Corynebacterium argentoratense, Corynebacterium matruchotii, Corynebacterium riegelii, Corynebacterium confusum, Corynebacterium cystidis, Corynebacterium diphtheria, Corynebacterium simulans, Corynebacterium sundvallense, Corynebacterium thomssensii, Corynebacterium freneyi, and Corynebacterium aurimucosum, nonlipophilic, nonfermentative Corynebacterium spp such as Corynebacterium afermentans afermentans, Corynebacterium auris, Corynebacterium pseudodiphtheriticum, and Corynebacterium propinquum and lipophilic Corynebacterium spp such as Corynebacterium jeikeium, Corynebacterium urealyticum, Corynebacterium afermentans lipophium, Corynebacterium accolens, Corynebacterium macginleyi, Corynebacterium tuberculostearum, Corynebacterium kroppenstedtii, Corynebacterium kutscheri, Corynebacterium pilosum, Corynebacterium bovis, CDC coryneform groups F-1 and G, and Corynebacterium lipophiloflavum, and other Corynebacterium spp such as Turicella, Arthrobacter, Brevibacterium, Dermabacter, Rothia, Oerskovia, Microbacterium, and Leifsonia aquatica. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Arcanobacteria spp. Examples of Arcanobacteria spp include A. haemolyticum, A. pyogenes (now known as Trueperella pyogenes, originally known as Actinomyces pyogenes), and A. bemardiae. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Rhodococcus spp. Examples of Rhodococcus spp include Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus fasciens, and Rhodococcus rhodochrous. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Gordonia spp.
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Tsukamurella spp.
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Acholeplasma spp.
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Actinobacteria such as Crossiella equi. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Bacillus spp. Examples of Bacillus spp include Bacillus anthracis, Bacillus cereus, Bacillus circulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus sphaericus, Bacillus subtilis, Brevibacillus brevis, Brevibacillus laterosporus, and Paenibacillus alvei. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Anaerobic Cocci. Examples of Anaerobic Cocci include Anaerococcus murdochii, Anaerococcus prevotii, Anaerococcus tetradius, Anaerococcus octavius, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus vaginalis, Atopobium parvulum, Finegoldia magna, Gallicola barnesae, Gemella asaccharolytica, Gemella bergeri, Gemella cuniculi, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis, Gemella sanguinis; Parvimonas micra, Peptococcus niger, Peptoniphilus asaccharolyticus, Peptoniphilus gorbachii, Peptoniphilus indolicus, Peptoniphilus harei, Peptoniphilus ivorii, Peptoniphilus lacrimalis, Peptoniphilus olsenii, Peptostreptococcus stomatis, Peptostreptococcus anaerobius, Ruminococcus productus, Slackia heliotrinireducens, and Staphylococcus saccharolyticus. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Anaerobic Gram-Positive Nonsporulating Bacilli. Examples of Anaerobic Gram-Positive Nonsporulating Bacilli include Alloscardovia omnicolens, Atopobium species (such as Atopobium minutum, Atopobium rimae, Atopobium parvulum, and Atopobium vaginae), Bifldobacteria (such as Bifidobacteria adolescentis, Bifidobacteria dentium, Bifidobacteria scardovii), Catabacter hongkongensis, Collinsella aerofaciens, Eggerthella (such as Eggerthella lenta, Eggerthella hongkongensis and Eggerthella sinensis), Eubacterium and related species (such as Eubacterium nodatum, Eubacterium tenue, Eubacterium brachy, Eubacterium infirmum, Eubacterium minutum, Eubacterium nodatum, Eubacterium saphenum, Eubacterium sulci, Filifactor alocis, Mogibacterium timidum, Mogibacterium vescum, Pseudoramibacter alactolyticus, Bulleidia extructa, and Solobacterium moorei), Lactobacillus species (such as Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus gassen, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus iners and Lactobacillus ultunensis), Mobiluncus species (such as Mobiluncus curtisii, Mobiluncus mulieris), Moryella indoligenes, Olsenella oral species (such as Olsenella uli and Olsenella profuse), Oribacterium sinus, Propionibacterium (such as Propionibacterium acnes and Propionibacterium propionicum), Slackia exigua, and Turicibacter sanguine. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Actinomyces spp. Examples of Actinomyces spp include Actinomyces israelii, Actinomyces naeslundii, Actinomyces viscosus, Actinomyces odontolyticus, Actinomyces meyeri, and Actinomyces gerencseriae (formerly Actinomyces israelii serotype II), Actinomyces europaeus, Actinomyces neuii, Actinomyces radingae, Actinomyces graevenitzii, Actinomyces hordeovulneris, Actinomyces turicensis, Actinomyces georgiae, Arcanobacterium (Actinomyces) pyogenes, Arcanobacterium (Actinomyces) bemardiae, Actinomyces funkei, Actinomyces lingnae, Actinomyces houstonensis, and Actinomyces cardiffensis. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Clostridium spp. Examples of Clostridium spp include Clostridium baratii, Clostridium bifermentans, Clostridium botulinum, Clostridium botulinum (types A, B, C, D, E, F, G), Clostridium butyricum, Clostridium difficile, Clostridium histolyticum, Clostridium novyi (type A), Clostridium novyi (type B), Clostridium perfringens, Clostridium perfringens (types A-E), Clostridium raemosum, Clostridium septicum, Clostridium sordelli, Clostridium sphenoides, Clostridium tedium, and Clostridium tetani. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Nocardia spp. Examples of Nocardia spp include Nocardia asteroides, Nocardia brasiliensis, Nocardia farcinica, Nocardia nova, Nocardia otitidiscaviarum, and Nocardia transvalensis. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Erysipelothrix spp, such as Erysipelothrix rhusiopathiae. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Listeria spp, such as Listeria monocytogenes. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Kytococcus spp, such as Kytococcus schroeteri. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Mycobacterium spp. Examples of Mycobacterium spp include Mycobacterium abscessus, Mycobacterium arupense, Mycobacterium asiaticum, Mycobacterium aubagnense, Mycobacterium avium complex, Mycobacterium boletii, Mycobacterium bolletii, Mycobacterium branderi, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacterium conspicuum, Mycobacterium elephantis, Mycobacterium farcinogenes, Mycobacterium florentinum, Mycobacterium fortuitum group, Mycobacterium genavense, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium heckeshomense, Mycobacterium heidelbergense, Mycobacterium houstonense, Mycobacterium immunogenum, Mycobacterium interjectum, Mycobacterium intracellulare, Mycobacterium senegalense, Mycobacterium africanum, Mycobacterium avium subsp paratuberculosis, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium mageritense, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium massiliense, Mycobacterium microti, Mycobacterium montefiorense (eels), Mycobacterium moracense, Mycobacterium mucogenicum, Mycobacterium nebraskense, Mycobacterium neoaurum, Mycobacterium novocastrense, Mycobacterium palustre, Mycobacterium parmense, Mycobacterium phlei, Mycobacterium phocaicum, Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacterium pseudoshottsii (fish), Mycobacterium pseudotuberculosis, Mycobacterium saskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senuense, Mycobacterium septicum, Mycobacterium simiae, Mycobacterium smegmatis, Mycobacterium szulgai, Mycobacterium terrae/chromogenicum complex, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium wolinskyi, and Mycobacterium xenopi. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, Trueperella spp. Examples of Trueperella spp include Trueperella abortisuis, Trueperella bemardiae, Trueperella bialowiezensis, Trueperella bonasi, Trueperella pyogenes (Arcanobacterium pyogenes).
In another embodiment, the bacterial agent is gram positive, gram negative or does not have a cell wall and selected from the group comprising, but not limited to, livestock pathogens. Examples of livestock pathogens include Actinobaculum suis, Actinomyces bovis, Arcanobacterium pyogenes, Bacillus anthracis, cereus, licheniformis, pumilus, melaninogenicus, subtilis, Clostridium botulinum, chauvoei, haemolyticum, novyi, perfringens, septicum, sordellii, tetani, colinum, Corynebacterium pseudotuberculosis, renale, Dermatophilus congolensis, Enterococcus spp (such as E. faecalis, E. faecium, E. durans, E. avium, E. hirae), Erysipelothrix rhusiopathiae, Listeria ivanovii, grayi, innocua, seeligeri, welshimeri, monocytogenes, Mycobacterium avium, bovis, paratuberculosis (Johne's Disease), Mycoplasma (such as capricolum subsp. capripneumoniae, subsp. capricolum, M. mycoides subsp mycoides, M. agalactiae, M. ovipneumoniae, M. conjunctivae, M. argini, M. bovis, and M. putrefaciens) Mycoplasma bovis, dispar, mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP) Mycoplasma gallisepticum (MG), iowae meleagridis (MM), synoviae (MS) Mycoplasma haemosuis (formerly Eperythrozoon suis), alkalescens, bovigenitalum, bovirhinis, bovoculi, californicum, canadense, cynos, equigenitalium, gateae, haemocanis, haemofelis, hyopneumoniae, hyorhinis, hyosynoviae, iowae, leachii, meleagridis, mycoides subsp capi, wenyoni, suis, Rhodococcus equi, Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus felis, Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus warneri, Staphylococcus haemolyticus, Staphylococcus sciuri, Staphylococcus saprophyticus, Staphylococcus hominis, Staphylococcus caprae, Staphylococcus cohnii subsp. cohnii, Staphylococcus cohnii subsp. urealyticus, Staphylococcus capitis subsp. capitis, Staphylococcus capitis subsp. urealyticus, Staphylococcus hyicus, Staphylococcus aureus, Staphylococcus pseudintermedius, Staphylococcus delphini, Staphylococcus schleiferi subsp. coagulans, Staphylococcus aureus subsp. anaerobius, Streptococcus uberis, Streptococcus canis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus pyogenes, Streptococcus bovis, Streptococcus equi subsp. Zooepidemicus, Streptococcus equinus, Streptococcus equi (Streptococcus equi subsp equi), Streptococcus equisimilis (Streptococcus dysgalactiae subsp equisimilis), porcinus, suis, zooepidemicus, Streptococcus zooepidemicus (Streptococcus equi subsp zooepidemicus), Streptococcus dysgalactiae subsp. equisimilis, Propionibacterium acnes, Propionibacterium granulosum, Eubacterium, Peptococcus indolicus, and Peptostreptococcus anaerobius; and various species of the following Gram negative genera: Actinobacillus, Aeromonas, Anaplasma, Arcobacter, Avibacterium, Bacteroides, Bartonella, Bordetella, Borrelia, Brachyspira, Brucella, Campylobacter, Capnocytophaga, Chlamydia, Chlamydophila, Chryseobacterium, Coxiella, Cytophaga, Dichelobacter, Edwardsiella, Ehrlichia, Escherichia, Flavobacterium, Francisella, Fusobacterium, Gallibacterium, Haemophilus, Histophilus, Klebsiella, Lawsonia, Leptospira, Mannheimia, Megasphaera, Moraxella, Neorickettsia, Nicoletella, Ornithobacterium, Pasteurella, Photobacterium, Piscichlamydia, Piscirickettsia, Poiphyromonas, Prevotella, Proteus, Pseudomonas, Rickettsia, Riemerella, Salmonella, Streptobacillus, Tenacibaculum, Vibrio, and Yersinia. 
In another embodiment, the bacterial agent is gram positive and selected from the group comprising, but not limited to, pathogens of companion animal species such as cats, dogs and horses. Examples of such pathogens include equine pathogens such as Streptococcus equi, Streptococcus zooepidemicus, Rhodococcus equi, Clostridium difficile, Clostridium perfringens, Corynebacterium pseudotuberculosis, Clostridium piliforme, Actinomyces bovis, Staphylococcus aureus, beta haemolytic Streptococcus spp, Dermatophilus congolense, Clostridium tetani, and Clostridium botulinum. Further examples include pathogens of dogs and cats such as Staphylococcus spp, Streptococcus spp, Clostridium spp, Actinomyces spp, Enterococcus spp, Nocardia spp, Mycoplasma spp, and Mycobacterium spp.
In another embodiment, the bacterial agent is gram negative and selected from the group consisting of the following representative families and species: Acetobacteraceae:—Roseomonas cervicalis; Roseomonas fauriae; Roseomonas gilardii. —Aeromonadaceae:—Aeromonas allosaccharophila; Aeromonas aquariorum; Aeromonas caviae; Aeromonas hydrophila (and subspecies); Aeromonas salmonicida; Aeromonas shubertii; Aeromonas veronii biovar sobria (Aeromonas sobria). —Alcaligenaceae:—Achromobacter xylosoxidans; Aicaligenes faecalis; Bordetella ansorpii; Bordetella avium; Bordetella bronchiseptica; Bordetella hinzii; Bordetella holmesii; Bordetella parapertussis; Bordetella pertussis; Bordetella petrii; Bordetella trematum; Oligella ureolytica; Oligella urethralis. —Anaplasmataceae:—Anaplasma phagocytophilum; Anaplasma platys; Anaplasma bovis; Anaplasma centrale; Anaplasma marginale; Anaplasma odocoilei; Anaplasma ovis; Ehrlichia canis; Ehrlichia chaffeensis; Ehrlichia ewingii; Ehrlichia muris; Ehrlichia ovina; Ehrlichia ruminantium; Neoehrlichia lotoris; Neoehrlichia mikurensis; Neorickettsia helminthoeca; Neorickettsia risticii; Neorickettsia sennetsu; Wolbachia pipientis. —Armatimonadaceae:—Armatimonas rosea. —Bacteroidaceae:—Bacteroides forsythus; Bacteroides fragilis; Bacteroides melaninogenicus; Bacteroides ruber, Bacteroides urealtyicus. —Bartonellaceae:—Bartonella alsatica; Bartonella australis; Bartonella bacilliformis; Bartonella birtlesii; Bartonella bovis; Bartonella capreoi; Bartonella chomelii; Bartonella clarridgeiae; Bartonella doshiae; Bartonella elizabethae; Bartonella grahamii; Bartonella henselae; Bartonella koehlerae; Bartonella peromysci; Bartonella phoceensis; Bartonella quintana; Bartonella rattimassiliensis; Bartonella rochalimae; Bartonella schoenbuchensis; Bartonella telpae; Bartonella tamiae; Bartonella taylorii; Bartonella tribocorum; Bartonella vinsonii subsp berkhoffii; Bartonella vinsonii subsp. arupensis; Bartonella vinsonii subsp. vinsonii. —Bdellovibrionaceae:—Bdellovibrio spp. —Brachyspiraceae:—Brachyspira spp including Brachyspira hampsonii, Brachyspira hyodysenteriae, Brachyspira murdochi, Brachyspira pilosicoi. —Brucellaceae:—Brucalla abortus; Brucalla canis; Brucalla ceti; Brucalla melitensis; Brucella ovis; Brucalla pinnipedialis; Brucalla suis; Ochrobactrum anthropi; Ochrobactrum intermedium. —Burkholderiaceae:—Burkholderia aboris; Burkholderia ambifana (genomovar VII); Burkholderia anthina (genomovar VIII); Burkholderia cenocepacia (genomovar III); Burkholderia cepacia (genomovar I); Burkholderia diffusa; Burkholderia dolosa (genomovar VI); Burkholderia latens; Burkholderia mallei; Burkholderia metallica; Burkholderia multivorans (genomovar II); Burkholderia pseudomallei; Burkholderia pyrrocinia (genomovar IX); Burkholderia seminalis; Burkholderia stabilis (genomovar IV); Burkholderia ubonensis (genomovar); Burkholderia vietnamiensis (genomovar IV; Cupriavidus pauculus; Cupriavidus gilardii; Ralstonia picketti; Ralstonia mannitoilytica; Sphaerotilus hippei, Sphaerotilus montanus; Sphaerotilus natans. —Campylobacteraceae:—Arcobacter spp including Arcobacter skirrowii; Campylobacter coli; Campylobacter concisus; Campylobacter curvus; Campylobacter fetus; Campylobacter gracilis; Campylobacter helveticus; Campylobacter hominis; Campylobacter hyointestinalis; Campylobacter insulaenigrae; Campylobacter jejuni; Campylobacter lanienae; Campylobacter lari; Campylobacter laridis; Campylobacter mucosalis; Campylobacter rectus; Campylobacter showae; Campylobacter sputorum; Campylobacter upsaliensis. —Candidatus:—Piscichlamydia salmonis. —Cardiobactenaceae:—Cardiobacterium hominis; Cardiobacterium valvarum; Dichelobacter nodosus. —Chlamydiaceae:—Chlamydia spp including Chlamydia avium, Chlamydia gallinacea, Chlamydia muridarum, Chlamydia suis, Chlamydia trachomatis; Chlamydophila spp including Chlamydophila pneumoniae, Chlamydophila pecorum, Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae, and Chlamydophila felis. —Chthonomonadaceae:—Chthonomonas celidirosea. —Comamonadaceae:—Comamonas testosteroni; Verminephrobacter spp. —Coxiellaceae:—Coxiella burnetii. —Cytophagaceae:—Cytophaga columnaris; Cytophaga hutchinsonii; Flexibacter echinicida; Flexibacter elegans; Flexibacter flexilis; Flexibacter litoraiis; Flexibacter polymorphus; Flexibacter roseolus; Flexibacter ruber. —Desulfovibrionaceae:—Bilophila wadsworthia; Lawsonia intracellularis. —Enterobacteraceae:—Cedecea davisae; Cedecea lapagei; Cedecea neteri; amalonaticus; Citrobacter diversus; Citrobacter freundii; Citrobacter koseri; Cronobacter condimenti; Cronobacter dublinensis; Cronobacter helveticus; Cronobacter malonaticus; Cronobacter muytjensii; Cronobacter pulveris, Cronobacter sakazakii; Cronobacter turicensis; Cronobacter universalis; Cronobacter zurichensis; Edwardsiella ictaluri; Edwardsiella tarda; Enterobacter aerogenes; Enterobacter agglomerans; Enterobacter cloacae; Enterobacter cowanii; Escherichia albertii; Escherichia coli, including AIEC=adherent invasive E. coli, EaggEC=enteroaggregative E. coli; EHEC=enterohemonhagic E. coli; EIEC=enteroinvasive E. coli; EPEC=enteropathogenic E. coli; ETEC=enterotoxigenic E. coli; ExPEC=extraintestinal pathogenic E. coli, NMEC=neonatal meningitis E. coli, NTEC=necrotoxigenic E. coli, UPEC=uropathogenic E. coli.; Escherichia fergusonii; Ewingella americana; Hafnia alvei; Hafnia paralvei; Klebsiella granulomatis; Klebsiella oxytoca; Klebsiella pneumoniae; Kluyvera ascorbata; Kluyvera cryocrescens; Morganella morganii; Pantoea (formerly Enterobacter) agglomerans; Photorhabdus asymbiotica; Plesiomonas shigeloides; Proteus mirabilis; Proteus penneri; Proteus vulgaris; Providencia alcalifaciens; Providencia rettgeri; Providencia stuartii; Raoultella electrica; Raoultella ornithinolytica; Raoultella planticola; Raoultella terrigena; Salmonella bongori, Salmonella enterica subspecies enterica (many serotypes); Serratia liquifaciens; Serratia marcesans; Shigella boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Yersinia enterocolitica; Yersinia pestis; Yersinia pseudotuberculosis; Yersinia ruckeri. —Fimbriimonadaceae:—Fimbriimonas ginsengisoli. —Flavobacteriaceae:—Bergeyella zoohelcum; Capnocytophaga canimorsus; Capnocytophaga cynodegmi; Capnocytophaga gingivalis; Capnocytophaga granulosa; Capnocytophaga haemolytica; Capnocytophaga leadbetteri; Capnocytophaga ochracea; Capnocytophaga sputigena; Chryseobacterium indologenes; Chryseobacterium piscicola; Elizabethkingia meningoseptica; Flavobacterium branchiophilum; Flavobacterium columnare; Flavobacterium oncorhynchi; Flavobacterium piscicida; Flavobacterium psychrophilum; Myroides odoratus; Myroides odoratimimus; Ornithobacterium rhinotracheale; Riemerella anatipestifer, Riemerella columbina; Riemerella columbipharyngis; Tenacibaculum dicentrarchi; Tenacibaculum discolour, Tenacibaculum gallaicum; Tenacibaculum maritimum; Tenacibaculum soleae; Weeksella virosa. —Francisellaceae:—Francisella tularensis subsp. tularensis; Francisella tularensis subsp. holarctica; Francisella tularensis subsp. novicida; Francisella philomiragia; Francisella noatunensis; Francisella noatunensis subsp. orientalis (also termed Francisella asiatica). —Fusobacteriaceae:—Fusobacterium spp. including Fusobacterium necrophorum, Fusobacterium nucleatum, Fuso-bacterium polymorphum. —Helicobacteraceae:—Helicobacter cinaedi; Helicobacter fennelliae; Helicobacter pylori. —Legionelaceae:—Legionella pneumophila and other species including; Legionella anisa; Legionella birminghamensis; Legionella bozemannii; Legionella cincinnatiensis; Legionella dumoffii; Legionella feeleii; Legionella gormanii; Legionella hackeliae; Legionella jordanis; Legionella lansingensis; Legionella longbeachae; Legionella maceachemii; Legionella micdadei; Legionella oakridgensis; Legionella parisiensis; Legionella sainthelens; Legionella tusconensis; Legionella wadswothii; Legionella waltersii. —Leptospiraceae:—Leptospira alexanderi (including Leptospira alexanderi serovar Hebdomadis, Leptospira alexanderi serovar Manhao 3); Leptospira alstoni (including Leptospira alstoni serovar Pingchang, Leptospira alstoni serovar Sichuan); Leptospira biflexa (including Leptospira biflexa serovar Ancona, Leptospira biflexa serovar Canela); Leptospira borgpetersenii (including Leptospira borgpetersenii serovar Hardjo, Leptospira borgpetersenii serovar Hardjo-bovis, Leptospira borgpetersenii serovar Pomona, Leptospira borgpetersenii serovar Tarassovi); Leptospira broomii (including Leptospira broomii serovar Hurstbridge); Leptospira fainei (including Leptospira fainei serovar Hurstbridge); Leptospira idonii; Leptospira inadai (including Leptospira inadai serovar Lyme, Leptospira inadai serovar Malaya); Leptospira interrogans (including Leptospira interrogans serovar Australis, Leptospira interrogans serovar Autumnalis, Leptospira interrogans serovar Bratislava, Leptospira interrogans serovar Canicola, Leptospira interrogans serovar Grippotyphosa, Leptospira interrogans serovar Hardjo, Leptospira interrogans serovar Hardjo-bovis, Leptospira interrogans serovar Icterohaemonrrhagiae, Leptospira interrogans serovar Pomona, Leptospira interrogans serovar Pyrogenes, Leptospira interrogans serovar Tarassovi); Leptospira kirschneri (including Leptospira kirschneri serovar Bulgarica, Leptospira kirschneri serovar Cynopten, Leptospira kirschneri serovar Grippotyphosa); Leptospira kmetyi; Leptospira licerasiae; Leptospira meyeri (including Leptospira meyeri serovar Sofia); Leptospira noguchii (including Leptospira noguchii serovar Panama, Leptospira noguchii serovar Pomona); Leptospira santarosai; Leptospira terpstrae; Leptospira vanthielii; Leptospira weilii (including Leptospira weilii serovar Celledoni, Leptospira weilii serovar Sarmin); Leptospira wolbachii; Leptospira wolffii; Leptospira yanagawae. —Leptotrichiaceae:—Leptotrichia buccalis; Streptobacillus moniliformis. —Methylobacteriaceae:—Methylobacterium extorquens group; Methylobacterium fujisawaense; Methylobacterium mesophilicum; Methylobacterium zatmanii. —Moraxellaceae:—Acinetobacter baumannii (genomic species 2); Acinetobacter baylyi; Acinetobacter bouvetii; Acinetobacter calcoaceticus (genomic species 1); Acinetobacter gerneri; Acinetobacter grimontii; Acinetobacter haemolyticus (genomic species 4); Acinetobacter johnsonii (genomic species 7); Acinetobacter junii (genomic species 5); Acinetobacter lwoffi (genomic species 89); Acinetobacter parvus; Acinetobacter radioresistens (genomic species 12); Acinetobacter schindleri; Acinetobacter tandoii; Acinetobacter tjernbergiae; Acinetobacter townenri; Acinetobacter ursingii; Acinetobacter venetianus; Moraxella atlantae; Moraxella boevrei; Moraxella bovis; Moraxella bovoculi; Moraxella canis; Moraxella caprae; Moraxella catarrhalis; Moraxella caviae; Moraxella cuniculi; Moraxella equi; Moraxella lacunata; Moraxella lincolnii; Moraxella macacae; Moraxella nonliquefaciens; Moraxella oblonga; Moraxella osloensis; Moraxella ovis; Moraxella phenylpyruvica; Moraxella pluranimalium; Moraxella porci. —Moritellaceae:—Moritella abyssi; Moritella dasanensis; Moritella japonica; Moritella marina; Moritella pro-funda; Moritella viscosa; Moritella yayanosi. —Neisseriacae:—Chromobacterium violaceum; Eikenella corrodens; Kingella denitrificans, Kingella kingae, Kingella oralis, Kingella potus; Neisseria cinema; Neisseria elongata; Neisseria flavescens; Neisseria gononrrhoeae; Neisseria lactamica; Neisseria meningitidis; Neisseria mucosa; Neisseria polysaccharea; Neisseria sicca; Neisseria subflava; Neisseria weaver Vitreoscilla spp. —Nitrosomonadaceae:—Nitrosomonas eutropha; Nitrosomonas halophila; Nitrosomonas oligotropha. —Pasteurellaceae:—Actinobacillus actinomycetemcomitans; Actinobacillus equuli; Actinobacillus lignieresii; Actinobacillus pleuropneumoniae; Actinobacillus seminis; Actinobacillus succinogenes; Actinobacillus ureae; Aggregatibacter actinomycetemcomitans, Aggregatibacter segnis, Aggregatibacter aphrophilus; Avibacterium avium; Avibacterium endocarditidis; Avibacterium gallinarum; Avibacterium paragallinarum; Avibacterium volantium; Bibersteinia trehalose; Gallibacterium anatis; Gallibacterium genomospecies 1; Gallibacterium genomospecies 2; Gallibacterium genomospecies 3; Gallibacterium group V; Gallibacterium melopsittaci; Gallibacterium salpingitidis; Gallibacterium trehalosifermentans; Haemophilus aegyptius; Haemophilus avium; Haemophilus ducreyi; Haemophilus haemolyticus; Haemophilus influenzae; Haemophilus parahaemolyticus; Haemophilus parainfluenzae; Haemophilus parasuis; Histophilus somni; Mannheimia caviae; Mannheimia glucosida; Mannheimia granulomatis; Mannheimia haemolytica; Mannheimia ruminalis; Mannheimia varigena; Nicoletella semolina; Pasteurella aerogenes; Pasteurella bettyae; Pasteurella caballi; Pasteurella canis; Pasteurella dagmatis; Pasteurella multocida (subspecies multocida, septicum, gallicida); Pasteurella pneumotropica; Pasteurella stomatis; Pasteurella trehalosi. —Piscirickettsiaceae:—Piscirickettsia salmonis. —Plesiomonadaceae:—Plesiomonas shigelloides. —Polyangiaceae:—Sorangium cellulosum. —Porphyromonadaceae:—Dysgonomonas capnocytophagoides; Dysgonomonas gadei; Dysgonomonas hofstadii; Dysgonomonas mossii; Dysgonomonas oryzarvi; Dysgonomonas wimpennyi; Porphyromonas gingivalis. —Prevotellaceae:—Prevotella spp. including Prevotella intermedia, Prevotella melaninogenica. —Pseudomonadaceae:—Chryseomonas luteola; Pseudomonas aeruginosa; Pseudomonas luteola; Pseudomonas fluorescens; Pseudomonas putida; Pseudomonas stutzeri; Pseudomonas oryzihabitans. —Rhizobiaceae:—Agrobacterium tumefaciens; Rhizobium radiobacter. —Rickettsiaceae:—Orientia chuto; Orientia tsutsugamushi; Rickettsia aeschlimannii; Rickettsia africae; Rickettsia akari; Rickettsia argasii; Rickettsia asiatica; Rickettsia australis; Rickettsia bellii; Rickettsia canadensis; Rickettsia conori; Rickettsia cooleyi; Rickettsia felis; Rickettsia heilongjiangensis; Rickettsia helvetica; Rickettsia honei; Rickettsia hoogstraalii; Rickettsia hulinensis; Rickettsia hulinii; Rickettsia japonica; Rickettsia marmionii; Rickettsia martinet; Rickettsia massiliae; Rickettsia monacensis; Rickettsia montanensis; Rickettsia monteiroi; Rickettsia moreli; Rickettsia parked; Rickettsia peacockii; Rickettsia philipii; Rickettsia prowazekii; Rickettsia raoultii; Rickettsia rhipicephali; Rickettsia rickettsii; Rickettsia sibirica subgroup; Rickettsia slovaca; Rickettsia tamurae; Rickettsia typhi. —Shewanellaceae:—Shewanella putrefaciens. —Sphingomonadaceae:—Sphingobactenum multivorum; Sphingobacterium spiritivorum; Sphingomonas paucimobilis. —Spirillaceae:—Spirillum minus; Spirillum volutans; Spirillum winogradskyi. —Spirochaetaceae:—Borrelia afzelii; Borrelia anserina; Borrelia bissettii; Borrelia burgdorferi; Borrelia coriaceae; Borrelia duttonii; Borrelia garinii; Borrelia hermsii; Borrelia hispanica; Borrelia japonica; Borrelia lonestari; Borrelia lusitaniae; Borrelia miyamotoi; Borrelia parked; Borrelia persica; Borrelia recurrentis; Borrelia spielmanii; Borrelia turicatae; Borrelia turicatae; Borrelia valaisiana; Treponema carateum; Treponema pallidum ssp. endemicum; Treponema pallidum ssp. pallidum; Treponema pallidum ssp. pertenue. —Succinivibrionaceae:—Anaerobiospirillum spp. —Sutterellaoeae:—Sutterella spp including Sutterella wadsworthia. —Thermaceae:—Meiothermus spp. —Thermotogaceae:—Thermotoga neapolitana. —Veillonellaceae:—Dialister spp; Megamonas spp; Megasphaera spp; Pectinatus spp; Pelosinus spp; Propionispora spp; Sporomusa spp; Veillonella spp.; Zymophilus spp. —Vibrionaceae:—Photobacterium damselae; Vibrio adaptatus; Vibrio alginolyticus; Vibrio azasii; Vibrio campbellii; Vibrio cholera; Vibrio damsel; Vibrio fluvialis; Vibrio fumisii; Vibrio hollisae; Vibrio metchnikovii; Vibrio mimicus; Vibrio parahaemolyticus; Vibrio vulnificus. —Wolbachieae:—Wolbachia spp. —Xanthomonadaceae:—Luteimonas aestuarii; Luteimonas aquatica; Luteimonas composti; Luteimonas lutimaris; Luteimonas marina; Luteimonas mephitis; Luteimonas vadosa; Pseudoxanthomonas broegbemrnensis; Pseudoxanthomonas japonensis; Stenotrophomonas maltophilia; Stenotrophomonas nitritireducens. 
Most preferably, the bacterial agent causing the bacterial infection is gram negative and is selected from the group comprising: Acinetobacter species, Aeromonas hydrophila, Citrobacter species, Enterobacter species, Escherichia coli, Klebsiella pneumoniae, Morganella morganii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia. 
In another preferred embodiment, the bacteria agent causing the bacterial colonisation or infection is resistant to a conventional antibiotic used to treat the colonisation or infection. In one preferred embodiment, the bacterial agent is resistant to a compound selected from the group comprising: one or more of aminoglycosides (for example gentamicin, tobramycin, amikacin, or netilmicin); anti-MRSA cephalosporins (for example ceftaroline); antipseudomonal penicillins+β-lactamase inhibitors (for example ticarcilin-clavulanic acid or piperacillin-tazobactam); carbapenems (for example ertapenem, imipenem, meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for example cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (for example cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefotetan); fluoroquinolones (for example ciprofloxacin); folate pathway inhibitors (for example trimethoprim-sulphamethoxazole); glycylcyclines (for example tigecycline); monobactams (for example aztreonam); penicillins (for example ampicillin); penicillins+β-lactamase inhibitors (for example amoxicillin-clavulanic acid or ampicillin-sulbactam); phenicols (for example chloramphenicol); phosphonic acids (for example fosfomycin); and polymyxins (for example colistin); tetracyclines (for example tetracycline, doxycycline or minocycline). Preferably, the bacterial agent resistant to these compounds is gram negative.
Preferably, the bacterial agent is resistant to a compound selected from the group comprising: penicillins, cephalosporins, carbapenems, monobactams and other β-lactam antibiotics, fusidanes, aminoglycosides, fluoroquinolones, streptogramins, tetracyclines, glycylcyclines, chloramphenicol and other phenicols, macrolides and ketolides, lincosamides, oxazolidinones, aminocyclitols, polymyxins, glycopeptides, lipopeptides, bacitracin, mupiricin, pleuromutilins, rifamycins, sulphonamides and trimethoprim. More preferably, the compound is selected from the group comprising: β-lactams, glycopeptides, lipopeptides, macrolides, oxazolidinones and tetracyclines. Preferably, the bacterial agent is resistant to the compound when the compound is at a concentration range selected from the following: 0.001 μg/mL-10,000 μg/mL; 0.01 μg/mL-1000 μg/mL; 0.10 μg/mL-100 μg/mL; and 1 μg/mL-50 μg/mL
Most preferably, the bacterial agent causing the bacterial infection is selected from the group comprising, but not limited to, gram positive bacteria. The bacterial agent is most preferably a Gram positive bacterial agent selected from the group comprising Staphylococcus aureus, Staphylococcus pseudintermedius, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, and Clostridium difficile. 
In one preferred embodiment, the bacterial agent has no cell wall. Preferably, the bacterial agent is selected from the group comprising: Mycoplasma spp, Mycoplasma agalactiae, Mycoplasma alkalescens, Mycoplasma amphoriforme, Mycoplasma arginini, Mycoplasma bovigenitalum, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasma buccale, Mycoplasma californicum, Mycoplasma canadense, Mycoplasma capricolum subsp. capricolum, Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma conjunctivae, Mycoplasma cynos, Mycoplasma dispar, Mycoplasma equigenitalium, Mycoplasma faucium, Mycoplasma felis, Mycoplasma fermentans (incognitus str.), Mycoplasma gallisepticum (MG), Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma haemocanis, Mycoplasma haemofelis, Mycoplasma haemosuis (formerly Eperythrozoon suis), Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma iowae meleagridis (MM), Mycoplasma iowae, Mycoplasma leachii, Mycoplasma lipophilum, Mycoplasma meleagridis, Mycoplasma mycoides subsp capri, Mycoplasma mycoides subsp mycoides, Mycoplasma mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP), Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma primatum, Mycoplasma putrefaciens, Mycoplasma salivarium, Mycoplasma spermatophilum, Mycoplasma suis, Mycoplasma synoviae (MS), Mycoplasma wenyonii, Mycoplasma, Ureaplasma spp, Ureaplasma parvum, Ureaplasma urealyticum, Ureaplasma, and Ureoplasma diversum. 
In another most preferred embodiment, the bacterial agent is Staphylococcus aureus. 
In another preferred embodiment, the bacterial agent is resistant to a compound selected from the group comprising: one or more of aminoglycosides (for example gentamicin); ansamycins (for example rifampicin); anti-MRSA cephalosporins (for example ceftaroline); anti-staphylococcal β-lactams (or cephamycins) (for example oxacillin or cefoxitin); carbapenems (for example ertapenem, imipenem, meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for example cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (for example cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefotetan); fluoroquinolones (for example ciprofloxacin or moxifloxacin); folate pathway inhibitors (for example trimethoprim-sulphamethoxazole); fucidanes (for example fusidic acid); glycopeptides (for example vancomycin, teicoplanin or telavancin); glycylcyclines (for example tigecycline); lincosamides (for example clindamycin); lipopeptides (for example daptomycin); macrolides (for example erythromycin); oxazolidinones (for example linezolid or tedizolid); phenicols (for example chloramphenicol); phosphonic acids (for example fosfomycin); streptogramins (for example quinupristin-dalfopristin); and tetracyclines (for example tetracycline, doxycycline or minocycline). Preferably, the bacterial agent resistant to these compounds is gram positive.
In another most preferred embodiment, the bacterial agent is Streptococcus pneumoniae. The Streptococcus pneumoniae may be a strain that is resistant to one or more of β-lactams and macrolides.
In another most preferred embodiment, the bacterial agent is Streptococcus pyogenes. 
In another most preferred embodiment, the bacterial agent is Streptococcus agalactiae. 
In another most preferred embodiment, the bacterial agent is either Enterococcus faecium or Enterococcus faecalis. The Enterococcus faecium or Enterococcus faecalis may be a strain that is resistant to one or more of aminoglycosides (for example gentamicin (high level) or streptomycin (for example streptomycin (high level)); carbapenems (for example imipenem, meropenem or doripenem); fluoroquinolones (for example ciprofloxacin, levofloxacin or moxifloxacin); glycopeptides (for example vancomycin or teicoplanin); glycylcyclines (for example tigecycline); lipopeptides (for example daptomycin); oxazolidinones (for example linezolid); penicillins (for example ampicillin); streptogramins (for example quinupristin-dalfopristin); tetracycline (for example doxycycline or minocycline).
In another most preferred embodiment, the bacterial agent is Clostridium difficile. 
The bacterial infection in the subject may cause a disease selected from the group comprising, but not limited to, nosocomial pneumonia caused by Staphylococcus aureus (MDR, XDR, PDR or methicillin-susceptible or -resistant strains), or invasive pneumococcal diseases such as pneumonia, bronchitis, acute sinusitis, otitis media, conjunctivitis, meningitis, bacteremia, sepsis, osteomyelitis, septic arthritis, endocarditis, peritonitis, pericarditis, cellulitis, and brain abscess caused by Streptococcus pneumoniae (including multi-drug resistant strains [MDRSP] such as those resistant to β-lactams and macrolides), complicated skin and skin structure infections, including diabetic foot infections, with or without concomitant osteomyelitis, caused by Staphylococcus aureus (methicillin-susceptible and -resistant strains), Streptococcus pyogenes, or Streptococcus agalactiae, uncomplicated skin and skin structure infections caused by Staphylococcus aureus (methicillin-susceptible and -resistant strains) or Streptococcus pyogenes, community-acquired pneumonia caused by Streptococcus pneumoniae (including multi-drug resistant strains [MDRSP], including cases with concurrent bacteraemia, or Staphylococcus aureus (methicillin-susceptible and -resistant strains) and Staphylococcus aureus bloodstream infections (bacteraemia), including those with right-sided infective endocarditis, caused by methicillin-susceptible and methicillin-resistant isolates, vancomycin-resistant Enterococcus infections, including cases with concurrent bacteraemia, and treatment of Clostridium difficile-associated diarrhea (CDAD).
Gram negative organisms are important causes of many infectious diseases in humans and other animal species. Bone and joint infections (Gram-negative organisms or mixed bacteria, are an important cause of vertebral osteomyelitis and septic arthritis), cardiovascular system infections (including endocarditis caused by the HACEK group—Haemophilus parainfluenzae, Haemophilus aphrophilus, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella conrrodens, Kingella kingae), central nervous system infections (the commonest causes of bacterial meningitis are Neisseria meningitidis, Streptococcus pneumoniae and, in nonvaccinated young children, Haemophilus influenzae type b (Hib), in neonates and infants less than 3 months of age, Streptococcus agalactiae (group B streptococcus), Escherichia coli and other aerobic Gram-negative rods are important pathogens, brain abscess or subdural empyema, the infecting organism(s) vary with the underlying predisposing cause but where the likely site of origin is the ear, enteric Gram-negative bacilli are commonly involved), eye infections (common pathogens include Haemophilus influenza, Neisseria gonorrhoeae or Chlamydia trachomatis), gastrointestinal tract infections (a wide range of pathogens are implicated including enterotoxigenic Escherichia coli (ETEC), Salmonella, Campylobacter, Shigella, Vibrio cholera and Yersinia enterocolitica), genital infections (bacterial vaginosis is a polymicrobial clinical syndrome with high concentrations of anaerobic (eg Mobiluncus species) and other fastidious bacteria (including Gardnerella vaginalis and Atopobium vaginae), and Mycoplasma hominis; non-sexually acquired pelvic inflammatory disease (PID) is usually caused by mixed vaginal flora, including anaerobes, facultative Gram-negative bacteria and Mycoplasma hominis, while sexually acquired PID is usually initiated by C. trachomatis or N. gonorrhoeae with growing evidence that M. genitalium infection is involved in a significant minority of cases), intra-abdominal infections (peritonitis due to perforated viscus is usually a polymicrobial infection with aerobic and anaerobic bowel flora while spontaneous bacterial peritonitis (SBP) is usually caused by enteric Gram-negative bacilli, such as Escherichia coli and Klebsiella species, Klebsiella pneumoniae is an increasingly identified cause of liver abscess), community-acquired pneumonia (Mycoplasma pneumoniae, Chlamydophila (Chlamydia) pneumoniae, Chlamydophila (Chlamydia) psittaci, Haemophilus influenza, aerobic Gram-negative bacilli including Klebsiella pneumonia, Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia pseudomallei), otitis externa (including acute diffuse) (bacterial cultures commonly yield Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus and Klebsiella species), otitis media (including acute) (common bacterial pathogens include Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis), sepsis (including severe) (including Acinetobacter baumannii, disseminated gonococcal sepsis, Gram-negative enteric bacteria, Neisseria meningitidis (meningococcal sepsis) and Pseudomonas aeruginosa), Systemic infections (Spotted fevers (Rickettsia) and scrub typhus (Orientia), Brucellosis, Cat-scratch disease and other Bartonella infections, Leptospirosis, Lyme disease, Melioidosis, Q fever, Typhoid and paratyphoid fevers (enteric fevers), urinary tract infections (acute cystitis, acute pyelonephritis, recurrent urinary tract infections and atheter-associated bacteriuria and urinary tract infections).
In humans, gram negative bacteria are common causes of intra-abdominal infections (IAIs), urinary tract infections (UTIs), hospital acquired pneumonia, and bacteraemia. Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) are important pathogens in the hospital setting, accounting for 27% of all pathogens and 70% of all Gram-negative pathogens causing healthcare-associated infections [Sievert D M, Ricks P, Edwards J R, et al. Antimicrobial-resistant pathogens associated with healthcare-associated infections: summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2009-2010. Infect Control Hosp Epidemiol. 2013; 34:1-14.].
Gram negative bacteria are showing rising rates of resistance to current therapies. The production of extended-spectrum β-lactamase (ESBL) enzymes is a common mechanism of resistance. Rates of ESBL-producing E. coli and K. pneumoniae have risen substantially, with the result that these bacteria are increasingly resistant to widely used antimicrobials.
P. aeruginosa is the most common Gram-negative cause of nosocomial pneumonia and the second most common cause of catheter-related UTIs in the U.S.
E. coli is the most common cause of UTIs. Cases of UTI caused by ESBL-producing E. coli and K. pneumonia as well as P. aeruginosa, including MDR strains, are increasing. ESBL-producing E. coli and K. pneumoniae are also frequently isolated in patients with complicated IAI (cIAI).
P. aeruginosa is a clinically challenging and virulent pathogen that can be a cause of common infections in humans such as nosocomial pneumonia, UTI, IAI, and bloodstream infections. P. aeruginosa is the most common Gram-negative organism causing ventilator associated pneumonia and the second most common cause of catheter-associated UTIs.
The increase in the number of infections caused by Gram-negative bacteria is being accompanied by rising rates of resistance. Treatment options to meet this challenge are increasingly limited. There is a critical need for new antibiotics to meet the needs of patients now and in the future.
In another preferred embodiment, robenidine, or a therapeutically acceptable salt thereof, is administered together with a compound or agent that removes or substantially removes or reduces the integrity of the cell wall of the bacterial agent. As an example, the compound is selected from the group consisting of: β-lactams, fosfomycin, lysozyme, polymyxins and chelating agents such as ethylenediaminetetraacetic acid (EDTA). As an example, the agent is an immunological agent (such as an antibody or vaccine) that reduces the integrity of the cell wall. In one preferred embodiment, robenidine, or a therapeutically acceptable salt thereof, is administered together with a compound that removes or substantially removes or weakens the integrity of the outer cell wall of a gram negative or positive bacterial agent.
According to another aspect of the invention, there is provided an antibacterial pharmaceutical composition comprising a therapeutically effective amount of robenidine, or a therapeutically acceptable salt thereof.
According to another aspect of the invention, there is provided an antibacterial veterinary composition comprising a therapeutically effective amount of robenidine, or a therapeutically acceptable salt thereof.
The method of treating or preventing a bacterial infection or colonisation in a subject, may also comprise the administration of the pharmaceutical or veterinary compositions of the invention.
The pharmaceutical composition may optionally include a pharmaceutically acceptable excipient or carrier. The veterinary composition may optionally include a veterinary acceptable excipient or carrier.
The pharmaceutical or veterinary composition of the invention preferably contains robenidine, or a pharmaceutically acceptable salt, at a concentration of selected from the group consisting of: 1 mg/g to 500 mg/g; 5 mg to 400 mg/g; 10 mg/g to 200 mg/g; 20 mg/g to 100 mg/g; 30 mg/g to 70 mg/g; and 40 mg/g to 60 mg/g.
In another embodiment, the pharmaceutical or veterinary composition comprises impurities, wherein the quantity of impurities as a percentage of the total weight of the composition is selected from the group consisting of: less than 20% impurities (by total weight of the composition); less than 15% impurities; less than 10% impurities; less than 8% impurities; less than 5% impurities; less than 4% impurities; less than 3% impurities; less than 2% impurities; less than 1% impurities: less than 0.5% impurities; less than 0.1% impurities. In one embodiment, the pharmaceutical or veterinary composition comprises microbial impurities or secondary metabolites, wherein the quantity of microbial impurities as a percentage of the total weight of the composition is selected from the group consisting of: less than 5%; less than 4%; less than 3%; less than 2%; less than 1%; less than 0.5%; less than 0.1%; less than 0.01%; less than 0.001%. In one embodiment, the pharmaceutical or veterinary composition is sterile and stored in a sealed and sterile container. In one embodiment, the pharmaceutical or veterinary composition contains no detectable level of microbial contamination.
Preferably, the robenidine is pharmaceutical or veterinary grade. Methods to synthesise commercial quantities of robendine are widely available in the art. Commercial quantities of pharmaceutical or veterinary grade robenidine are available from Zhejiang Esigma Animal Health Co., Ltd, Haining City, Peoples Republic of China.
The pharmaceutical or veterinary composition of the invention may comprise a further antimicrobial agent. The further antimicrobial agent may be an antifungal agent or antibacterial agent. The method of treating or preventing a bacterial infection or colonisation in a subject, may also comprise the administration of robenidine with a further antimicrobial agent.
In one embodiment, the antifungal agent is selected from the group comprising, but not limited to naturally occurring agents including Echinocandins (Anidulafungin, Caspofungin, Micafungin), Polyenes (Amphotericin B, Candicidin, Filipin, Fungichromin (Pentamycin), Hachimycin, Hamycin, Lucensomycin, Mepartricin, Natamycin, Nystatin, Pecilocin, Perimycin), and other naturally occurring antifungal agents including Griseofulvin, Oligomycins, Pyrrolnitrin, Siccanin, and Viridin. The antifungal agent may be a synthetic compound selected from the group comprising, but not limited to Allylamines (Butenafine, Naftifine, Terbinafine) Imidazoles (Bifonazole, Butoconazole, Chlormidazole, Climbazole, Croconazole (Cloconazole), Clotrimazole, Eberconazole, Econazole, Enilconazole, Fenticonazole, Flutrimazole, Fosfluconazole, Isoconazole, Ketoconazole, Lanoconazole, Luliconazole, Miconazole, Neticonazole, Omoconazole, Oxiconazole Nitrate, Parconazole, Sertaconazole, Sulconazole, Tioconazole), Thiocarbamates (Liranaftate, Tolciclate, Tolindate, Tolnaftate), Triazoles (Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Saperconazole, Terconazole, Voriconazole), and other synthetic agents such as Acrisorcin, Amorolfine, Bromosalicylchloranilide (Bromochlorosalicylanilide), Buclosamide, Calcium Propionate, Chlorphenesin, Ciclopirox, Cloxyquin (Cloxiquine), Coparaffinate, Exalamide, Flucytosine, Haloprogin, Hexetidine, Loflucarban, Nifuratel, Nifuroxime, Piroctone, Potassium Iodide, Propionic Acid, Pyrithione, Salicylanilide, Sodium Parachlorobenzoate, Sodium Propionate, Sulbentine, Tenonitrozole, Triacetin, Trimetrexate, Undecylenic Acid (Undecenoic Acid), and Zinc Propionate.
The composition of the invention may comprise an antibiotic adjunct selected from the group comprising, but not limited to, β-Lactamase Inhibitors (Avibactam, Clavulanic Acid, Sulbactam, Sultamicilin, Tazobactam), Renal Dipeptidase Inhibitors (Cilastatin), and Renal Protectant (Betamipron).
In one embodiment, the composition of the invention comprises a further antibiotic selected from the group comprising, but not limited to, 2,4-DIAMINOPYRIMIDINES, including Baquiloprim, Brodimoprim, Iclaprim, Ormetoprim, Pyrimethamine, Tetroxoprim, Trimethoprim; AMINOCOUMARINS, including Novobiocin; AMINOCYCLITOLS, including Spectinomycin, AMINOGLYCOSIDES, including Amikacin, Apramycin, Arbekacin, Bekanamycin, Butirosin, Dibekacin, Dihydrostreptomycin, Etimicin, Fortimicins (Astromicin), Framycetin, Gentamicin, Hygromycin B, Isepamicin, Kanamycin, Micronomicin, Neomycin, Netilmicin, Paromomycin, Plazomicin, Ribostamycin, Sisomicin, Streptomycin, Tobramycin, Verdamicin; AMINOMETHYLCYCLINES, including Omadacycline; AMPHENICOLS, including Azidamfenicol, Chloramphenicol, Florfenicol, Thiamphenicol; ANSAMYCINS, including Rifabutin, Rifamide, Rifampin (Rifampicin), Rifamycin, Rifapentine, Rifaximin; ANTISEPTIC AGENTS, including Acridine derivatives (including acriflavine, aminoacridine, ethacridine, proflavine), Bispyridines (including octenidine dihydrochloride), Brominated salicylanilides (including bromsalans), Chlorhexidine, Phenol derivatives (including thymol and triclosan), Quarternary ammonium compounds (including Alkyldimethylethylbenzyl Ammonium Chloride, benzalkonium chloride, cetylpyridinium chloride, benzethonium chloride, cetrimonium); ANTITUBERCULAR AGENTS, including Cycloserine, Delamanid, Ethambutol, Ethionamide, Isoniazid (Ftivazide), Morinamide, p-Aminosalicylic Acid (PAS), Protionamide, Pyrazinamide, Terizidone, Thioacetazone, Tiocarlide; ARSENICALS, including Arsanilic Acid, Roxarsone; BACTERIOCINS, including Nisin, Brilacidin (PMX-30063); β-LACTAM CARBACEPHEMS, including Loracarbef; B-LACTAM CARBAPENEMS, including Biapenem, Doripenem, Ertapenem, Faropenem, Imipenem, Meropenem, Panipenem, Razupenem, Ritipenem, Sulopenem, Tebipenem, Tomopenem; B-LACTAM CEPHALOSPORINS, including Cefacetrile, Cefaclor, Cefadroxil, Cefalexin, Cefaloglycin, Cefalonium, Cefaloridine, Cefalothin, Cefamandole, Cefapirin, Cefatrizine, Cefazaflur, Cefazedone, Cefazolin, Cefcapene, Cefdinir, Cefditoren, Cefepime, Cefetamet, Cefixime, Cefmenoxime, Cefodizime, Cefonicid, Cefoperazone, Ceforanide, Cefoselis, Cefotaxime, Cefotiam, Cefovecin, Cefozopran, Cefpimizole, Cefpiramide, Cefpirome, Cefpodoxime, Cefprozil, Cefquinome, Cefradine, Cefroxadine, Cefsulodin, Ceftaroline, Ceftazidime, Cefteram, Ceftezole, Ceftibuten, Ceftiofur, Ceftizoxime, Ceftobiprole, Ceftolozane, Ceftradine, Ceftrezole, Ceftriaxone, Ceftroxadine, Cefuroxime, Cefuzonam, Pivcefalexin; B-LACTAM CEPHAMYCINS, including Cefbuperazone, Cefmetazole, Cefminox, Cefotetan, Cefoxitin; B-LACTAM MONOBACTAMS, including Aztreonam, Carumonam, Tigemonam; B-LACTAM OXACEPHEMS, including Flomoxef, Latamoxef, Moxalactam; B-LACTAM PENICILLINS, including Amdinocillin (Mecillinam), Amoxicillin, Ampicillin, Apalcillin, Aspoxicillin, Azidocillin, Azlocillin, Bacampicillin, Carbenicillin, Carindacillin, Ciclacillin, Clemizole Penicillin, Clometocillin, Cloxacillin, Cyclacillin, Dicloxacillin, Epicillin, Fenbenicillin, Floxacillin (Flucloxacillin), Hetacillin, Lenampicillin, Mecillinam, Metampicillin, Methicillin Sodium, Meziocillin, Nafcillin, Oxacillin, Penamecillin, Penethamate Hydriodide, Penicillin G, Penicillin G Benzathine, Penicillin G Procaine, Penicillin N, Penicillin O, Penicillin V, Phenethicillin Potassium, Piperacillin, Pivampicillin, Pivmecillinam, Propicillin, Quinacillin, Sulbenicillin, Sultamicillin, Talampicillin, Temocillin, Ticarcillin; BICYCLOMYCINS, including Bicozamycin; BORON CONTAINING ANTIBACTERIAL AGENTS, including AN3365 (aminomethylbenzoxaboroles), GSK2251052 (leucyl-tRNA synthetase inhibitors); CYCLIC ESTERS, including Fosfomycin; FATTY ACID SYNTHESIS INHIBITORS (FabI), AFN-1252, MUT056399, FAB-001; FLUOROQUINOLONES, including Avarofloxacin, Balofloxacin, Besifloxacin, Chinfloxacin, Cinoxacin, Ciprofloxacin, Clinafloxacin, Danofloxacin, Delafloxacin, Difloxacin, Enoxacin, Enrofloxacin, Finafloxacin, Fleroxacin, Flumequine, Garenoxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Ibafloxacin, Levofloxacin, Lomefloxacin, Marbofloxacin, Miloxacin, Moxifloxacin, Nadifloxacin, Norfloxacin, Ofloxacin, Orbifloxacin, Pazufloxacin, Pefloxacin, Pradofloxacin, Prulifloxacin, Rosoxacin, Rufloxacin, Sarafloxacin, Sitafloxacin, Sparfloxacin, Temafloxacin, Tosufloxacin, Trovafloxacin, Zabofloxacin; FUSIDANES, including Fusidic Acid; GLYCOLIPODEPSIPEPTIDE, including Ramoplanin; GLYCOPEPTIDES, including Avoparcin, Dalbavancin, Norvancomycin, Oritavancin, Teicoplanin, Telavancin, Vancomycin; GLYCOPHOSPHOLIPIDS, including Bambermycins (bambermycin, moenomycins, flavophospholipol); GLYCYLCYCLINES, including Tigecycline; HYBRIDS, Cadazolid (Oxazolidinone-quinolone), TD-1792 (glycopeptide-cephalosporin); LINCOSAMIDES, including Clindamycin, Lincomycin, Pirlimycin; LIPOPEPTIDES, including Daptomycin, Surotomycin; MACROLIDES, including Azithromycin, Carbomycin, Cethromycin, Clarithromycin, Dirithromycin, Erythromycin, Fidaxomicin, Flurithromycin, Gamithromycin, Josamycin, Kitasamycin, Leucomycin, Meleumycin, Midecamycins, Miokamycin, Mirosamycin, Oleandomycin, Primycin, Rokitamycin, Rosaramicin, Roxithromycin, Sedecamycin, Solithromycin, Spiramycin, Telithromycin, Terdecamycin, Tildipirosin, Tilmicosin, Troleandomycin, Tulathromycin, Tylosin, Tylvalosin; NITROFURANS, including Furaltadone, Furazidin, Furazolidone, Furazolium Chloride, Nifuratel, Nifurfoline, Nifuroxazide, Nifurpirinol, Nifurtoinol, Nifurzide, Nitrofural, Nitrofurantoin, Nitrofurazone; NITROIMIDAZOLES, including Dimetridazole, Metronidazole, Omidazole, Ronidazole, Secnidazole, Tinidazole; OLIGOSACCHARIDES, including Avilamycin, Eveminomicin; OTHER ANTIBACTERIAL AGENTS, including Auriciosene, Chloroxine, Chlorquinaldol, Clioquinol, Clofoctol, Halquinol, Lotilibcin, Mandelic Acid, Methenamine (hexamine), Nitazole, Nitroxoline, Perchlozone, Taurolidine, Thenoic Acid, Xibomrnol; OXAZOLIDINONES, including Eperezolid, Linezolid, Posizolid, Radezolid, Sutezolid, Tedizolid (Torezolid); PEPTIDE DEFORMYLASE INHIBITORS, including GSK1322322; PEPTIDES, including Omiganan, Pexiganan; PLEUROMUTILINS, including Retapamulin, Tiamulin, Valnemulin; POLYETHER IONOPHORES, including Laidlomycin, Lasalocid, Maduramicin, Monensin, Narasin, Salinomycin, Semduramicin; POLYMYXINS, including Colistin, Polymyxin B; POLYPEPTIDES, including Amphomycin, Bacitracin, Capreomycin, Enduracidin, Enramycin, Enviomycin, Fusafungine, Gramicidin(s), Iseganan, Magainins, Nosiheptide, Ristocetin, Thiostrepton, Tuberactinomycin, Tyrocidine, Tyrothricin, Viomycin; PSEUDOMONIC ACIDS, including Mupirocin; QUINOLONES, including Nalidixic Acid, Nemonoxacin, Oxolinic Acid, Ozenoxacin, Pipemidic Acid, Piromidic Acid; QUINOXAUNES, including Carbadox, Olaquindox; RIMINOFENAZINES, including Clofazimine; STATINS, including Atorvastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Pravastatin, Rosuvastatin, Simvastatin; STREPTOGRAMINS, including Dalfopristin, Flopristin, Linopristin, Pristinamycin, Quinupristin, Virginiamycin; STREPTOTHRICINS, including Nourseothricin; SULFONAMIDES, including Acetyl Sulfamethoxypyrazine, Chloramine-B, Chloramine-T, Dichloramine T, Formosulfathiazole, Mafenide, N4-Sulfanilylsulfanilamide, Noprylsulfamide, N-Sulfanilyl-3,4-xylamide, Ormaosulfathiazole, Phthalylsulfacetamide, Phthalylsulfathiazole, Salazosulfadimidine, Succinylsulfathiazole, Sulfabenzamide, Sulfacarbamide, Sulfacetamide, Sulfachlorpyridazine, Sulfachrysoidine, Sulfaclozine, Sulfacytine, Sulfadiazine, Sulfadicramide, Sulfadimethoxine, Sulfadimidine, Sulfadoxine, Sulfaethidole, Sulfaguanidine, Sulfaguanole, Sulfalene, Sulfaloxic Acid, Sulfamerazine, Sulfameter, Sulfamethazine, Sulfamethizole, Sulfamethomidine, Sulfamethoxazole, Sulfamethoxypyridazine, Sulfamethylthiazole, Sulfametopyrazine, Sulfametrole, Sulfamidochrysoidine, Sulfamonomethoxine, Sulfamoxole, Sulfanilamide, Sulfanilylurea, Sulfaperine, Sulfaphenazole, Sulfaproxyline, Sulfapyrazine, Sulfapyridine, Sulfaquinoxaline, Sulfathiazole, Sulfathiourea, Sulfatroxazole, Sulfisomidine, Sulfisoxazole (Sulfafurazole); SULFONES, including Acediasulfone, Dapsone, Glucosulfone Sodium, p-Sulfanilylbenzylamine, Succisulfone, Sulanilic Acid, Sulfoxone Sodium, Thiazolsulfone; TETRACYCLINES, including Chlortetracycline, Clomocycline, Demeclocycline, Doxycycline, Eravacycline, Guamecycline, Lymecycine, Meciocycline, Methacycline, Minocycline, Oxytetracycline, Penimepicycline, Pipacycline, Rolitetracycline, Sarecycline, Tetracycline.
The composition of the invention may further comprise an excipient selected from the group comprising, but not limited to, binders and compression aids, coatings and films, colouring agents diluents and vehicles disintegrants, emulsifying and solubilising agents, flavours and sweeteners, repellents, glidants and lubricants, plasticisers, preservatives, propellants, solvents, stabilisers, suspending agents and viscosity enhancers.
According to a further aspect of the invention, there is provided a medical device when used in a method of treating or preventing a bacterial infection in the subject.
According to further aspect of the invention, there is provided a medical device comprising the composition of the invention. The composition of the invention may be any slow release form, and/or in the form of a coating of the medical device.
The medical device may be in a form selected from the group comprising: an implant, a plaster, a bandage, and other dressing applied to a bacterial infection in a subject.
According to further aspect of the invention, there is provided a method of killing bacteria, the method including the step of contacting the bacteria with robenidine, or a therapeutically acceptable salt thereof.
According to further aspect of the invention, there is provided the use of robenidine, or a therapeutically acceptable salt thereof, to kill bacteria, said use comprising the step of contacting the bacteria with robenidine, or a therapeutically acceptable salt thereof.
Terms used herein will have their customary meanings in the art unless specified.



BRIEF DESCRIPTION OF THE DRAWINGS
Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawings in which:
FIG. 1 shows a table of the Minimum Inhibitory Concentrations for the individual Staphylococcus aureus isolates according to example 1;
FIG. 2 shows a table of the Minimum Inhibitory Concentrations for the individual Enterococcus isolates according to example 1;
FIG. 3 shows a table of the Minimum Inhibitory Concentrations for the individual Streptococcus pneumoniae isolates according to example 1;
FIG. 4 shows a table of the NCL812 MIC50, MIC90, MIC mode and MIC range for Australian isolates of MRSA, VRE and Str. pneumoniae according to example 1. Comparative MIC values for ampicillin are shown in parenthesis;
FIG. 5 shows a table of the Minimum Inhibitory Concentrations values for NCL812 (robenidine) and linezolid against Staphylococcus aureus ATCC29213 according to example 2;
FIG. 6 shows a graph of the effect of NCL812 on DNA macromolecular synthesis in Staphylococcus aureus according to example 2;
FIG. 7 shows a graph of the effect of NCL812 on RNA macromolecular synthesis in Staphylococcus aureus according to example 2;
FIG. 8 shows a graph of the effect of NCL812 on protein macromolecular synthesis in Staphylococcus aureus (ATCC29213) according to example 2;
FIG. 9 shows a graph of the effect of NCL812 on cell wall macromolecular synthesis in Staphylococcus aureus (ATCC29213) according to example 2;
FIG. 10 shows a graph of the effect of NCL812 on lipid macromolecular synthesis in Staphylococcus aureus (ATCC29213) according to example 2;
FIG. 11 shows a graph summarising the effect of NCL812 on macromolecular synthesis in Staphylococcus aureus (ATCC29213) according to example 2;
FIG. 12 shows a graph of the effect of NCL812 on ATP release from Staphylococcus aureus (ATCC29213) according to example 3;
FIG. 13 shows a table of Staphylococcus aureus clone/isolate name, type, source, antibiogram, clindamycin resistance status, multi-locus sequence type (MLST), staphylococcal cassette chromosome (SCCmec) type, clonal complex, Panton-Valentine leukocidin status (PVL), and spa type for isolates used according to example 4. MSSA; methicillin-susceptible S. aureus. HA-MRSA; hospital-acquired methicillin-resistant S. aureus. CA-MRSA; community-associated methicillin-resistant S. aureus. M. B; M. Barton (University of South Australia). G; Gribbles pathology (South Australia). J. P.; J. Perry (University of Adelaide). VIMP; Nares of students from Veterinary Immunology, Microbiology, & Public Health (University of Adelaide). S. P.; S. Polyak (University of Adelaide). G. C.; Geoff Coombs (PathWest Laboratory Medicine, Western Australia). Em; Erythromycin. Ci; Ciprofloxacin. Gn; Gentamicin. Tm; Trimethoprim. Te; Tetracycline. FA; Fusidic Acid. Rf; Rifampicin. Mp; Mupirocin;
FIG. 14 shows a table of the percentage of presumptively identified S. aureus isolates reporting positive to selected phenotypic and genotypic tests according to Example 4. HA-MRSA; hospital-acquired S. aureus. CA-MRSA; community-associated S. aureus. S. aureus isolates were identified as testing positive to protein A latex agglutination (Protein A), slide coagulase, Voges-Proskauer and polymyxin B resistance tests, as well as testing positive for polymerase chain reaction (PCR) and real-time PCR amplification of the spa gene. Methicillin-resistant S. aureus isolates were identified as isolates testing positive to the criteria described above, as well as positive for PCR and real-time PCR of the mecA gene;
FIG. 15 shows a table of the resistance of S. aureus isolates to antibacterial agents using the Kirby-Bauer disc diffusion method according to Example 4. HA-MRSA; hospital-acquired methicillin-resistant S. aureus. CA-MRSA; community-associated methicillin-resistant S. aureus; 
FIG. 16 shows a table of the number and percentage of identified mec gene complexes in 20 S. aureus strains classified as methicillin-resistant according to Example 4. Respective staphylococcal cassette chromosome (SCCmec) complexes and types expressing phenotypic resistance to oxacillin and cefotetan are indicated as well as real-time mecA status, and the average negative dF/dT peak obtained from melting point analysis from real-time PCR of the mecA gene. Figures in parentheses indicate percentages;
FIG. 17 shows a graph showing the average melting point peaks for the negative derivative plot −dF/dT after real-time polymerase chain reaction of the mecA gene in methicillin-resistant S. aureus isolates grouped by mec gene complexes, A (n=4), B (n=10), C2 (n=4) and unclassified (n=2). Groups indicated with different superscripts are significantly different (P<0.05), according to Example 4;
FIG. 18 shows a table of the characteristics of antibacterial NCL812 and the β-lactam antibacterial ampicillin according to Example 4, detailing antibacterial solubility in dimethyl sulfoxide (DMSO), solubility in cation-adjusted Mueller-Hinton II broth (CAMHB), and average minimum inhibitory concentrations (MIC) (μg/ml at 24-h) against methicillin-resistant S. aureus (MRSA) determined from preliminary studies and those determined during this present study. ATCC 49775; methicillin-susceptible S. aureus isolate and ATCC control strain. MRSA580; methicillin-resistant S. aureus isolate #580. MRSA698; methicillin-resistant S. aureus isolate #698;
FIG. 19 shows a table of in vitro activities of the novel antibacterial NCL812 and the 1-lactam antibacterial ampicillin against S. aureus clinical isolates according to example 4. HA-MRSA; hospital-acquired methicillin-resistant S. aureus. CA-MRSA; community-associated methicillin-resistant S. aureus. MIC; minimum inhibitory concentration (μg/ml). MBC; minimum bactericidal concentration (μg/ml). MIC/MBCrange; minimum and maximum MIC/MBC for all isolates. MIC/MBC50; MIC/MBC at which 50% of isolates are inhibited. MIC/MBC90; MIC/MBC at which 90% of isolates are inhibited;
FIG. 20 shows a graph of the optical densities of the unsupplemented growth control, ampicillin and different concentrations of antibacterial agent NCL812 against methicillin-susceptible S. aureus ATCC 49775 using broth microdilution methodology according to example 4. The concentrations of NCL812 tested were at the MIC and four times the MIC determined under test conditions, up to 24-h incubation. Ampicillin was tested at the MIC. Bactericidal activity was tested at 0-, 1-, 2-, 4-, 8-, 12-, and 24-h for antibacterials;
FIG. 21 shows a graph of kill kinetic curves for methicillin-susceptible S. aureus ATCC 49775 demonstrating bactericidal activity of NCL812 using the Clinical and Laboratory Standards Institute macrodilution methodology in a 10-mi vial according to example 4. The concentrations of antibacterials tested were at 1× and 4× the MIC determined under test conditions. Bactericidal activity was determined at 0-, 1-, 2-, 4-, 8-, 12- and 24-h after antibacterial addition. Bactericidal activity was defined as a 3-log 10 (99.9%) decrease in the number viable bacteria from the initial inoculum size;
FIG. 22 shows a table of the antibacterial susceptibility of 20 S. pneumoniae isolates for six different antibacterials according to example 5;
FIG. 23 shows a graph indicating the change of pH during macro-broth dilution assay for S. pneumoniae strain D39 exposed to 4 μg/ml in NCL812 and 0.0023 μg/ml ampicillin according to example 5;
FIG. 24 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with NCL812 according to example 5;
FIG. 25 shows a graph illustrating in the 14-hour time-kill of S. pneumoniae strain D39 treated with NCL812 according to example 5;
FIG. 26 shows a graph illustrating the 14-hour time-kill of S. pneumoniae strain D39 treated with ampicillin according to example 5;
FIG. 27 shows a graph illustrating the 12-hour time-kill of S. pneumoniae strain D39 treated with NCL812, adopted from FIG. 40 according to example 5;
FIG. 28 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with ampicillin according to example 5;
FIG. 29 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with erythromycin according to example 5;
FIG. 30 shows a graph illustrating the 48-hour time-kill of S. pneumoniae strain D39 treated with NCL812 and 5% choline chloride according to example 5;
FIG. 31 shows a graph illustrating the 12-hour time-kill of S. pneumoniae strain D39 treated with NCL812 and 5% choline chloride according to example 5;
FIG. 32 shows a graph illustrating the relative minimum bactericidal concentration (MBC) of S. pneumoniae strain D39 treated with ampicillin over a 48-hour time period according to example 5;
FIG. 33 shows a graph illustrating the relative MBC for S. pneumoniae strain D39 treated with erythromycin over a 48-hour time period according to example 5;
FIG. 34 shows a graph illustrating the viable count (log10 CFU/ml) of S. pneumoniae strain D39 treated with NCL812 from a macro-broth dilution of time-kill over 24 hours according to example 5;
FIG. 35 shows a graph illustrating the viable count (log10 CFU/ml) of S. pneumoniae strain D39 treated with ampicillin from a macro-broth dilution of time-kill over 24 hours according to example 5;
FIG. 36 is a bar graph illustrating the mean cell membrane thickness of treated and untreated D39 according to example 5;
FIG. 37 is a bar graph illustrating the mean width of periplasmic space of treated (16 μg/ml NCL812) and untreated D39 samples according to example 5;
FIG. 38 is a table showing the Staphylococcus pseudintermedius isolates tested according to example 6;
FIG. 39 is a table showing the antibiotic resistance profile of the Staphylococcus pseudintermedius isolates tested according to example 6.
FIG. 40 is a graph illustrating the effectiveness of NCL812 against gram-negative E. coli spheroplasts according to example 7;
FIG. 41 is a graph illustrating the cumulative release of NCL812 from Formulation B according to example 9;
FIG. 42 shows the kill kinetics assay of Staphylococcus aureus KC01 at different concentrations of NCL812, up to 24 h incubation according to example 11; and
FIG. 43 shows the kill kinetics assay of Enterococcus faecalis USA01 at different concentrations of NCL812, up to 24 h incubation according to example 11.



DESCRIPTION OF EMBODIMENTS
General
Before describing the present invention in detail, it is to be understood that the invention is not limited to particular exemplified methods or compositions disclosed herein. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.
All publications referred to herein, including patents or patent applications, are incorporated by reference in their entirety. However, applications that are mentioned herein are referred to simply for the purpose of describing and disclosing the procedures, protocols, and reagents referred to in the publication which may have been used in connection with the invention. The citation of any publications referred to herein is not to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In addition, the carrying out of the present invention makes use of, unless otherwise indicated, conventional microbiological techniques within the skill of the art. Such conventional techniques are known to the skilled worker.
As used herein, and in the appended claims, the singular forms “a”, “an”, and “the” include the plural unless the context clearly indicates otherwise.
Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar to, or equivalent to, those described herein may be used to carry out the present invention, the preferred materials and methods are herein described.
The invention described herein may include one or more ranges of values (e.g. size, concentration, dose etc). A range of values will be understood to include all values within the range, including the values defining the range, and values adjacent to the range that lead to the same or substantially the same outcome as the values immediately adjacent to that value which define the boundary of the range.
The pharmaceutical for veterinary compositions of the invention may be administered in a variety of unit dosages depending on the method of administration, target site, physiological state of the patient, and other medicaments administered. For example, unit dosage form suitable for oral administration include solid dosage forms such as powder, tablets, pills, and capsules, and liquid dosage forms, such as elixirs, syrups, solutions and suspensions. The active ingredients may also be administered parenterally in sterile liquid dosage forms. Gelatin capsules may contain the active ingredient and inactive ingredients such as powder carriers, glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate, and the like.
The phrase “therapeutically effective amount” as used herein refers to an amount sufficient to inhibit bacterial growth associated with a bacterial infection or colonisation. That is, reference to the administration of the therapeutically effective amount of robenidine according to the methods or compositions of the invention refers to a therapeutic effect in which substantial bacteriocidal or bacteriostatic activity causes a substantial inhibition of bacterial infection. The term “therapeutically effective amount” as used herein, refers to a sufficient amount of the composition to provide the desired biological, therapeutic, and/or prophylactic result. The desired results include elimination of bacterial infection or colonisation or reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. In relation to a pharmaceutical or veterinary composition, effective amounts can be dosages that are recommended in the modulation of a diseased state or signs or symptoms thereof. Effective amounts differ depending on the composition used and the route of administration employed. Effective amounts are routinely optimized taking into consideration pharmacokinetic and pharmacodynamic characteristics as well as various factors of a particular patient, such as age, weight, gender, etc and the area affected by disease or disease causing microbes.
As referred to herein, the terms “treatment” or “treating” refers to the full or partial removal of the symptoms and signs of the condition. For example, in the treatment of a bacterial infection or colonisation, the treatment completely or partially removes the signs of the infection. Preferably in the treatment of infection, the treatment reduces or eliminates the infecting bacterial pathogen leading to microbial cure.
As referred to herein, the term “bacteria” refers to members of a large domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals and can be present as individual cells or present in linear chains or dusters of variable numbers and shape. Preferably the terms “bacteria” and its adjective “bacterial” refer to bacteria such as the Gram positive Staphylococcus spp, Streptococcus spp, Bacillus spp, Enterococcus spp, Listeria spp, Mycoplasma spp, and anaerobic bacteria; Gram negative Escherichia coli, Enterobacter spp, Klebsiella spp and Pseudomonas spp; and the cell wall free bacteria such as Mycoplasma spp and Ureaplasma spp. The terms may refer to an antibiotic-sensitive strain or an antibiotic-resistant strain. In a preferred embodiment, the terms refer to MRSA or MRSP. In another preferred embodiment, the terms refer to MDR Staphylococcus spp, Streptococcus spp, Enterococcus spp, Clostridium difficile, Escherichia coli, Enterobacter spp, Klebsiella spp and Pseudomonas spp.
Referred to herein, the term “methicillin-resistant bacteria” (such as methicillin-resistant Staphylococcus) refers a bacteria isolate that demonstrates resistance at any dose to all β lactams including penicillins, carbapenems and first to fourth generation cephalosporins, but not to the fifth generation anti-MRSA cephalosporins (for example ceftaroline). Multidrug-resistant (MDR) is defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, extensively drug-resistant (XDR) is defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and pandrug-resistant (PDR) is defined as non-susceptibility to all agents in all antimicrobial categories currently available.
An example of susceptible, MDR, XDR and PDR bacteria includes the following. Wild type, antibacterial unexposed isolates of Staphylococcus aureus that are likely to be susceptible to all of the following antibacterial categories (and agents): aminoglycosides (for example gentamicin); ansamycins (for example rifampicin); anti-MRSA cephalosporins (for example ceftaroline); anti-staphylococcal β-lactams (or cephamycins) (for example oxacillin or cefoxitin); carbapenems (for example ertapenem, imipenem, meropenem or doripenem); non-extended spectrum cephalosporins; 1st and 2nd generation cephalosporins (for example cefazolin or cefuroxime); extended-spectrum cephalosporins; 3rd and 4th generation cephalosporins (for example cefotaxime or ceftriaxone); cephamycins (for example cefoxitin or cefotetan); fluoroquinolones (for example ciprofloxacin or moxifloxacin); folate pathway inhibitors (for example trimethoprim-sulphamethoxazole); fucidanes (for example fusidic acid); glycopeptides (for example vancomycin, teicoplanin or telavancin); glycylcyclines (for example tigecycline); lincosamides (for example clindamycin); lipopeptides (for example daptomycin); macrolides (for example erythromycin); oxazolidinones (for example linezolid or tedizolid); phenicols (for example chloramphenicol); phosphonic acids (for example fosfomycin); streptogramins (for example quinupristin-dalfopristin; and tetracyclines (for example tetracycline, doxycycline or minocycline). Isolates that are non-susceptible to more than one agent in more than three antimicrobial categories are classified as MDR (all MRSA, for example, meet the definition of MDR). Isolates that are non-susceptible to more than one agent in all but one or two antimicrobial categories are classified as XDR. Isolates that are non-susceptible to all listed antibacterial agents are PDR.
Pharmaceutically and veterinary acceptable salts include salts which retain the biological effectiveness and properties of the compounds of the present disclosure and which are not biologically or otherwise undesirable. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto. Acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as by way of example only, alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di(substituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di(substituted alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amines, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amines, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group.
Pharmaceutically and veterinary acceptable acid addition salts may be prepared from inorganic and organic acids. The inorganic acids that can be used include, by way of example only, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. The organic acids that can be used include, by way of example only, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like.
The pharmaceutically or veterinary acceptable salts of the compounds useful in the present disclosure can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Company, Easton, Pa. (1985), p. 1418, the disclosure of which is hereby incorporated by reference. Examples of such acceptable salts are the iodide, acetate, phenyl acetate, trifluoroacetate, acryl ate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, γ-hydroxybutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, hexyne-1,6-dioate, caproate, caprylate, chloride, cinnamate, citrate, decanoate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, propanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like.
The pharmaceutical or veterinary compositions of the invention may be formulated in conventional manner, together with other pharmaceutically acceptable excipients if desired, into forms suitable for oral, parenteral, or topical administration. The modes of administration may include parenteral, for example, intramuscular, subcutaneous and intravenous administration, oral administration, topical administration and direct administration to sites of infection such as intraocular, intraaural, intrauterine, intranasal, intramammary, intraperitoneal and intralesional.
The pharmaceutical or veterinary compositions of the invention may be formulated for oral administration. Traditional inactive ingredients may be added to provide desirable colour, taste, stability, buffering capacity, dispersion, or other known desirable features. Examples include red iron oxide, silica gel, sodium laurel sulphate, titanium dioxide, edible white ink, and the like. Conventional diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as sustained-release compositions for the continual release of medication over a period of time. Compressed tablets may be in the form of sugar coated or film coated tablets, or enteric-coated tablets for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain colouring and/or flavouring to increase patient compliance. As an example, the oral formulation comprising NCL812 may be a tablet comprising anyone, or a combination of, the following excipients: calcium hydrogen phosphate dehydrate, microcrystalline cellulose, lactose, hydroxypropyl methyl cellulose, and talc.
The compositions described herein may be in the form of a liquid formulation. Examples of preferred liquid compositions include solutions, emulsions, injection solutions, solutions contained in capsules. The liquid formulation may comprise a solution that includes a therapeutic agent dissolved in a solvent. Generally, any solvent that has the desired effect may be used in which the therapeutic agent dissolves and which can be administered to a subject. Generally, any concentration of therapeutic agent that has the desired effect can be used. The formulation in some variations is a solution which is unsaturated, a saturated or a supersaturated solution. The solvent may be a pure solvent or may be a mixture of liquid solvent components. In some variations the solution formed is an in situ gelling formulation. Solvents and types of solutions that may be used are well known to those versed in such drug delivery technologies.
The composition described herein may be in the form of a liquid suspension. The liquid suspensions may be prepared according to standard procedures known in the art. Examples of liquid suspensions include micro-emulsions, the formation of complexing compounds, and stabilising suspensions. The liquid suspension may be in undiluted or concentrated form. Liquid suspensions for oral use may contain suitable preservatives, antioxidants, and other excipients known in the art functioning as one or more of dispersion agents, suspending agents, thickening agents, emulsifying agents, wetting agents, solubilising agents, stabilising agents, flavouring and sweetening agents, colouring agents, and the like. The liquid suspension may contain glycerol and water.
The composition described herein may be in the form of an oral paste. The oral paste may be prepared according to standard procedures known in the art.
The composition is described herein may be in the form of a liquid formulation for injection, such as intra-muscular injection, and prepared using methods known in the art. For example, the liquid formulation may contain polyvinylpyrrolidone K30 and water.
The composition is described herein may be in the form of topical preparations. The topical preparation may be in the form of a lotion or a cream, prepared using methods known in the art. For example, a lotion may be formulated with an aqueous or oily base and may include one or more excipients known in the art, functioning as viscosity enhancers, emulsifying agents, fragrances or perfumes, preservative agents, chelating agents, pH modifiers, antioxidants, and the like. For example, the topical formulation comprising NCL812 may be a gel comprising anyone, or a combination of, the following excipients: PEG 4000, PEG 200, glycerol, propylene glycol. The NCL812 compound may further be formulated into a solid dispersion using SoluPlus (BASF, www.soluplys.com) and formulated with anyone, or a combination of, the following excipients: PEG 4000, PEG 200, glycerol, propylene glycol.
For aerosol administration, the composition is of the invention they be provided in finely divided form together with a non-toxic surfactant and a propellant. The surfactant is preferably soluble in the propellant. Such surfactants may include esters or partial esters of fatty acids.
The compositions of the invention may alternatively be formulated using nanotechnology drug delivery techniques such as those known in the art. Nanotechnology-based drug delivery systems have the advantage of improving bioavailability, patient compliance and reducing side effects.
The formulation of the composition of the invention includes the preparation of nanoparticles in the form of nanosuspensions or nanoemulsions, based on compound solubility. Nanosuspensions are dispersions of nanosized drug particles prepared by bottom-up or top-down technology and stabilised with suitable excipients. This approach may be applied to robenidene which has poor aqueous and lipid solubility in order to enhance saturation solubility and improve dissolution characteristics. An example of this technique is set out in Sharma and Garg (2010) (Pure drug and polymer-based nanotechnologies for the improved solubility, stability, bioavailability, and targeting of anti-HIV drugs. Advanced Drug Delivery Reviews, 62: p. 491-502). Saturation solubility will be understood to be a compound-specific constant that depends on temperature, properties of the dissolution medium, and particle size (<1-2 μm).
The composition of the invention may be provided in the form of a nanosuspension. For nanosuspensions, the increase in the surface area may lead to an increase in saturation solubility. Nanosuspensions are colloidal drug delivery systems, consisting of particles below 1 μm. Compositions of the invention may be in the form of nanosuspensions including nanocrystalline suspensions, solid lipid nanoparticles (SLNs), polymeric nanoparticles, nanocapsules, polymeric micelles and dendrimers. Nanosuspensions may be prepared using a top-down approach where larger particles may be reduced to nanometer dimensions by a variety of techniques known in the art including wet-milling and high-pressure homogenisation. Alternatively, nanosuspensions may be prepared using a bottom-up technique where controlled precipitation of particles may be carried out from solution.
The composition of the invention may be provided in the form of a nanoemulsion. Nanoemulsions are typically clear oil-in-water or water-in-oil biphasic systems, with a droplet size in the range of 100-500 nm, and with compounds of interest present in the hydrophobic phase. The preparation of nanoemulsions may improve the solubility of robenidine described herein, leading to better bioavailability. Nanosized suspensions may include agents for electrostatic or steric stabilisation such as polymers and surfactants. Compositions in the form of SLNs may comprise biodegradable lipids such as triglycerides, steroids, waxes and emulsifiers such as soybean lecithin, egg lecithin, and poloxamers. The preparation of a SLN preparation may involve dissolving/dispersing drug in melted lipid followed by hot or cold homogenisation. If hot homogenisation is used, the melted lipidic phase may be dispersed in an aqueous phase and an emulsion prepared. This may be solidified by cooling to achieve SLNs. If cold homogenisation is used, the lipidic phase may be solidified in liquid nitrogen and ground to micron size. The resulting powder may be subjected to high-pressure homogenisation in an aqueous surfactant solution.
Robenidine as described herein may be dissolved in oils/liquid lipids and stabilised into an emulsion formulation. Nanoemulsions may be prepared using high- and low-energy droplet reduction techniques. High-energy methods may include high-pressure homogenisation, ultrasonication and microfluidisation. If the low-energy method is used, solvent diffusion and phase inversion will generate a spontaneous nanoemulsion. Lipids used in nanoemulsions may be selected from the group comprising triglycerides, soybean oil, safflower oil, and sesame oil. Other components such as emulsifiers, antioxidants, pH modifiers and preservatives may also be added.
The composition may be in the form of a controlled-release formulation may include a degradable or non-degradable polymer, hydrogel, organogel, or other physical construct that modifies the release of the polyether ionophore. It is understood that such formulations may include additional inactive ingredients that are added to provide desirable colour, stability, buffering capacity, dispersion, or other known desirable features. Such formulations may further include liposomes, such as emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use in the invention may be formed from standard vesicle-forming lipids, generally including neutral and negatively charged phospholipids and a sterol, such as cholesterol.
The formulations of the invention may have the advantage of increased solubility and/or stability of NCL812, particularly for those formulations prepared using nanotechnology techniques. Such increased stability and/or stability of NCL812 may improve bioavailability and enhance drug exposure for oral and/or parenteral dosage forms.
Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
EXAMPLES
Example 1
The Minimum Inhibitory Concentrations (MIC) for NCL812 in Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococcus Spp. (VRE) and Streptococcus pneumoniae 
In this example and other examples in the specification, the term NCL812 is used to indicate robenidine.
This study was undertaken to determine minimum inhibitory concentrations (MIC) for a new antibacterial agent, NCL812. The antibacterial agent represents a potentially new class of drug with a perceived narrow spectrum of activity against bacteria and a novel mechanism of action. This study focused on recent isolates of three major opportunistic pathogens of humans where the development of antibacterial resistance to existing antibacterial classes is problematic: methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp. (VRE) and Streptococcus pneumoniae. 
Materials and Methods
Bacterial Isolate Collection and Identification
Sixty one test isolates were sourced from clinical diagnostic microbiology laboratories. The MRSA isolates were originally cultured on selective Brilliance MRSA Chromogenic Agar (Oxoid). Suspect colonies were selected on the basis of their colony appearance on this agar and identification as Staphylococcus aureus was determined using colony characteristics on non-selective Sheep Blood Agar and phenotypic characteristics such as Gram stain, positive catalase test, positive coagulase test (tube coagulase test using rabbit plasma) and clumping factor (agglutination with the Oxoid Staphytect latex test), positive Voges Proskauer test, and the ability to produce acid from trehalose. A positive cefoxitin resistance screen confirmed the isolates as MRSA. All Enterococcus isolates underwent a standard biochemical identification. Biochemical profiling provisionally identified four of the VRE isolates as Enterococcus faecalis and the remainder as Enterococcus faecium, however this is not 100% reliable for human Enterococcus strains and full biochemical profiling using API-ZYM profiling will be undertaken to 100% confirm identity. All Str. pneumoniae isolates were identified on the basis of standard biochemical profiling.
Preparation of Antimicrobials
Analytical grade NCL812 (batch 20081214) with a defined potency of 1000 mg/g (ie 100%) was obtained The powder was stored at a temperature of −20° C. at the study site, in a locked freezer. Aliquots (1 ml) of stock solution (25.6 mg/ml) were prepared in DMSO and stored at −80° C. and defrosted immediately before use.
Minimum Inhibitory Concentration Assay
Minimum inhibitory concentration tests were performed according to CLSI Standards (CLSI 2008). 90 μL of one of the test compound solutions, or ampicillin, was added to the end column of a 96 well plate that contained 90 μL of CAMHB in each well. The solutions were then serially diluted across the row, leaving 2 columns for positive and negative controls. A bacterial suspension was prepared by adding fresh colonies obtained from an overnight culture on Sheep Blood Agar (SBA) to a 9.1 g/L saline solution. This suspension was adjusted to a concentration of between 4×108 and 5×108 CFU/mL. Concentration of the suspension was determined by measuring optical density (OD) using a spectrophotometer at a wavelength of 600 nm where the correct concentration was determined to have an optical density of between 1.00 and 1.20. One milliliter of this suspension was added to 9 mL of saline before being added to all wells, excluding the negative control wells, in 10 μL volumes giving a final concentration of between 4×105 and 5×105 CFU/mL in each well. The tests were then incubated for 24 hours at 37° C. and then assessed both visually and using OD readings from a microplate reader at a wavelength of 600 nm. These tests were performed in duplicate but repeated if discrepancies were observed.
The minimum inhibitory concentration (MIC) was determined to be the lowest concentration of antibiotic that prevented growth of bacteria both visually and using OD readings. Direct statistical comparisons between the test compounds and ampicillin could not be performed in light of confidential information restrictions, such as restrictions on disclosure of information relating to the compound structure, such as molecular weight. Instead, MIC values were collated and used to determine the lowest concentration of each compound that was effective against 50% and 90% of isolates, referred to as the MIC50 and MIC50 respectively. These values, as well as the range of MIC values, were then used for direct comparisons between test compounds and for general comparisons with ampicillin.
Results
Ampicillin MIC values obtained for the ATCC control strains were within the normal range expected on the basis of CLSI recommendations. The NCL812 and ampicillin MIC values for each isolate are indicated in FIG. 1 (MRSA isolates), FIG. 2 (VRE isolates) and FIG. 3 (Str. pneumoniae isolates).
The pooled MIC50, MIC90, MIC mode and MIC range for NCL812 for each of the species of bacteria tested are shown in FIG. 4.
NCL812 MIC values were remarkably consistent within and between each of the three species. MIC50 and MIC90 values were both equal (4 μg/ml) for MRSA, VRE and Str. pneumoniae isolates, with less than 10% of isolates showing MIC values either 1-2 dilutions below or only one dilution above this figure.
Example 2
Effect of NCL812 on Staphylococcus aureus Macromolecular Synthesis
Materials and Methods
Test Compounds
Test compound NCL812 was transported to the experimental facility under conditions of ambient temperature and then stored at 2-8° C. until assayed. Stock solutions were made by dissolving NCL812 dry powder in 100% DMSO to a concentration of 6,400 μg/ml.
Minimal Inhibitory Concentration Testing
The MIC assay method followed the procedure described by the Clinical and Laboratory Standards Institute, or CLSI (Clinical and Laboratory Standards Institute). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Eighth Edition. CLSI document M07-A8 [ISBN 1-56238-689-1]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-19898 USA, 2009), and employed automated liquid handlers to conduct serial dilutions and liquid transfers. The medium employed for the MIC assay was Mueller Hinton II Broth (MHB II—Becton Dickinson, Sparks, M D; Cat No 212322; Lot 9044411). S. aureus ATCC 29213 served as the quality control strain, and linezolid was utilized as the quality control antibiotic to validate the assay. NCL812 and linezolid were both dissolved in 100% DMSO before addition to the growth medium.
Macromolecular Synthesis Assays
Bacteria and Growth Conditions
The effect of NCL812 on whole cell DNA, RNA, cell wall, protein and lipid synthesis was investigated using S. aureus ATCC 29213. Cells were grown at 35° C. overnight on Trypticase Soy agar. A colony from the plate was used to inoculate 10 ml of Mueller Hinton broth II (MHBII), and the culture was grown to early exponential growth phase (OD600=0.2 to 0.3) while incubating in a shaker at 35° C. and 200 rpm.
DNA, RNA, and Protein Synthesis
When cells reached early exponential phase, 100 μl of culture was added to triplicate wells containing various concentrations of test compound or control antibiotics (5 μl) at 20× the final concentration in 100% DMSO. A 5% DMSO treated culture served as the “no drug” control for all experiments. Cells were added in MHBII at 105% to account for the volume of drug added to each reaction or in M9 minimal medium for protein synthesis reactions. Following 15 min incubation at room temperature, either [3H] thymidine (DNA synthesis), [3H] uridine (RNA synthesis) or [3H] leucine (protein synthesis) was added at 0.5-1.0 μCi per reaction, depending on the experiment. Reactions were allowed to proceed at room temperature for 15-30 min and then stopped by adding 12 μl of cold 5% trichloroacetic acid (TCA) or 5% TCA/2% casamino acids (protein synthesis only). Reactions were incubated on ice for 30 min and the TCA precipitated material was collected on a 25 mm GF/A filter. After washing three times with 5 ml of cold 5% TCA, the filters were rinsed two times with 5 ml 100% ethanol, allowed to dry, and then counted using a Beckman LS3801 liquid scintillation counter.
Cell Wall Synthesis
Bacterial cells in early exponential growth phase were transferred to M9 minimal medium and added to 1.5 ml eppendorf tubes (100 μl/tube) containing various concentrations of test compound or control antibiotics (5 μl) at 20× the final concentration in 100% DMSO as described above. Following a 5 min incubation at 37° C., [14C]N-acetylglucosamine (0.4 μCi/reaction) was added to each tube and incubated for 45 min in a 37° C. heating block. Reactions were stopped through the addition of 100 μl of 8% SDS to each tube. Reactions were then heated at 95° C. for 30 min in a heating block, cooled, briefly centrifuged, and spotted onto pre-wet HA filters (0.45 μM). After washing three times with 5 ml of 0.1% SDS, the filters were rinsed two times with 5 ml of deionized water, allowed to dry, and then counted using a Beckman LS3801 liquid scintillation counter.
Lipid Synthesis
Bacterial cells were grown to early exponential growth phase in MHBII broth and added to 1.5 ml eppendorf tubes (in triplicate) containing various concentrations of test compound or control antibiotics as described above. Following a 5 min incubation at room temperature, [3H]glycerol was added at 0.5 μCi per reaction.
Reactions were allowed to proceed at room temperature for 15 min and then stopped through the addition of 375 μl chloroform/methanol (1:2) followed by vortexing for 20 seconds after each addition. Chloroform (125 μl) was then added to each reaction, vortexed, followed by the addition of 125 μl dH2O and vortexing. Reactions were centrifuged at 13,000 rpm for 10 min, and then 150 μl of the organic phase was transferred to a scintillation vial and allowed to dry in a fume hood for at least 1 hr. Samples were then counted via liquid scintillation counting.
Results
Susceptibility testing was conducted with NCL812 and S. aureus ATCC 29213 to determine the concentrations of drug needed in the macromolecular synthesis assays.
FIG. 5 shows that the MIC for NCL812 was 4 μg/mL, while the quality control agent linezolid was within the CLSI-established quality control range (Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Nineteenth Informational Supplement. CLSI document M100-S20 [ISBN 1-56238-716-2]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pa. 19087-1898 USA, 2010). Precipitation of NCL812 was observed at ≧8 μg/mL in plates that were prepared in an identical fashion, but did not receive an inoculum of S. aureus. Macromolecular synthesis inhibition studies were performed using concentrations of NCL812 that were equivalent to 0, 0.25, 0.5, 1, 2, 4 or 8-fold the MIC value (4 μg/ml) for S. aureus ATCC 29213 (FIGS. 6-11).
FIG. 6 shows the effect of NCL812 on DNA synthesis. NCL812 demonstrated no inhibition at 0.25-fold the MIC, 40% inhibition at 0.5-fold, and approximately 95% inhibition at the MIC. This is compared to the control ciprofloxacin which showed approximately 51% at 8-fold the MIC (0.5 μg/ml).
The results for NCL812 inhibition of RNA synthesis were very similar to the DNA synthesis study, with rifampicin serving as the positive control (FIG. 7). It should be noted that precipitation was observed at 4 to 8-fold the MIC in the Mueller Hinton broth II utilized in the DNA and RNA synthesis assays.
Protein synthesis was inhibited in a dose-dependent manner at 0.25, 0.5, and 1-fold the MIC value of NCL812 showing up to 97% inhibition at the MIC (FIG. 8). Linezolid demonstrated approximately 61% inhibition of protein synthesis at 8-fold the MIC (2 μg/ml). Precipitation of NCL812 occurred at 4 and 8-fold the MIC in the protein synthesis assay.
In FIG. 9, NCL812 also showed a somewhat dose-dependent inhibition of cell wall synthesis, though there was a large increase in inhibition from 1 to 2-fold the MIC. However, inhibition dropped to approximately 68% and 52% at 4-fold and 8-fold the MIC, respectively. Precipitation of NCL812 occurred at 2, 4, and 8-fold the MIC in the M9 minimal medium used for the cell wall synthesis assay, and that is the likely cause of the decline in inhibition. In comparison, the positive control vancomycin showed 96% inhibition at 8-fold the MIC (2 μg/ml).
NCL812 demonstrated a similar inhibition profile against lipid synthesis as that shown for DNA and RNA synthesis, reaching approximately 90% inhibition at the MIC (FIG. 10). The positive control inhibitor cerulenin demonstrated 72% inhibition at 8-fold the MIC (32 μg/ml).
FIG. 11 represents a composite of all five macromolecular synthesis reactions. It can be observed that the inhibition curves were similar for each pathway, suggesting a global inhibition of several pathways simultaneously by NCL812. It is possible that NCL812 targets the cell membrane, causing leakage of essential ions and/or metabolites, thereby leading to a global shutdown of the cell synthesis pathways.
In summary, NCL812 inhibited DNA, RNA, protein, cell wall, and lipid pathways in a growing culture of S. aureus. Though some instances of dose-dependent inhibition of pathways was observed, all five macromolecular synthesis reactions were similarly sensitive to NCL812.
Example 3
Effect of NCL812 on ATP Release from Staphylococcus aureus 
Materials and Methods
Test Compounds
The test compound NCL812 was shipped under conditions of ambient temperature and then stored at 2-8° C. until assayed. Stock solutions were made by dissolving NCL812 dry powder in 100% DMSO to a concentration of 1,600 μg/ml. The comparator agent was polymyxin B (Sigma, P-4932 (Lot 044K11905)).
Test Organism
S. aureus ATCC 29213 was originally acquired from the American Type Culture Collection (Manassas, Va.).
ATP Release Assay
The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was utilized to measure the leakage of ATP from bacteria. Cultures were grown to early exponential phase (0.2-0.3 optical density units at 600 nm) in Mueller-Hinton Broth II and then treated with seven different concentrations of either NCL812 or polymyxin B (positive control) utilizing the MIC for each compound as a guide (0, 0.25, 0.5, 1, 2, 3, 4, or 8-fold the MIC). The negative control received 2% DMSO, which represented the final DMSO concentration in each assay. After a 30 min exposure to drug, cells were sedimented by centrifugation and the supernatant was analyzed for the presence of ATP. Results were expressed as ATP concentration released to the medium (μM).
Results
The MIC for NCL812 has been previously determined to be 4 μg/ml. The ATP release assay is conducted by growing S. aureus to exponential phase and then adding the antibiotic at multiples of the MIC in an effort to detect a dose-dependent response.
As shown in FIG. 12, the positive control polymyxin B released ATP from S. aureus cells in a dose-dependent fashion with maximal release of approximately 0.34 μM ATP at 8-fold the MIC (256 μg/ml). ATP release in the presence of NCL812 was dose-dependent at 0.5-1 fold the MIC, resulting in maximal release (0.33 μM) observed at the MIC (4 μg/ml). ATP release actually decreased thereafter at 2 to 8-fold the MIC. It should be noted that in previous studies, precipitation of NCL812 was observed at 4 to 8-fold the MIC in Mueller Hinton broth II.
In summary, NCL812 demonstrated dose-dependent release of ATP from actively growing S. aureus cells. ATP release from the cells into the growth medium reached maximum levels at the MIC value, and this was followed by a decrease in ATP release at higher doses. The data indicated that NCL812 may interact with the cell membrane of S. aureus, causing leakage of vital metabolites such as ATP.
Example 4
In Vitro Antibacterial Activity of NCL812 Against Methicillin-resistant and Methicillin-susceptible Staphylococcus aureus 
Materials and Methods
Antimicrobial Agents
Aliquots of stock solution of NCL812 (25.6-mg/ml) was prepared in dimethyl sulfoxide, stored at −80° C. and defrosted immediately before use. Ampicillin stock was obtained from Sigma-Aldrich (Australia). Antimicrobial discs were obtained from Thermo Fisher Scientific (Australia).
Microorganisms
Twenty nine clinical isolates of MRSA were obtained (FIG. 13), along with S. aureus control organism ATCC 49775. Isolate identification was confirmed by conventional phenotypic methodologies, including the slide coagulase test, Vogues-Proskauer test, polymyxin B sensitivity (300-units), and Staphytect Plus Protein A latex slide agglutination (Thermo Fisher Scientific Australia). Bacteria were stored at −80° C. in 40% glycerol broth and routinely grown from stock on sheep blood agar (SBA) incubated at 37° C. In subsequent experiments, only fresh cultures <24-h were used.
Isolate Resistotyping
Antibiotic-susceptibility profiling of the isolate collection was undertaken using Kirby-Bauer disc diffusion, 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 in physiological saline. Turbidity was adjusted to a 0.5 McFarland standard and suspensions were spread over the medium. Antibiotic discs were transferred onto the inoculated medium and analysed after 24-h incubation at 37° C. Isolates labelled as MRSA that were not β-lactam-resistant on the basis of the Kirby-Bauer test were grown from stock on plate count agar supplemented with 5-μg/ml ampicillin and subject to repeat testing, as Penicillin Binding Protein2a expression can be induced by exposure to β-lactam antimicrobials.
Molecular Detection of the Protein A and mecA Genes to Confirm MRSA Status
Isolate identities were confirmed genotypically using a novel, duplex conventional polymerase chain reaction (PCR) test targeting the spa (protein A) and mecA (methicillin resistance) genes. In addition, the isolates were tested in a mecA and spa Sybr green real-time PCR. Approximately ten colonies of each overnight bacterial subculture was suspended in 1× phosphate buffered saline (pH 7.4) and vortexed. Isolates were subject to DNA extraction using the QIAampO DNA Mini Kit (Qiagen, Australia) following the manufacturers protocols. Template DNA was eluted in 50-μl of elution buffer and either used directly in PCR, or stored at −20° C. prior to DNA amplification using the spa forward (5′-TGATACAGTAAATGACATTG-3′) and reverse (5′-TTCTTATCAACAACAAGTTC-3′) primers and mecA forward (5′-TTCGTGTCTTTTAATAAGTGAGG-3′) and reverse (5′-ATGAAGTGGTAAATGGTAATATCG-3′) primers (Invitrogen, Australia). Conventional PCR amplification was performed in a 20-μl volume containing 10-μl HotStarTaq Plus Master Mix (Qiagen, Australia), 0.5-μM of each spa primer, 0.2-1 μM of each mecA primer, and 3-μl of extracted DNA. An automated thermal cycler (T100 Thermal Cycler, Bio-Rad) was used for PCR amplification of the spa and mecA genes.







TABLE 1







PCR and RT PCR reaction conditions











Temperature
Time




(° C.)
(seconds)
Number of cycles














PCR stage





Enzyme activation
95
300
1


Amplification:
94
30


Denaturation


Annealing
50
30
38 


Extension
72
38


Cooling
20
∞
1


Real-time PCR stage


Enzyme activation
95
300
1



95
15


Amplification:
50
20
40 


Denaturation


Annealing
70
40
Single acquisition



95
5


Melting curve
55
20
1



95
0
Continuous acquisition


Cooling
40
30
1









The mecA and spa amplified products of 325- and 120-bp, respectively, were detected by GelRed staining followed by electrophoresis in 2% agarose gels.
Minimum Inhibitory Concentration Testing
The in vitro activities of NCL812, and ampicillin as a positive control, were determined by broth microdilution as recommended by the CLSI in cation-adjusted Mueller-Hinton II broth. Microtiter plates containing two-fold dilutions of each antimicrobial agent were inoculated with  ̃105-CFU/ml of each isolate in a 100-μl final volume. Plates were incubated for 24-h at 37° C. Turbidity (absorbance at OD600) was measured using a Bio-Rad Benchmark Plus microplate spectrophotometer in Microplate Manager® version 5.2.1 (Bio-Rad). Minimum inhibitory concentration (MIC) endpoints were defined as the lowest antimicrobial concentration assessed by the spectrophotometer that inhibited bacterial growth. ATCC 49775 was included in the isolate collection as a control organism using breakpoints defined by the CLSI. The MIC50, MIC90 (concentrations that inhibited growth of the lower 50% and 90% of total organisms, respectively), and MIC range (minimum and maximum) were calculated to profile the antimicrobial susceptibility of the isolate collection.
Bactericidal Activity
The bactericidal activity of NCL812 was established by determination of the minimum bactericidal concentration (MBC) and time-kill analyses using CLSI guidelines. The MBC was defined as the lowest drug concentration at which 99.95% of the original inoculum was eliminated.
Time-kill assays for ATCC 49775 were performed in cation-adjusted Mueller-Hinton II broth in Microtiter plates and again in 10-ml volumes for macrodilution assays at antimicrobial concentrations equivalent to 1× and 4× the MIC. Bactericidal activity in macrodilution assays was identified as a 3-log10 decrease from the initial inoculum size. Bacteria were cultured overnight at 37° C. on SBA. Colonies were suspended in broth and the turbidity was adjusted to a 0.5 McFarland standard to obtain a bacterial suspension of  ̃105-CFU/ml. Bacterial suspensions were incubated at 37° C. with shaking. Aliquots were removed at 0-, 1-, 2-, 4-, 8-, 12-, and 24-h after antimicrobial addition, diluted, plated onto SBA and incubated for 48-h at 37° C. for viable count determination. Turbidimetric growth curves for S. aureus were obtained for Microtiter plate assays by monitoring optical density changes using a Bio-Rad Benchmark Plus microplate spectrophotometer at 600 nm. Optical densities were measured at 0-, 1-, 2-, 4-, 8-, 12, and 24-h after antimicrobial addition.
Statistical Methodology
Microbiological data was interpreted using CLSI guidelines. Data was examined using the student's t-test, Fisher's exact test, analysis of variance, and a generalized linear model for tests of between-subjects effects where appropriate. Differences were considered significant at the 0.05 level in IBM SPSS® version 19.0 (University of Adelaide).
Results
Confirmation of Staphylococcus aureus Identity and mecA Status
Latex agglutination tests confirmed that all 30 isolates were protein A positive. The isolates tested positive for coagulase activity using slide agglutination. Voges-Proskauer and polymyxin B resistance tests confirmed that all isolates were S. aureus except for a single methicillin-susceptible isolate; MSSA DE-25 (FIG. 14). Based on spa gene PCR amplification, this isolate was not identified as a S. aureus isolate despite testing positive in the protein A latex agglutination and slide coagulase tests. This canine-origin Staphylococcus spp. was identified as Staphylococcus pseudintermedius based on biochemical characteristics. The mecA conventional and real-time PCR results confirmed that 66.66% of the isolates were classified as methicillin-resistant on the basis of possession of the mecA gene. There were no significant differences between the ability of conventional and real-time PCR to detect the mecA gene (P>0.05).
Staphylococcus aureus Antimicrobial Susceptibility Profiles
Antimicrobial susceptibility assays revealed that HA-MRSA isolates had the highest mean prevalence of resistance to multiple antimicrobial classes (P<0.000). CA-MRSA isolates were next most resistant (P<0.007), followed by methicillin-susceptible staphylococci (P<0.037) (FIG. 15). Oxacillin resistance was expressed in only 80.00% and 10.00% of HA-MRSA and CA-MRSA isolates, respectively. Cefotetan resistance was expressed in 80.00% and 20.00% of HA-MRSA and CA-MRSA isolates, respectively. Although oxacillin and cefotetan did not significantly differ in their ability to detect MRSA (P>0.05), detection was significantly improved when using the mecA PCR when compared to disc diffusion (P<0.013). The majority of HA-MRSA isolates expressed resistance to amoxicillin-clavulanic acid, cefotetan, cephalexin, clindamycin, erythromycin, oxacillin, and penicillin-G, whereas the majority of CA-MRSA isolates were resistant to only clindamycin, erythromycin, and penicillin-G. None of the isolates tested were vancomycin resistant. Overall, the most prevalent resistance phenotypes were penicillin-G (83.33%), erythromycin (73.33%), and clindamycin (43.33%), whilst only single isolates (3.33%) were resistant to trimethoprim-sulfamethoxazole and rifampicin.
mec Gene Complex Interactions
All MRSA isolates belonging to mec gene complex A expressed resistance to both oxacillin and cefotetan (FIG. 16). However, only 20% of mec gene complex B MRSA isolates were phenotypically resistant to these antimicrobials. Of the MRSA isolates belonging to mec gene complex C2, only a single isolate expressed methicillin resistance to oxacillin and only two isolates expressed resistance to cefotetan. Unclassified MRSA isolates expressed full resistance to oxacillin and cefotetan.
Melting point peaks for the mecA real-time PCR negative derivative plot −dF/dT differed between mec gene complex (P<0.003) (FIG. 17). On average, mec gene complex B and unclassified isolates demonstrated higher melting point peaks than other SCCmec types (P<0.012).
Physical Properties and MIC of NCL812
Initial tests of NCL812 showed that it was soluble in DMSO, but produced a cloudy solution when dissolved in cation-adjusted Mueller-Hinton II broth (CAMHB) (FIG. 18). In initial testing, NCL812 was found to have consistent MIC values (FIG. 18).
In Vitro Antibacterial Activities: Minimum Inhibitory Concentrations
MIC50 and MIC90 values for compound NCL812 (4- and 4-8-μg/ml) are shown in FIG. 19. MIC values differed by S. aureus classification (susceptible, HA- or CA-MRSA) (P<0.005). In many cases, NCL812 had significantly increased activity against CA-MRSA and methicillin-susceptible staphylococci by one dilution when compared to HA-MRSA (P<0.002 and P<0.020, respectively), however there were no significant differences between MIC values for methicillin-susceptible staphylococci and CA-MRSA (P>0.05). Ampicillin MIC values were within the normal range expected on the basis of CLSI guidelines.
In Vitro Antibacterial Activities: Minimum Bactericidal Concentrations
The MBCs determined from NCL812 were equivalent to the MIC for 93.33% and 83.33% of S. aureus isolates, respectively (FIG. 19). In all remaining cases, MBCs were one dilution higher. For NCL812, MBCs ranged from 2-8-μg/ml and 4-16-μg/ml, respectively.
Time-kill Studies
In comparison to the turbidimetric growth curve of ATCC 49775, no visible bacterial growth was observed when ATCC 49775 was inoculated into cation-adjusted Mueller Hinton II broth supplemented with NCL812 at 1× and 4× the MIC in microdilution assays (P<0.033 and P<0.038, respectively) (FIG. 20).
When analysed in 10-mi macrodilution assays, broth supplemented with antimicrobials at 1× and 4× the MIC and inoculated with ATCC 49775 displayed significantly reduced viable counts for both NCL812 when compared to the growth control (0.000<P<0.008) (FIG. 21). Additionally, the time-kill profiles of NCL812 did not significantly differ (P>0.05). Both antimicrobials remained bactericidal until approximately 8-12-h after antimicrobial addition, where bacterial regrowth was observed. Considerable variation in the killing activity of NCL812 was observed from 8-24-h. Although NCL812 was no longer bactericidal by 24-h, viable counts observed at 1× the MIC remained significantly lower than those obtained from unsupplemented broth (P<0.046).
In summary, the example set out above demonstrates bactericidal activity by NCL812 against both methicillin-susceptible staphylococci and MRSA. MIC and MBC values were consistently low across the selection of isolates (MICrange 2-8-μg/ml). NCL812 retained good in vitro antimicrobial activity against common, multidrug-resistant MRSA isolates, including the epidemic UK EMRSA-15, EMRSA-16, and EMRSA-17, Irish EMRSA-1, AUS EMRSA-3, NY/JAPAN HA-MRSA, and predominant CA-MRSA clones. NCL812 was also active against one S. pseudintermedius isolate that was originally identified as a S. aureus strain.
Preliminary testing suggests that NCL812 targets the S. aureus cell membrane, causing dose-dependent release of vital metabolites such as adenosine-5′-triphosphate. Disruption of the bacterial membrane bilayer or proteins that are integral to membrane function in bacteria is a target for numerous large antimicrobials which are ubiquitous in nature; including glycolipids, lipopeptides, lipoproteins, fatty acids, neutral lipids, phospholipids, and biosurfactants. Although NCL812 is a low molecular mass (≦500-Da) synthetic compound, it does appear to exert bactericidal activity in a similar manner to other antimicrobials which target the Gram-positive cell membrane, including the high molecular weight cyclic lipodepsipeptide antimicrobial agent daptomycin, or the low molecular mass quinolone-derived HT61, whose chemical structure is not currently available. Many of these lipophilic antibacterial agents are not effective against Gram-negative microorganisms due to the presence of the outer lipid bilayer membrane, which contains narrow porin channels reducing the net penetration of some compounds into the cell.
The insolubility of NCL812 at even low concentrations in microbiological media may reflect the amphipathic and oligomeric nature of this antimicrobial and suggests that the real MIC may be much lower than observed, as it is likely that it is only NCL812 in solution that is biologically active. In time-kill studies, NCL812 exerted rapid in vitro bactericidal activity against ATCC 49775.
Importantly, the apparent short in vitro half-life of this antimicrobial resulted in bacterial regrowth observed at 12-h after antimicrobial addition. This suggests that if a viable bacterial population survives the initial exposure to NCL812 prior to antimicrobial inactivation, bacterial regrowth will occur. The development of resistance to NCL812 in these studies was ruled out as test bacteria remained susceptible to NCL812 following harvesting, washing and MIC testing. Whilst the apparent short in vitro half-life of NCL812 may be a desirable characteristic for future in vivo application, it does suggest that NCL812 may need to be administered every 8-h in future in vivo safety and efficacy experiments to maintain adequate systemic concentrations, though it would appear from the time-kill profile that the NCL compound series are concentration-dependent rather than time-dependent antimicrobials.
To overcome the methicillin-susceptible MRSA phenotype, extending disc diffusion incubation time from 24- to 48-h compensates for the slow derepression of the mecR gene. Although the effects of longer incubation were not examined, and the small sample size of MRSA isolates prevented further investigation into mec complex interactions; genetic techniques were of significantly improved sensitivity when compared to phenotypic methods for confirmation of the mecA status of the isolates in this study. Although genetic techniques are not always employed as a routine method for detecting MRSA, real-time PCR identification of the presence of the mecA gene in a Staphylococcus spp. isolate remains the diagnostic gold standard.
Example 5
In Vitro Pharmacodynamics of a New Antimicrobial Agent for Streptococcus pneumoniae 
Materials and Methods
Pneumococcal Antimicrobial Susceptibility
Pneumococcal Strains and Growth Conditions
Twenty pneumococcal isolates comprised of eight characterised laboratory strains and 12 clinical isolates were obtained.







TABLE 2







Characterisation of Isolates Tested













Infectious Dose


Strain
Serotype
Reference
(ID50) by IP





D39
2
Avery et al. 2010
102 *


(NCTC

Nature Reviews Microbiology


7466)

8: 260-271


A66.1
3
Francis et al. 2001.
8 × 103 (10, 98)




Infect Immun. 69: 3350-2358


EF3030
19F
Briles et al. 2003
≧105* 




J. Infec. Diseases. 188:




339-348


L82016
 6B
Briles et al. 2000
≧105* 




Infect Immun. 68: 796-800


P9
 6A
This study
104 *


P21
3
This study
≦101 * 


TIGR4
4
Tettlelin et al. 2001
104 *




Science 293: 498-506


WU2
3
Briles et al. 1981
 5 × 1012 *




J. J Exp Med. 153: 694-705


WCH16
 6A
This study
5 × 104 *


WCH43
4
This study
102 *


WCH46
4
This study
104 *


WCH57
8
This study
104 *


WCH77
5
This study
104 *


WCH86
4
This study
104 *


WCH89
7
This study
≧105 * 


WCH92
4
This study
≦104 * 


WCH137
 6A
This study
Not determined





to date


WCH158
19F
This study
105 *


WCH184
19F
This study
108 (14)


WCH211
11 
This study
5 × 106 *









The National Collection of Type Cultures (NCTC) control strain D39 was used as a growth control for all MIC and MBC assays. D39 was later designated for kill kinetics, point of resistance assays and transmission electron microscopy (TEM) studies as it is a well documented laboratory strain with a defined in vivo pathogenesis that displayed consistent NCL812 MICs and MBCs.
For all in vitro assays, fresh pneumococcal isolates were grown overnight (O/N) on horse blood agar (HBA) plates (39 g/L Columbia blood agar base [Oxoid] 5% [v/v] defribinated horse blood [Oxoid] at 37° C. with 5% supplemented CO2. Mueller-Hinton blood agar with 5% defibrinated sheep blood (MHSBA Roseworthy Media and Blood Service) was used for disk diffusion analysis as directed by Clinical Laboratory Standards Institute (CLSI) standards. Pneumococci were routinely grown in broth consisting of 4% lysed horse blood (LHB) with Cation Adjusted Mueller Hinton Broth (CAHMB, [Difco]) at 37° C. with 5% supplemented CO2. Horse serum broth (HSB, 10% (v/v) donor 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 assays. Isolates were stored in HSB at −80° C.
Antibiotic Stocks and Reagents
NCL812 was provided in dry powder form by Neoculi. A total of 256 mg was dispensed into 10 ml of 100% dimethyl sulphoxide (DMSO) to make a stock of 25.6 mg/ml, which was then diluted 1:100 in CAHMB to make a final working stock of 256 μg/ml. Ampicillin dry powder was from Sigma A0166. The original 25.6 mg/ml stock was diluted in saline 1:100, 1:4, 1:20 and finally 1:16 in CAMHB to make a final working stock of 0.18 μg/ml. Erythromycin was from Sigma E077 and choline chloride was from Roche Diagnostics. Twenty micro liters of 0.05 μg/ml erythromycin was diluted 1:25 in 4.980 mls of CAMHB to give a final working stock of 0.2 μg/ml. Choline chloride (0.5%) was added to 4% LHB:CAMHB for specific kill kinetic assays.
Defining Antimicrobial Susceptibility of Pneumococcal Isolates
Isolate susceptibility to 12 different antimicrobials (FIG. 22) were determined by CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) methods. Antimicrobials were selected based upon the CLSI and EUCAST guidelines. Zone diameters for antimicrobials other than Ciprofloxacin for S. pneumoniae were determined by CLSI standards; whilst Zone diameters for Ciprofloxacin antimicrobial susceptibility to S. pneumoniae were determined by EUCAST.







TABLE 3







Interpretive Standards for Zone Diameters









Interpretive Standards for



Zone Diameters (mm)













Resis-
Inter-



Antibiotic

tant
mediate
Sensi-


Class
Antimicrobial (μg)
(R)
(I)
tive





β-lactam
Oxacillin (1 μg)°
≦20
≦20
≧20



Ampicillin (10 μg)°
≦20
≦20
≧20



Amoxicillin-clavulanate
≦20
≦20
≧20



(20/10 μg) °


Fluoroquin-
Ciprofloxacin (5 μg)*
≦22
≦22
≧22


olone


Folate pathway
Trimethoprim-
≦15
16-18
≧19


inhibitor
sulphamethoxazole



(1.25/23.75 μg)°


Glycopeptide
Vancomycin (30 μg)°
—
—
≦17


Lincosamide
Clindamycin (2 μg) °
≦15
16-18
≧19


Macrolide
Erythromycin (15 μg) °
≦15
16-20
≧21



Clarithromycin (15 μg) °
≦16
17-20
≧21


Phenicol
Chloramphenicol (30 μg) °
≦20
—
≧21


Rifamycin
Rifampin (5 μg) °
≦16
17-18
≧19


Tetracycline
Tetracycline (30 μg) °
≦18
19-22
≧23









Standardised bacterial suspensions were spread onto MHSBA using a sterile cotton swab. [Bacterial suspensions from of Streptococcus pneumoniae were standardised to an OD600nm between 0.08 and 0.1 using a spectrophotometer and then diluted 1:20. Bacterial colonies were taken from an O/N horse blood agar plate. To ensure the purity of the 1:20 bacterial suspension, 50 μL was spread plated onto horse blood agar and incubated O/N at 37° C. with 5% CO2. The CFU was calculated and compared to the initial plate counts.] Antibiotic disks (Sigma) were placed using a disk dispenser (Oxoid) according to CLSI standards. MHSBA plates were incubated for 16 hrs-24 hrs at 37° C. in 5% CO2. Zones of complete inhibition were measured in triplicate to the nearest millimeter using a ruler on natural light-reflected growth, and the mode was represented as the diameter for each isolate. Pneumococcal isolates were categorised as sensitive, intermediate (I) or resistant (R) by CLSI standards and quality control (QC) ranges (FIG. 22).
Determination of NCL812 MIC50, MIC90, MIC Range and MBC50, MBC90, MBC Range
MICs for NCL812 for all isolates listed in Table 2 were determined by measuring optical density at 600 nm (OD600 nm) (Spectramax spectrophotometer, Molecular Devices Corporation) as an indicator of bacterial growth using 96-well microtitre trays after incubation for 24 hrs at 37° C. in 5% CO2. [Micro-broth dilutions and 96-well trays are prepared by the following method: 90 μL of 4% LHB:CAMHB is aliquotted into all wells using a multichannel pipette. 90 μl of working antimicrobial stocks were no serial diluted down the tray by a 1:2 dilution. Negative broth controls and dilution control were taken into account when planning the set up of a 96-well tray.] 10 μL of bacterial suspension was then added to the appropriate wells in the 96 well tray. Appropriate positive (no antimicrobial), negative (no antimicrobial or bacteria) and negative dilution (a serial dilution control of antimicrobial and broth) controls were included in each assay. MBC and plate counts for kill kinetic assays were determined by aliquotting 20 μL from each well of the 96-well microtitre tray onto HBA, and incubating at 37° C. with 5% C02. The MBC was determined by a 99.95% inhibition of S. pneumoniae, taking into account the dilution factor. MICs and MBCs were determined in quadruplicate and the mode was taken as the representative value. The MIC50, MIC90 and MIC range and MBC50, MBC90 and MBC range were determined according to CLSI standards. The MIC50 and MIC90, or MBC50 and MBC90, are defined by the lowest concentrations which, when all the MICs and MBCs of the isolates are arranged from lowest to highest, inhibited the 50th and 90th percentile of the total amount of isolates, respectively.
Micro-broth Dilution Time Kill Studies with NCL812 Using Strain D39
Bacterial suspensions were added in triplicate to a 96-well microtitre tray containing NCL812 with a starting concentration of 128 μg/ml and serially diluted 1:2 sequentially to a concentration of 0.25 μg/ml. Negative dilution controls were subtracted from the median growth value to obtain a suitable indicator of overall bacterial production. The 96-well tray was incubated at 37° C. in 5% CO2 and read every 2 hrs for the first 12 hrs followed by final reads at 24 and 48 hrs at 600 nm. To further supplement this data, a separate experiment in which a 96-well tray was read automatically at half hourly intervals using a spectrophotometer (Spectramax spectrophotometer, Molecular Devices Corporation) for 14 hrs was performed to confirm the trends in growth curves observed from original micro-broth dilution studies.
MBC Time Kill Studies with NCL812 Using Strain D39
MBC kill kinetics assays involved the preparation of three 96-well microtitre trays. At specific time points, aliquots obtained from these trays provided viable counts following incubation at 37° C. in 5% CO2 on HBA, and the MBC was determined after 24 hrs of growth.
Macro-broth Dilution Time Kill Studies of D39 with NCL812
Bacterial suspensions and working antibiotic stocks were prepared as described above. [For preparing macro-broth dilutions, 20 ml tubes were filled each with 9 mls of 4% LHB:CAMHB. 9 mls of a working antimicrobial stock was diluted 1:2 when added to one of the tubes, and then serial diluted down from a high to low concentration of antimicrobial. 1 ml of S. pneumoniae bacterial suspension was added to the appropriate tubes, including the positive control. Tubes were incubated at 37° C. with 5% CO2 with gentle manual tilting of the tubes treated with NCL812 every 10 mins for the first 12 hrs. At every 2-3 hrs during the first 12 hrs of growth and then at 24 hrs and 48 hrs, 50 μL of each bacterial suspension was spread plated onto HBA and incubated at 37° C. with 5% CO2 for 16-24 hrs.]







TABLE 4







Concentration of antimicrobials









Serial dilution
NCL-812 (μg/ml)
Ampicillin (μg/ml)












1
128
0.09


2
64
0.045


3
32
0.023


4
16
0.011


5
8
0.0065


6
4


7
2









Cultures were incubated at 37° C. in 5% CO2 with gentle manual tilting every 10 mins for the first 12 hrs. Viable counts from 50 μL aliquots of each concentration were read following incubation at 37° C. in 5% CO2 for 24 hrs. The pH of each sample was measured at specific time points using pH indicator strips. Confluent growth was defined when more than 1000 colonies were counted per plate. A bactericidal effect was defined as a 3-log 10-unit reduction (99.9%) of the original cell suspension determined at 24 hrs for each concentration.
Point of Resistance Assay for NCL812
Macro-broth dilutions were prepared as above. Broth cultures of strain D39 (10 ml) were incubated in the presence of 2 μg/ml and 4 μg/ml of NCL812, and 0.022 μg/ml of Ampicillin for 6 hrs at 37° C. in 5% CO2. Samples were centrifuged at a relative centrifugal force (RCF) of 101.45×g for 10 mins and washed in 50 mls of phosphate buffered saline (PBS) twice to remove any residual antimicrobial, and/or bacterial end products and media. Washed bacteria were resuspended and MICs were performed.
Effect of NCL812 on D39 Cell Membrane Ultra-structure
Transmission Electron Microscopy
Morphological appearance and morphometric analysis of the cell membrane was determined using transmission electron microscopy (TEM). Bacterial suspensions and 10 ml cultures of D39 were prepared as before. Samples were incubated at 37° C. in 5% CO2 with gentle manual tilting of the cultures every 10 mins. Cultures were exposed to either 1 μg/ml, 4 μg/ml or 16 μg/ml of NCL812 and harvested at 6 or 12 hrs by centrifugation at 101.45×g for 20 mins and washed twice in 50 mls of PBS. Critical time points for TEM work were determined by analysing trends in the growth curves produced from the kill kinetics studies. Samples were resuspended in PBS containing 20% glycerol and stored at −80° C. until required. Before fixation, 20% glycerol was removed by centrifugation and washing on ice three times in 50 mls of PBS.
Samples were fixed using modified protocols defined by a previous study examining cell wall ultrastructure of S. pneumoniae (Hammerschmidt, S. et al. 2005. Infect Immun 73:4653-4667). A lysine-acetate-based formaldehyde-glutaraldehyde ruthenium red-osmium fixation procedure involved fixing the bacterial pellets with a cacodylate buffer solution containing 2% formaldehyde, 2.5% glutaraldehyde, 0.075% ruthenium red and 0.075 M of lysine acetate for 1 hr. After washing with cacodylate buffer containing 0.075% ruthenium red three times, a second fixation in cacodylate buffer solution containing 2% formaldehyde, 2.5% glutaraldehyde and 0.075% ruthenium red was undertaken for 1.5 hrs. Cells were subsequently washed three times with cacodylate buffer containing 0.075% ruthenium red and underwent a final fixation in 1% osmium tetroxide in cacodylate containing 0.075% ruthenium red for 1 hr. The samples were then washed three times in cacodylate buffer containing 0.075% ruthenium red only.
Samples were washed and dehydrated using a graded series of ethanol (70, 90, 95 and 100%) for 10-20 min, two times for each step. Samples were infiltrated using 50:50 LR White resin in 100% ethanol for 1 hr, and subsequently washed with 100% LR White resin for 1 hr and left O/N in a third change of 100% LR white to ensure adequate infiltration of resin. The samples were then embedded in fresh LR White resin and incubated at 50° C. for 48 hrs. Sections were cut to 1 μm using a glass knife, stained with Toluidene Blue and viewed under a light microcrope at 400× to identify the presence of stained pneumococci. At least four ultra-thin sections were then cut to 90 nm using a diamond knife and placed on matrix grids, one section per grid. Ultra-thin sections were then stained with uranyl acetate and lead citrate alternatively at 5 min intervals, followed by three washes with distilled water in-between each exposure. Stained sections were then placed on grids and viewed between 25000× and 130000× on a Philips CM100 Transmission Electron Microscope. Images were obtained at 130000× magnification and analysed using analySIS [Olympus Soft Imaging Systems].
Statistical Analysis
Statistical analyses were conducted using statistics program GraphPad Prism (5th ed, GraphPad Software Inc.) 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 dilution studies where multiple replicates could not be obtained due to the high costs involved in this assay. Two tailed, unpaired t-tests were performed.
Results
Pharmacodynamics of NCL812 in S. pneumoniae 
Quality Control Disk Diffusion Analysis for 20 S. pneumoniae Isolates
Although nine out of the 12 antimicrobials used for disk diffusion analysis had established QC ranges by EUCAST, QC ranges were not defined for amoxicillin-clavulanate, clarithromycin and clindamycin (Table 3).
WCH16 and WCH184 were both resistant to at least two antimicrobials whereas EF3030 and WCH137 were intermediate and resistant to trimethoprim-sulphamethoxazole respectively (FIG. 22). The other remaining 16 isolates were sensitive to all 12 antimicrobials. Sensitivity to ampicillin was confirmed for each isolate, enabling the use of ampicillin as a positive control in later micro-broth dilution assays (FIG. 22).
Solubility and Activity of NCL812 in Different Media
NCL812 was brought in 100% DMSO but developed turbidity when it was further diluted into CAMHB or PBS.







TABLE 5







Visual analysis of NCL812 and ampicillin solubility










NCL812
Ampicillin















Diluent





CAMHB
Turbid
Transparent



DMSO
Transparent
Transparent



PBS
Precipitate
Transparent



Media



4% LHB:CAMHB
Turbid
Transparent



10% horse serum-
Precipitate
Transparent



supplemented broth










Growth of S. pneumoniae strain D39 in an MIC assay for NCL812 using 10% HSB (220 mls of horse serum is filtered to 10% in 180 mls of Lemco nutrient broth) resulted in a threefold increase in the MIC for D39 treated with NCL812 with a twofold increase for the positive ampicillin control.







TABLE 6







Difference in activity of NCL812 in different media.









Relative MIC with media type for D39












10% horse serum-
Fold-


Antimicrobial
4% LHB:CAMHB
supplemented broth
increase













NCL812
4
32
3


Ampicillin
0.023
0.09
2









There was no change in MIC for D39 with differing storage conditions of pre-prepared 96-well microtitre trays.







TABLE 7







Storage conditions of prepared micro


titre trays for micro broth dilution.










Storage condition












Antimicrobial
−2° C.
4° C.















NCL812
8
8



Ampicillin
0.023
0.023










During macro-broth dilutions, the pH of the media did not change compared to appropriate controls (FIG. 23).
Determination of S. pneumoniae In Vitro Susceptibility to NCL812
Determination of NCL812 MIC50, MIC90, MIC range
NCL812 exhibited a MIC50 and MIC90 of 8 μg/ml and MIC range of 4-8 μg/ml when tested against all 20 strains. The MIC for ampicillin was comparable to recent published findings using micro-broth dilution as an endpoint for antimicrobial resistance in pneumococcal isolates, thus confirming the accuracy of MICs obtained for NCL812.







TABLE 8







MIC and MBC valuesfor isolates treated


with NCL812 and ampicillin










NCL812 (μg/ml)
Ampicillin (μg/ml)















MIC50
8
0.023



MIC90
8
0.023



MIC Range
4-8
0.011-0.09



MBC50
8
0.023



MBC90
8
0.023



MBC Range
4-8
0.011-0.09










Determination of NCL812 MBC50, MBC90, MBC Range

Minimum bactericidal concentrations (MBC50, MBC90 and MBC range respectively) were determined for NCL812 and ampicillin for all twenty isolates.







TABLE 9







MIC and MBC values for NCL812 and ampicillin


for each pneumococcal isolate












NCL812

Ampicillin













MIC
MBC
MIC
MBC

















D39
4
8
0.023
0.023



EF3030
8
8
0.023
0.023



A66.1
8
8
0.045
0.045



TIGR4
4
8
0.023
0.023



WU2
4
8
0.023
0.023



L82016
8
8
0.023
0.023



P9
8
8
0.023
0.023



P21
4
8
0.023
0.023



WCH158
4
8
0.023
0.023



WCH89
4
4
0.023
0.023



WCH57
8
8
0.023
0.023



WCH77
4
8
0.023
0.023



WCH46
4
4
0.023
0.045



WCH86
4
8
0.023
0.023



WCH137
4
8
0.023
0.023



WCH184
4
4
0.045
0.045



WCH16
8
autolysis
0.023
Autolysis



WCH43
4
8
0.023
0.023



WCH92
8
8
0.09
0.09 



WCH211
4
8
0.023
0.023










Micro-broth Dilution Time Kill Studies of D39 Treated with NCL812

D39 exposed to sub-inhibitory concentrations (≦2 μg/ml) of NCL812 grew similar to unexposed controls over a 48 hour period (FIG. 24). Higher concentrations of NCL812 (≧16 μg/ml) resulted in no bacterial growth for 48 hrs (FIG. 24). These growth characteristics were validated by a micro-broth kill kinetic study using a Spectramax spectrophotometer, which measured growth (represented as OD600) at half-hourly intervals for 14 hrs for NCL812 and ampicillin (FIGS. 25 and 26). The commencement of exponential growth for D39 treated with NCL812 is shown in FIG. 27.
The growth of D39 treated with NCL812 was compared to D39 treated with ampicillin or erythromycin over 48 hrs (FIGS. 28 and 29). D39 treated with ampicillin exhibited similar growth to D39 exposed to NCL812 over 48 hrs (FIG. 28). Erythromycin-treated D39 produced very different growth curves from NCL812 where a larger difference in growth between concentrations was observed (FIG. 29). The addition of 5% choline chloride to the media over a 48 hour period resulted in no significant difference in growth for NCL812 compared to positive and growth controls (FIGS. 30 and 31).
Point of Resistance Testing
D39 treated with ≦4 μg/ml NCL812 entered a log phase of growth at 6 hrs (FIG. 24), as shown in four independent experiments. The possibility of antimicrobial resistance to NLC812 between 5 and 6 hrs was investigated by determining further MICs on D39 exposed to 2 μg/ml NCL812, 4 μg/ml NCL812 and 0.0225 μg/ml ampicillin for 6 hrs. Results showed no significant increase in MIC for all samples of D39 exposed to NCL812 compared to growth controls, and ampicillin







TABLE 10







MIC and MBC values of D39 exposed to 2


ug/ml or 4 ug/ml of NCL812 for 6 hours













MIC of D39

MBC of D39




following

following



Original
exposure to
Original
exposure to



MIC of
NCL812 for
MBC of
NCL812 for



D39
6 hrs.
D39
6 hrs















D39 exposed to 2
4
8
8
8


μg/ml NCL812


D39 exposed to 4
4
8
8
8


μg/ml NCL812


D39 exposed
0.023
0.045
0.023
0.023


0.023 μg/ml


Ampicillin


D39 growth *
8
8
8
8


D39 growth2 **
8
8
8
8





* D39 growth control: S. pneumoniae strain D39 grown for 6 hrs in 4% LHB:CAMHB.


** D39 growth2 control: S. pneumoniae strain D39 on HBA O/N, resuspended in saline (0.1 OD600) and diluted 1/20 in sterile saline.






Micro-broth Dilutions by Measuring Relative MBC at Specific Time Points

Relative MBCs were determined at specific time intervals from using broth dilution assays incubated for 48 hrs for NCL812 and control antimicrobials ampicillin and erythromycin (FIGS. 32 and 33). MICs of ampicillin (0.023 μg/ml) and erythromycin (0.00275 μg/ml) for D39 were similar in range to published findings for other pneumococcal isolates. A comparative difference in growth between NCL812, ampicillin, and erythromycin was observed (FIGS. 32 and 33). Ampicillin and erythromycin demonstrated a time-dependent reduction in bacteria. NCL812 exhibited fast bactericidal action, evidenced by an approximate 3-fold decrease in MBC within 5 hrs. A consistent bactericidal concentration (8 μg/ml) was maintained for the full 48 hrs for NCL812.
Macro-broth Dilution Time Kill Studies of D39 with NCL812
Viable counts for each time point were represented as a log 10 CFU/ml reduction for NCL812 (FIG. 34) and ampicillin (FIG. 35). Consistent confluent growth (determined by a limit of 2×104 CFU) was observed for unexposed controls and 2 μg/ml NCL812. Complete bactericidal activity (defined by a 3-log reduction in CFU) for 128 μg/ml of NCL812 was observed by a 4-log reduction of colony forming units (CFU) in 3 hrs and concentrations between 16 μg/ml and 64 μg/ml NCL812 were effective at eliminating bacterial growth within 8 hrs (FIG. 34). NCL812 at 4 μg/ml and 8 μg/ml appeared to be inactivated at 11 hrs post-exposure, as increased growth of strain D39 after this time point was observed (FIG. 34).
Transmission Electron Microscopy
Morphometric analysis revealed significant changes to the cell membrane in strain D39 exposed to 16 μg/mL NCL812 for 6 hrs compared to growth controls. Samples treated with 4 μg/ml as well as 12 hr cultures were not considered for morphemetric analysis due to the lack of bacterial cells available in each section. Treated samples possessed significantly thicker cell membranes (6.43±0.29 nm) compared to untreated samples (4.35±0.24 nm) (p<0.0001) (FIG. 36). The periplasmic space (intracellular space between the cell membrane and the cell wall) of D39 treated with 16 μg/ml NCL812 was significantly wider (4.54±0.096 nm) compared to untreated samples (3.91±0.14 nm) (p<0.001) (FIG. 37).







TABLE 11







Mean cell membrane thickness and periplasmic space












Treatment (16 μg/ml




Growth control
NCL812 for 6 hrs)









Statistical test













Unpaired t-test



Mean ± SEM
Mean ± SEM
(P value)














Cell membrane
4.35 ± 0.24 nm,
6.43 ± 0.29 nm,
P < 0.0001



n = 12
n = 13


Periplasmic
3.91 ± 0.14 nm,
4.54 ± 0.096 nm,
P < 0.001 


space
n = 11
n = 11









In summary, NCL812 produced highly consistent MICs and equivalent MBCs for the S. pneumoniae strain collection, confirming that it is bactericidal against this organism. In kill kinetics experiments, which measured the relative MBC over a 48 hr period, a consistent bactericidal effect was elicited in D39 after 6 hrs from initial exposure to NCL812.
This demonstration of bactericidal activity is the first to be observed in S. pneumoniae. This demonstrates that NCL812 is effective against pneumococcal in vitro.
Competitive binding between components in blood, serum or broth decreased the antimicrobial activity of NCL812. This was reflected in the increase of MIC observed between different broth types and diluents. Following the completion of these studies, recent independent research confirmed precipitation of NCL812 in PBS and reported complete solubility in water containing 4% DMSO, following initial dilution in 100% DMSO. A water-soluble NCL812 will greatly improve in vivo bioavailability and negative interaction between blood or serum proteins.
Based on the findings of this study, NCL812 exhibits a mechanism of action against S. pneumoniae that is different from lactam or macrolide classes, as it appears to exhibit concentration-dependent bactericidal activity as opposed to time-dependant qualities. Identifying the maximum pharmacokinetic serum concentration of NCL812 in vivo will assist confirmation of its concentration-dependant pharmacodynamic activity. Furthermore, the addition of choline chloride to the media confirmed that the mechanism of action for NCL812 is not associated with the affinity to cell wall choline binding proteins, and therefore may not be cell wall associated.
Morphometric analysis of the cell membrane and periplasmic space of D39 treated with 16 μg/ml NCL812 for 6 hrs showed that the cell membrane and periplasmic space was larger in treated samples, compared to control samples. The apparent increase in membrane size could be due to an 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 cell membrane, potentially by depolarisation or ATP inhibition. The mechanism of action of NCL812 may not be calcium-dependant as it appears that no competitive binding between NCL812 and ruthenium red, a calcium channel inhibitor of lipid bilayers, was observed in electron micrographs.
In conclusion, this in vitro study has demonstrated that NCL812 has many desirable characteristics as a fast-acting concentration-dependent bactericidal antimicrobial that appears to target the cell membrane of S. pneumoniae. These characteristics are desirable to treat acute pneumococcal infections. As NCL812 may possess a mechanism of action that targets the cell membrane, it will act much more quickly than time-dependent antimicrobials such as β-lactams and macrolides and potentially could be more effective than other bactericidal concentration-dependent antimicrobials such as fluoroquinolones which have intracellular targets.
Example 6
Characterization of Methicillin-susceptible and Methicillin-resistant Isolates of Staphylococcus Pseudintermedius from Australia and Preliminary In Vitro Efficacy of a New Anti-staphylococcal Compound
Materials and Methods
Sample Collection and Identification of Methicillin Susceptible Staphylococcus Pseudintermedius (MSSP) and Methicillin Resistant Staphylococcus Pseudintermedius (MRSP)
A total of 23 Staphylococcus pseudintermedius isolates were obtained from dogs (FIG. 38).
Ten methicillin susceptible and 13 methicillin resistant Staphylococcus pseudintermedius were collected for the study. Isolates were phenotypically classified as methicillin resistant on the basis of in vitro resistance to oxacillin and genetically for the presence of mecA gene according to standard procedures.
Oxacillin and cefoxitin susceptibility testing using disk diffusion technique and Epsilometer testing were performed. Identification of mecA gene was performed using polymerase chain reaction (PCR)
CLSI disk diffusion susceptibility testing was performed on the 23 Staphylococcus pseudintermedius isolates for the following antimicrobials: penicillin, amoxicillin, erythromycin, gentamicin, clindamycin, ciprofloxacin, cephalexin, chloramphenicol, tetracycline, oxytetracycline, vancomycin, cefotetan, moxifloxacin and rifampin (FIG. 39).
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) testing was undertaken using CLSI methodology for NCL812 and included ampicillin as a control. Anti-staphylococcal compounds were then tested against all 23 isolates and minimum inhibitory concentrations (MIC) were determined according to standard protocols. After the MICs were determined, the minimum bactericidal concentrations were performed to determine if these compounds are bacteriostatic or bacteriocidal.
Results
The mecA gene was present in 13 isolates of MRSP and negative in 10 MSSP. All MRSP isolates were resistant to oxacillin based on disc diffusion (<=17 mm) and E-test MIC (>=0.5 mg/L).
When cefoxitin resistance breakpoint was set at <=24 mm, 3/13 (23%) and 5/13 (38%) of MRSP tested were susceptible to cefoxitin. When cefoxitin resistance break point was set at <=30 mm, only 1/13 (7.7%) of MRSP tested at Veterinary Diagnostic Laboratory was susceptible.
The MRSP isolates were resistant to multiple antibiotic classes. Of the 13 MRSP isolates, all 13 were susceptible to rifampin. 3/13 (23%) were susceptible to chloramphenicol; 10/13 (77%) were susceptible to vancomycin.
Interestingly, 3/13 (23%) of the MRSP isolates were susceptible to amoxicillin; 8/13 (62%) were susceptible to cephalothin; 12/13 (92%) susceptible to cefotetan and 12/13 (92%) susceptible to moxifloxacin,
All 23 isolates were susceptible to NCL812 based on MICs. In addition, NCL812 has been shown to be bactericidal based on minimal bactericidal concentrations (MBC).
The MIC range of NCL812 against the Staphylococcus pseudintermedius isolates was found to be between 1 μg/mL and 4 μg/mL







TABLE 12







MIC range of NCL812 against Staphylococcus pseudintermedius


isolates











Isolate
AMP
NCL812















S1P1
128
4



S2P2
128
2



S3P3
128
2



S4P4
128
1



S5P5
16
2



S6P6
64
2



S7P7
128
2



S8P8
128
2



S9P9
32
2



S10P10
64
2



S11P11
128
4



S12P12
32
2



S13P13
0.25
2



S14P14
1
2



S15P15
4
4



S16P16
0.25
2



S17P17
1
2



S18P18
4
4



S19P19
0.5
4



S20P20
4
4



S21P21
0.1
2



S22P22
8
4



S23P23
32
2










The MIC 50 and MIC 90 of NCL812 against the Staphylococcus pseudintermedius isolates was found to be 2 μg/mL and 4 μg/mL respectively. The MIC mode and MIC range of NCL812 against the Staphylococcus pseudintermedius isolates was found to be 2 μg/mL and 1-4 μg/mL respectively.







TABLE 13







Combined MIC values of NCL812 against


Staphylococcus pseudintermedius isolates











Column1
AMP
NCL812















MIC50 (μg/ml)
32
2



MIC90 (μg/ml)
128
4



MIC mode (μg/ml)
128
2



MIC range (μg/ml)
0.1-128
1-4










Methicillin resistant Staphylococcus pseudintermedius (MRSP) is an emerging problem in dogs, cats and horses. Two major clonal MRSP lineages have been reported from dogs in Europe (ST 71) and North America (ST 68). There have also been reports of MRSP affecting dogs in Japan and a single case of MRSP in a veterinary worker in Hong Kong.
In this study, MRSP isolates were determined using a combination of presence of mecA gene and in vitro resistance to oxacillin. Cefoxitin susceptibility has been used as a substitute for oxacillin for methicillin resistant Staphylococcus aureus. However, cefoxitin disk diffusion tests using interpretive guidelines recommended for human isolates of methicillin resistant Staphylococcus aureus and coagulase negative staphylococci are unreliable in identifying MRSP. A cefoxitin breakpoint resistance of <=30 mm=resistant and >=31=susceptible has been proposed by Bemis et a, 2012. This study is in agreement that this breakpoint may be more reliable in predicting methicillin resistant Staphylococcus pseudintermedius. MRSP isolates are generally resistant to multiple antibiotic classes. Bacterial culture and antibiotic susceptibilities are therefore recommended for all suspect MRSP infections to allow appropriate selection of antibiotics. A limitation noted in this study is the apparent in vitro susceptibility of MRSP isolates to amoxicillin and cephalosporins (cephalothin and cefotetan). NCL812 was effective against all 23 isolates of both MSSP and MRSP. A larger scale study is warranted to confirm the effectiveness of NCL812 against Staphylococcus pseudintermedius as it may provide a safe alternative antibiotic option for emerging MRSP infections in domestic animals.
Example 7
Activity of NCL812 Against Gram-negative Organisms
The aim of this study was to determine if a target of the antibacterial activity of NCL812 is present within gram-negative cells. To determine if a target of NCL812 is within the gram-negative cell, the outer membrane, and most of the cell wall, was removed using ampicillin, then the modified cells (known as spheroplasts) were treated with NCL812 at various concentrations.
Induction of Spheroplast State
E. coli ATCC 25922 was grown overnight on agar at 37° C. Two colonies from the overnight incubation were used to inoculate  ̃20 ml of cation adjusted mueller hinton broth. Inoculated broth was incubated for 18 hours at 37° C. Six ml of overnight broth culture was added to 20 ml of supplemented cation adjusted mueller hinton broth (supplemented with 50 mg/ml ampicillin, 0.4 M sucrose, 8 mM MgSO4) and incubated overnight at 37° C. Formation of spheroplasts was checked using a phase contrast microscope.
Activity of NCL812
Three ml of spheroplast culture was added to each test tube and 50 μl of DMSO containing the appropriate concentration of NCL812 was added to each test tube. Fifty microliters of DMSO only was added to the control test tube. Spheroplasts were incubated for 24 hours with 20 μl samples taken at 0, 2, 4, 6, 8 and 24 hours. Twenty microliter samples were serially diluted 1:10 and 10 ul samples of appropriate dilutions were spotted in triplicate onto brain heart infusion agar. Brain heart infusion agar was incubated at 37° C. for 48 hours and colonies were counted at 24 and 48 hours to determine the number of colony forming units.
Imaging of NCL812 Treated Spheroplasts
Samples were taken after 24 hours of exposure to the NCL812 compound, stained with Trypan blue and imaged.
Results
A spheroplast induction rate above 99% was consistently observed. A target of NCL812 was found to be present within E. coli cells with a significant decrease in the number of colony forming units observed over time as the concentration of NCL812 increased ≧32 μg/ml (FIG. 40). The experiment was repeated in triplicate with one representative shown in FIG. 40. Images of the spheroplasts taken after 24 hours of exposure to the NCL812 compound showed the development of pleomorphic cells increasing in frequency as the concentration of NCL812 increased (data not presented). These results show the effectiveness of NCL812 as an antibacterial agent against gram-negative bacteria.
Example 8
Formulations of NCL812
The following formulations were prepared using standard methods in the art.
Formulation a—Topical Formulation—PEG-based Gel with NCL812
4.0 g PEG 4000;
3.5 g PEG 200;
0.6 propylene glycol;
1.9 g water; and
0.204 g of NCL812.
PEG 4000, PEG 200 and propylene glycol were mixed and heated to 150° C. and until all solid crystals were dissolved. NCL812 was added to water and sonicated for 30 minutes until fully suspended. The NCL812 solution and gel solutions were mixed and allowed to cool and solidify. Formulation A demonstrated acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.
Formulation B—Topical Formulation—PEG-based Gel with NCL812
3.0 g PEG 4000;
1.0 g PEG 8000;
3.0 g PEG 200;
1.0 g propylene glycol;
1.9 g water and
0.202 g of NCL812.
PEG 4000, PEG 8000, PEG 200 and propylene glycol were mixed and heated to 150° C. and until all solid crystals were dissolved. NCL812 was added to water and sonicated for 30 minutes until fully suspended. The NCL812 solution and gel solutions were mixed and allowed to cool and solidify. Formulation B demonstrated acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.
Formulation C—Topical Formulation—PEG-based Gel with NCL812-Soluplus
2.5 g PEG 4000;
4.0 g PEG 200;
2.5 g propylene glycol;
1.0 g water; and
1.8 g solid dispersion of NCL812-SoluPlus.
Soluplus was purchased from BASF (www.soluplus.com). NCL812-SoluPlus was prepared using standard methods in the art.
PEG 4000, PEG 200, NCL812-SoluPlus and propylene glycol were mixed and heated to 150° C. and until all solid crystals were dissolve. Water was added and then the solution was sonicated. The solution was allowed to cool and solidify. Formulation C demonstrated acceptable viscosity, ease of skin application, uniform suspension and consistent and fine texture.
Formulation D—Tablet Formulation
30 mg Calcium hydrogen phosphate dehydrate;
80 mg Microcrystalline cellulose;
50 mg Lactose;
8 mg Hydroxypropyl methyl cellulose
1.5 mg Talc
10 mg of NCL812
The excipients were weighed and mixed for 5 minutes. The mixture was fed into a feed hopper of a tablet press machine and the machine was operated according to standard procedures in the art.
Formulation D demonstrated acceptable tablet hardness, disintegration and frability.
Formulation E—Oral Suspension
2.0 ml Glycerol;
1.5 ml Absolute ethanol;
600 mg NCL812; and
To 60 ml Vehicle (Ora Sweet and Ora Plus, 1:1).
NCL 812 powder was sieved through a 75 μm sieve. 600 mg of sieved NCL 812 was mixed with 2.0 ml glycerol and 1.5 ml absolute ethanol. The mixture was placed in a mortar and manually milled until all NCL 812 was suspended uniformly. The suspension was sonicated for 30 minutes. Vehicle (55 ml of Ora Sweet and Ora Plus mixture) was then added to the suspension and milled for another 10 minutes. Volume was made up with the Ora plus and Ora sweet mixture to 60 ml by transferring to a measuring cylinder
Formulation E demonstrated acceptable suspension and demonstrated acceptable short term stability.
Formulation F—Intramuscular Injection
20 mg/ml Polyvinylpyrrolidone K30 (PVPK30);
0.09 mg/ml NCL812; and
50 ml water.
Two percent of w/v PVP K30 solution was prepared by the addition of 1.0 g of PVP K30 to 50 ml of MilliQ water. The solution was then placed in a sonicator for 30 minutes to equilibrate and 4.5 mg of NCL 812 was added to the PVP solution and placed on an incubator shaker at a maximum speed of 10 rpm over a period of 24 hours, with controlled temperature of 25±1° C. Solution was transferred to 5 ml vials and checked for clarity, appearance, pH and short-term stability. The pH of solution was 7.25.
Formulation F demonstrated acceptable transparency and short term stability.
Example 9
Release of NCL812 from Formulation B
The objective of this study was to measure the release of NCL812 from the Formulation B prepared in example
Franz diffusion cells were utilized to quantify the release rate of NCL 812 from its topical formulations. Five milliliters of absolute ethanol, which was chosen as the desired release medium, was loaded into the receptor chamber. Temperature of the receptor fluid was kept constant, at 32±1° C. using a water jacket. Acetyl cellulose membranes, with pore size of 0.45 um (Pall Corporation) was selected and placed between donor and receptor chamber. Followed by that, a number of test samples (Formulation B) were loaded into the donor chamber. One milliliter of receptor fluid was collected at regular time intervals of 0.25, 0.50, 0.75, 1, 2, 3, 4, 5, 6, 7, 8 and 24 hours through the sampling port. One milliliter of fresh absolute ethanol was immediately returned to the receptor chamber. UV-HPLC was utilized to analyse the content of the receptor fluids attained.
FIG. 41 presents the cumulative release of NCL812 over time. This study demonstrates that Formulation B provides an acceptable release profile for NCL812.
Example 10
Synergy Studies with Other Classes of Antimicrobial Agent
Methods
The checkerboard method (Gunics et al., 2000 Int. J. Antimicrob. Agents. 14:239-42) was used to find interactions (synergy, antagonism, no effect) of NCL812 in combination with tetracycline, chloramphenicol, erythromycin (macrolide), ampicillin (β-lactam broad-spectrum), gentamicin (aminoglycoside), ciprofloxacin (fluoroquinolone), sulfamethoxazole (sulphonamide), or penicillin G (β-lactam narrow-spectrum). For initial experiments, a laboratory strain of Staphylococcus aureus T3-129 was used, however this strain gave inconsistent results for some of the antimicrobials and a new strain of Staphylococcus spp. designated MK1 (definitive species identification currently in progress) that was sensitive to all tested antimicrobials was used in subsequent tests.
Firstly, the MIC of each antibiotic alone was determined in accordance to CLSI standard guidelines. Secondly, the combination of NCL812 with each of above antibiotics was tested in duplicate. To evaluate the effect of the combination the fractional inhibitory concentration (FIC) was calculated for each antibiotic as follows: FIC of tested antibiotic=MIC of tested antibiotic in combination/MIC of antibiotic alone; FIC of NCL812=MIC of NCL812 in combination/MIC of NCL812 alone; and FICI=FIC index=FIC of NCL812+FIC of each tested antibiotic.
According to the checkerboard guidelines, Synergy (S) was defined as an FICI<0.5. No effect (NE) was defined as 0.5<FICI<4. Antagonism (A) was defined as a 4<FICI.
Results
MICs, FICs, FICI and the interaction between NCL812 and eight antibiotics is shown in Table 14. None of the eight tested compounds, representing distinct classes of antimicrobial agent showed either positive (synergism) or negative (antagonism) interaction with NCL812 consistent with an additive effect when antibacterial agents are added to NCL812.







TABLE 14







MICs, FICs, FICI and the interaction between NCL812 and eight antibiotics.











Antibiotic
NCL812













MIC (μg/ml)

MIC (μg/ml)





















With


With






Antibiotic Name
Experiment
Repeat
NCL812
Alone
FIC1
Antibiotic
Alone
FIC2
FICI
Result




















Tetracycline1
1
1st
0.25
0.5
0.5
1
4
0.25
0.75
NE




2nd
0.25
0.5
0.5
1
8
0.125
0.62
NE



2
1st
0.031
0.25
0.125
4
8
0.5
0.625
NE




2nd
0.031
0.25
0.125
4
8
0.5
0.625
NE


Chloramphenicol1
1
1st
4
8
0.5
1
4
0.25
0.75
NE




2nd
2
4
0.5
2
8
0.25
0.75
NE



2
1st
4
8
0.5
2
8
0.25
0.75
NE




2nd
0.5
8
0.0625
4
8
0.5
0.562
NE


Erythromycin1
1
1st
0.031
0.125
0.25
2
4
0.5
0.75
NE




2nd
0.007
0.125
0.063
2
4
0.5
0.562
NE



2
1st
0.007
0.25
0.25
2
8
0.25
0.5
NE




2nd
0.007
0.25
0.031
4
8
0.5
0.531
NE


Ampicillin1
1
1st
0.125
0.25
0.5
1
4
0.25
0.75
NE




2nd
0.25
0.5
0.5
0.125
4
0.031
0.53
NE



2
1st
0.062
0.125
0.5
2
8
0.25
0.75
NE




2nd
0.125
0.25
0.5
2
8
0.25
0.75
NE


Gentamicin2
1
1st
0.062
0.125
0.5
0.5
4
0.125
0.625
NE




2nd
0.062
0.125
0.5
1
4
0.25
0.75
NE



2
1st
0.5
1
0.5
1
4
0.25
0.75
NE




2nd
0.007
0.5
0.0156
2
4
0.5
0.515
NE


Ciprofloxacin2
1
1st
0.062
0.125
0.5
2
4
0.5
0.75
NE




2nd
0.003
0.125
0.025
4
2
0.5
0.525
NE



2
1st
0.125
0.25
0.5
0.5
4
0.125
0.625
NE




2nd
0.125
0.25
0.5
0.25
4
0.0625
0.562
NE


Sulfamethoxazole2
1
1st
4
8
0.5
1
4
0.25
0.75
NE




2nd
4
8
0.5
2
4
0.5
1
NE



2
1st
4
8
0.5
1
4
0.25
0.75
NE




2nd
4
8
0.5
2
4
0.5
1
NE


Penicillin G2
1
1st
0.062
0.125
0.5
2
4
0.5
1
NE




2nd
0.062
0.125
0.5
2
4
0.5
1
NE



2
1st
0.062
0.125
0.5
2
4
0.5
1
NE




2nd
0.031
0.25
0.125
2
4
0.5
0.625
NE





1S. aureus strain T3-29


2Staphylococcus spp. Strain MK1


FIC1 = MIC of anitbiotic in combination with NCL812/MIC of antibiotic alone


FIC2 = MIC of NCL812 in combination with antibiotic/MIC of NCL812 alone


FICI = FIC index






Example 11
The Effects of NCL812 on Antimicrobial Sensitive Isolates of Staphylococcus aureus and Enterococcus faecalis 
Materials and Methods
Strain Information
Two Staphylococcus aureus isolates were used in the following experiments; S. aureus MK01 a human skin strain, and S. aureus KC01 an equine skin strain. These isolates were identified by Gram stain and biochemical methods, including the Remel Staphaurex commercial kit. One Enterococcus faecalis isolate (USA01), was not identified as a VRE strain. As this isolate has previously been speciated, it was not subjected to further testing, except for observation of pure, characteristic growth on blood agar.
Investigation of Minimum Bactericidal Concentration (MBC)
CLSI Methodology
As in previous experiments, 10 μL of the contents of each well starting at the MIC was inoculated on to a Columbia SBA plate and incubated at 37° C. for 48 h. Plates were examined at 24 and 48 h and the MBC was recorded as the lowest concentration of NCL812 at which no colonies of bacteria were observed on the plate (or significant inhibition of growth was observed compared to the control) (CLSI 2005).
Kill Kinetics Assays for S. aureus KC01 & E. faecalis USA01 Method
S. aureus KC01 and E. faecalis USA01, not determined to be MRSA or VRE, respectively, were grown overnight on Columbia SBA at 37° C. A few colonies of bacteria were then suspended in CAMHB (cation-adjusted Mueller Hinton broth) and adjusted to OD600 of 0.08 to 0.10. The bacterial suspension was diluted 1:10. One milliliter of the bacteria were added to 9 mL of CAMHB containing various concentrations (up to 4×MIC) of NCL, to achieve a final bacterial concentration of 1 to 3×106 CFU/mL. The tubes were incubated at 37° C., with constant shaking. In order to determine the number of viable bacteria present at various time points, a 100 μL aliquot was removed from each tube and diluted. Then, 100 μL of each dilution were spread onto colony count agar, in duplicate, and incubated for 48 h at 37° C. After 24 h the numbers of colonies present on each plate were counted and therefore the number of viable bacteria present in the original suspension enumerated. Plates were re-checked after 48 hours.
Results
Minimum Inhibitory Concentration (MIC)
The NCL812 MIC for isolates S. aureus MK01 and KC01, and E. faecalis USA01 was investigated. The results were: S. aureus MK01=4-8 μg/mL, S. aureus KC01=2 μg/mL, E. faecalis USA 01=4 μg/mL.
S. aureus isolates MK01 and KC01 were investigated and no growth, or growth only at low concentrations of NCL812 (2 μg/ml), was observed, indicating that NCL812 is bactericidal against S. aureus. For the E. faecalis isolate tested (USA01) however, growth of bacteria was observed at all concentrations of NCL812 tested. There was an obvious reduction in the number of bacteria with increasing concentration, but growth was present compared with no growth for S. aureus. A summary of these results can be seen in Table 15. Table 15 shows the results for NCL812 MBC tests on two non-MRSA S. aureus isolates and one non-VRE E. faecalis isolate. Each MBC test was performed in duplicate. No change in the results was observed at 48 h. Table 16 shows NCL812 MBC values (μg/mL) for 20 MRSA isolates. Each MBC test was performed in duplicate starting from NCL812 MIC concentration to 16 times of MIC. Table 17 shows NCL812 MBC values (μg/ml) for 10 VRE isolates. Each MBC test was performed in duplicate starting from NCL812 MIC concentration to 32 times the MIC.







TABLE 15







NCL812 MBC tests on two non-MRSA Staphylococcus aureus


isolates and one non-VRE Enterococcus faecalis isolate.









NCL812 MBC














Organism/
2
4
8
16
32
64
128


Sample No.
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml


















S. aureus
1st
+
0
0
0
0
0
0


(KC01)
2nd
+
+
+
0
0
0
0


S. aureus
1st
0 (5)
0
0
0 (N)
0 (N)
0 (N)
0 (N)


(MK01)
2nd
0
0
0
0
0
0
0


E. faecalis
1st
N
+ (488)
+
+
+ (7) 
+ (1) 
+


(USA01)
2nd
N
+
+
+
+
+
+





+ = Growth on Sheep Blood Agar;


0 = No Growth on Sheep Blood Agar;


N = Not Cultured;


Numbers in Parenthesis are the Number of Bacteria Growing after 24 hours per ml of sample (CFU/ml)













TABLE 16







NCL812 MBC values (μg/ml) for 20 MRSA isolates.









NCL812 MBC












Organism/
4
8
16
32
64


Sample No.
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml
















MRSA 1
- 1st
0
0
0
0
 N**



- 2nd
0
0
0
0
N


MRSA 2
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 3
- 1st
0
 GB*
0
0
N



- 2nd
0
0
0
0
N


MRSA 4
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 5
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 6
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 7
- 1st
0
0
0
0
N



- 2nd
0
GB
0
0
N


MRSA 8
- 1st
GB
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 9
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 10
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 11
- 1st
0
0
0
0
N



- 2nd
GB
0
0
0
N


MRSA 12
- 1st
0
0
0
0
N



- 2nd
0
GB
0
0
N


MRSA 13
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 14
- 1st
0
0
0
0
N



- 2nd
0
0
0
0
N


MRSA 516
- 1st
0
0
0
0
0



- 2nd
0
0
0
0
0


MRSA 570
- 1st
0
0
0
0
0



- 2nd
0
0
0
0
0


MRSA 580
- 1st
0
0
0
0
0



- 2nd
0
0
0
0
0


MRSA 606
- 1st
0
0
0
0
GB



- 2nd
0
0
0
0
0


MRSA 610
- 1st
0
0
0
0
0



- 2nd
0
GB
0
0
0





GB = Bacterial Growth on Sheep Blood Agar


N** = Not cultured on Sheep Blood Agar













TABLE 17







NCL812 MBC values (μg/ml) for 10 VRE isolates.









NCL812 MBC














Organism/
2
4
8
16
32
64
128


Sample No.
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml
μg/ml


















VRE
- 1st
90*
20
4000
M
M
M
M


26c(dc)
- 2nd
0
70
3500
M
M
M
M


VRE
- 1st
500 
100
20
250 
M
M
M


37c
- 2nd
M
50
100
1100 
1400 
M
M


VRE
- 1st
0
0
0
720 
 0
 0
 0


35t
- 2nd
0
0
0
 0
 10
 20
10


VRE
- 1st
90 
330
0
M
M
M
M


16c(dc)
- 2nd
200 
0
20
M
M
M
M


VRE
- 1st
0
120
20
10
M
M
M


23c
- 2nd
0
0
0
 0
570
M
M


VRE
- 1st
0
0
M
M
M
M
M


25c
- 2nd
20 
20
M
M
M
M
M


VRE
- 1st
10 
820
980
M
M
M
M


16c
- 2nd
M
790
890
M
M
M
M


VRE
- 1st
0
0
0
180 
 10
110
M


19t
- 2nd
30 
0
0
70
 40
M
M


VRE
- 1st
10 
0
10
 0
180
970
M


14t
- 2nd
0
0
0
40
780
M
M


VRE
- 1st
0
0
0
M
M
M
M


12c
- 2nd
0
M
300
M
M
M
M





*Number of bacteria growing after 24 hours per ml of sample (CFU/ml);


M = many bacteria growing on the plate (too many to count)






Kill Kinetics Assays for S. aureus KC01 & E. faecalis USA01 Method

Colony counts were performed at t=0, 120, 240, and 360 min, then again at 24 h. At the 2 h time point S. aureus KC01 showed a minimum of a 2.5 log10 reduction in bacterial numbers from initial numbers, and greater than a 3 log10 reduction in comparison to the control at the same time point. A minimum of a 2 log10 reduction was still evident at 6 h incubation, however after 24 h the numbers of bacteria present had increased and this was not significantly different to the control.
Similar results were obtained with E. faecalis USA01, however the reduction in bacterial numbers observed was less than for S. aureus KC01. A 2 log10 reduction in CFU/mL was observed at 2 h, compared to the growth control. However, the reduction in CFU/mL compared to the original bacterial numbers was only just greater than 1 log10. At concentrations of 4-16 μg/mL of NCL812 this reduction in bacterial numbers remained consistent until the 6 h time point. At concentrations of 32 and 64 μg/mL however, there was approximately a 1 log10 rise in bacterial numbers over the same time period. At 24 h bacterial numbers at all concentrations had increased to almost the same level as the growth control.
The results observed with these strains of S. aureus and E. faecalis are consistent with the results observed for the kill kinetics assay for all MRSA and VRE isolates tested. The kill kinetics assay of Staphylococcus aureus KC01 at different concentrations of NCL812, up to 24 h incubation are shown in FIG. 42. The kill kinetics assay of Enterococcus faecalis USA01 at different concentrations of NCL812, up to 24 h incubation are shown in FIG. 43.
Example 12
A Method of Treating Bacterial Infection In Vivo by the Administration of NCL812
The objective of this study was to determine the efficacy of an Investigational Veterinary Product containing NCL812 in the treatment of a skin infection in mice
Summary of the Model
A useful animal model system should be clinically relevant, experimentally robust, ethically acceptable, convenient to perform and should provide reliable and reproducible results. There are many animal models of topical skin infection that have been described including the croton oil-inflamed skin model (Akiyama, H., H. Kanzaki, Y. Abe, J. Tada and J. Arata (1994). “Staphylococcus aureus infection on experimental croton oil-inflamed skin in mice.” Journal of Dermatological Science 8(1): 1-10), the burnt skin model (Stieritz, D. D., A. Bondi, D. McDermott and E. B. Michaels (1982). “A burned mouse model to evaluate anti-pseudomonas activity of topical agents.” Journal of Antimicrobial Chemotherapy 9(2): 133-140), the skin suture-wound model (McRipley, R. J. and R. R. Whitney (1976). “Characterization and Quantitation of Experimental Surgical-Wound Infections Used to Evaluate Topical Antibacterial Agents.” Antimicrobial Agents and Chemotherapy 10(1): 38-44), the skin tape-stripping model (Kugelberg, E., T. NorstrOm, T. K. Petersen, T. Duvoid, D. I. Andersson and D. Hughes (2005). “Establishment of a Superficial Skin Infection Model in Mice by Using Staphylococcus aureus and Streptococcus pyogenes.” Antimicrobial Agents and Chemotherapy 49(8): 3435-3441) and the linear full thickness scalpel cut method (Guo, Y., R. I. Ramos, J. S. Cho, N. P. Donegan, A. L. Cheung and L. S. Miller (2013). “In Vivo Bioluminescence Imaging To Evaluate Systemic and Topical Antibiotics against Community-Acquired Methicillin-Resistant Staphylococcus aureus-Infected Skin Wounds in Mice.” Antimicrobial Agents and Chemotherapy 57(2): 855-863).
Preliminary studies prior to the conduct of the current study established a new method of skin infection arising from a detailed study of the models mentioned above. Briefly, study mice are anaesthetised, a patch of dorsal skin is clipped to reveal the skin and a circular area of skin is removed with a hand held punch, leaving a wound on the dorsum with a central cavity. The wound is infected with a known number of the challenge organism. Approximately four to six hours after infection, the wound is either treated topically with a vehicle formulation or an active formulation. The infected skin wound is retreated every 12 hours for a total of 14 treatments. Mice are humanely euthanased, the area of the original infected wound is dissected and removed and its bacterial content quantified by standard microbiologic tests. In this way, the change in bacterial concentration due to treatment with the active formulation can be readily determined by examining the reduction in bacterial burden compared with the vehicle control.
Materials and Methods
Preparation of Infection Inoculum
Fresh cultures of bacteria (Staphylococcus aureus) were grown on Sheep Blood Agar at 37° C. for 16-18 hours. A few typical colonies were selected and suspended in 10 ml of Tryptic Soy Broth and incubated overnight in a shaking incubator (240 rpm) at 37° C. The overnight suspension was vortexed and diluted (1:100) in fresh Tryptic soy broth (100 μl [0.1 ml] in 9.9 ml broth). The fresh suspension was incubated for 3 hours in a shaking incubator (as above) in order to obtain mid-logarithmic phase bacteria. Bacteria were pelleted through centrifugation at 7,500 rpm for 10 mins. Broth supernatant was removed and bacteria suspended in 10 ml Phosphate Buffered Saline (PBS). These steps were repeated a further two times. The density of the suspension was checked by measuring absorbance at 600 nm, using a spectrophotometer with saline as a blank, to confirm the target density at a reading of approximately 0.100, consistent with a bacterial density of 2.5×107 CFU/ml. The suspension was placed into a rack placed into a lockable transport box with an ice brick to maintain refrigeration during transport, followed by storage in cool room upon arrival at the mouse skin infection laboratory. Final suspension was mixed thoroughly before inoculating the skin wounds created in mice.
In order to ensure the purity and accuracy of the suspension, the following steps were performed prior to placement into lock box.
Purity of bacterial suspension ensured by spreading 100 μl of the final suspension onto a SBA (sheep blood agar) plate which was incubated at 37° C. for 18 hours and examined to confirm uniform growth of one colony type. Viable counts were performed on final suspension by prepping saline in Eppendorf tubes (approximately 900 ul per tube), removing 100 μl sample and adding to first Eppendorf tube, vortexing the mixture and repeating using 2nd Eppendorf tube containing saline. This process was continued for 5-6 tubes. Finally, 100 μl of 5th and 6th dilutions were plated out on plate count agar, incubated at 37° C. for 18 hours and colony counts performed to confirm that the CFU/ml was approximately 2.5×107. Following inoculation of the wounds, this process was repeated to ensure that no contamination or decrease in viable counts had occurred during the time of the surgery.
Skin Wound Surgical Procedure
Each mouse was placed into induction chamber and anaesthesia induced using 2% isoflurane. Eyes of each anaesthetised mouse were covered with veterinary eye lubricant in order to prevent corneal dehydration. Each mouse removed from induction chamber and placed onto surgical area, in front of individual aesthetic nose cone. While under anaesthesia each mouse was monitored for assessment of depth of anaesthesia (response to pain, blink reflex, skeletal muscle tone) and respiratory and cardiac function. Back skin hair was shaved from surgical area with mechanical clippers. Shaved area was cleaned using 70% ethanol applied to paper towel followed by 10% w/v povidone-iodine solution. Once the iodine solution was dry, a subcutaneous injection of the nonsteroidal anti-inflammatory agent meloxicam was administered. Dorsal skin was pinched gently to allow creation of a circular full-thickness wound using ear punch/biopsy punch. Vehicle control and NCL812 treated mice had wounds inoculated with 10 μl of bacterial suspension using a micropipette (2.5×105 CFU/10 μl). Once the bacterial suspension was dry, mice were placed into individual recovery boxes labelled with the mouse number. The time of inoculation was recorded. Initial body weights of each mouse were recorded on the appropriate score sheet. Mice recovered to full consciousness within 5 minutes. Recovered mice were returned to individual housing and monitored hourly for post-surgical or anaesthetic complications.
Post-Surgical Care (4 Hours Post-surgery)
Mice were assessed for post-surgical complications and observations were recorded on clinic record sheet. Each mouse was carefully removed from IVC and placed into an assessment container, avoiding excessive handling or touching of the surgical site. Once the mouse was inside assessment container, it was assessed and the observations recorded on the post-surgical clinical record sheet. Whenever the suggested wellness breakpoints were reached, post-operative analgesia was administered and recorded on the clinical record sheet.
Animal Monitoring and Daily Care
Antibiotic Administration (7 am and 6 pm). The first administration of vehicle or NCL812 ointment occurred 4 hours post-surgically. Each ointment container was weighted prior to administration and the weight recorded. Each mouse was carefully restrained. Ointment (vehicle or NCL812) was applied to the lesion area and the treated mouse was returned to IVC where each mouse was observed to ensure ointment was not immediately removed by grooming. The weight of the ointment container post-administration was recorded. The vehicle and active NCL products 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 Example 8) contained robenidine at a concentration of 20 mg/g. Approximately 0.1-0.2 g of ointment was applied on each occasion, delivering a total topical dose of NCL812 between 28 and 56 mg to mice weighing between 18 g and 25 g.
Daily Monitoring. Monitoring of each mouse took place once daily at around 12 pm. Each mouse carefully removed from IVC and placed into observation container, avoiding excessive handling or touching surgical site. The coat, posture, eyes, behaviour, vocalisation and activity whilst in the container were carefully assessed and observations recorded on assessment sheet. Mouse faeces (either on floor of cage or in container) were checked for consistency and observations recorded. The weight of each mouse was determined whilst it was in the container and change in body weight calculated and recorded. The observation container was disinfected with ethanol and set aside to dry while a fresh container was used for the next mouse. For every second day, mice were again anaesthetised using 2% isoflurane and photographed using a ruler for size referencing. These photos were used to assess lesion size and infection progression during the trial period.
Tissue Analysis and Assessment of Antibacterial Efficacy
At the end of the 7 day skin wound assessment period, all test mice were euthanased prior to wound collection for post mortem examination. The skin wound was dissected from the dorsum of each mouse. The sample was placed in a sample tube and weighed before 1 ml PBS and sterile tissue homogenisation beads were added. Tissue samples were homogenised for 10 mins using a tissue homogeniser (Next Advance Bulet Blender) and then vortexed for approximately 30 seconds. 100 μl of supernatant was removed and placed into an Eppendorf tube containing 900 μl of PBS. This procedure was repeated using serial dilutions for a total of 8 dilutions. Finally, 100 μl of each dilution was pipetted onto a plate count agar in duplicate and incubated overnight at 37° C. Ten microliters of original suspension was placed onto sheep blood agar to assess culture purity and incubated overnight at 37° C. The following day, viable counts were performed using incubated plate count agar plates and the identity of staphylococcus aureus (the challenge organisms) as the harvested strain was confirmed.
Results
The mean colony count per gram of tissue observed in vehicle treated group was 5,888,436 (6.77 log 10). The mean colony count per g of tissue observed in NCL812 group was 141,254 (5.15 log 10). The log 10 colony forming units per gram of tissue and % reduction are summarised in the following table.







TABLE 15







Log10 colony forming units per gram of tissue and percentage reduction


following topical administration of vehicle and treatment











Treatment
Log10(CFU/g)
% reduction







Vehicle
6.77




NCL812
5.15
97.6










It is clear from this table that treatment with NCL812 leads to high level reduction in the number of infecting Staphylococcus aureus. These results demonstrate effective treatment of a bacterial colonisation or infection in vivo.

## Claims

1. A method of treating or preventing a bacterial colonization or infection by a bacterial agent in a subject, the method comprising the step of:
administering to the subject a therapeutically effective amount of robenidine or a therapeutically acceptable salt thereof, wherein the bacterial agent is selected from the group consisting of: Mycoplasma spp, Ureaplasma spp, and Mycobacterium spp wherein the robenidine or a therapeutically acceptable salt thereof is administered to the subject at a dose in the range of 0.1 mg/kg to 250 mg/kg bodyweight.

2. The method according to claim 1, wherein the subject is selected from the group consisting of: human, canine, feline, bovine, ovine, caprine, porcine, avian, piscine and equine species.

3. The method according to claim 1, wherein the bacterial agent is selected from the group consisting of Mycoplasma spp, including Mycoplasma agalactiae, Mycoplasma alkalescens, Mycoplasma amphoriforme, Mycoplasma arginini, Mycoplasma bovigenitalum, Mycoplasma bovirhinis, Mycoplasma bovis, Mycoplasma bovoculi, Mycoplasma buccale, Mycoplasma californicum, Mycoplasma canadense, Mycoplasma capricolum subsp. capricolum, Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma conjunctivae, Mycoplasma cynos, Mycoplasma dispar, Mycoplasma equigenitalium, Mycoplasma faucium, Mycoplasma felis, Mycoplasma fermentans(incognitus str.), Mycoplasma gallisepticum (MG), Mycoplasma gateae, Mycoplasma genitalium, Mycoplasma haemocanis, Mycoplasma haemofelis, Mycoplasma haemosuis(formerly Eperythrozoon suis), Mycoplasma hominis, Mycoplasma hyopneumoniae, Mycoplasma hyorhinis, Mycoplasma hyosynoviae, Mycoplasma iowae meleagridis (MM), Mycoplasma iowae, Mycoplasma leachii, Mycoplasma lipophilum, Mycoplasma meleagridis, Mycoplasma mycoidessubsp capri, Mycoplasma mycoides subsp mycoides, Mycoplasma mycoides subsp. mycoides (such as Contagious bovine pleuropneumonia CBPP), Mycoplasma orale, Mycoplasma ovipneumoniae, Mycoplasma ovis, Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma pneumoniae, Mycoplasma primatum, Mycoplasma putrefaciens, Mycoplasma salivarium, Mycoplasma spermatophilum, Mycoplasma suis, Mycoplasma synoviae (MS), Mycoplasma wenyonii.

4. The method according to claim 1, wherein the bacterial agent is selected from the group consisting of Ureaplasma spp, including Ureaplasma parvum, Ureaplasma urealyticum, Ureaplasma, and Ureoplasma diversum.

5. The method according to claim 1, wherein the bacterial agent is selected from the group consisting of Mycobacterium spp, including Mycobacterium abscessus, Mycobacterium arupense, Mycobacterium asiaticum, Mycobacterium aubagnense, Mycobacterium avium complex, Mycobacterium bolletii, Mycobacterium bolletii, Mycobacterium bovis, Mycobacterium branderi, Mycobacterium canettii, Mycobacterium caprae, Mycobacterium celatum, Mycobacterium chelonae, Mycobacterium chimaera, Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacterium conspicuum, Mycobacterium elephantis, Mycobacterium farcinogenes, Mycobacterium florentinum, Mycobacterium fortuitum group, Mycobacterium genavense, Mycobacterium goodii, Mycobacterium haemophilum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium houstonense, Mycobacterium immunogenum, Mycobacterium interjectum, Mycobacterium intracellulare, Mycobacterium senegalense, Mycobacterium africanum, Mycobacterium avium subsp paratuberculosis, Mycobacterium kansasii, Mycobacterium lacus, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium mageritense, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium massiliense, Mycobacterium microti, Mycobacterium montefiorense (eels), Mycobacterium moracense, Mycobacterium mucogenicum, Mycobacterium nebraskense, Mycobacterium neoaurum, Mycobacterium novocastrense, Mycobacterium palustre, Mycobacterium parmense, Mycobacterium phlei, Mycobacterium phocaicum, Mycobacterium pinnipedii, Mycobacterium porcinum, Mycobacterium pseudoshottsii (fish), Mycobacterium pseudotuberculosis, Mycobacterium saskatchewanense, Mycobacterium scrofulaceum, Mycobacterium senuense, Mycobacterium septicum, Mycobacterium simiae, Mycobacterium smegmatis, Mycobacterium szulgai, Mycobacterium terrae/chromogenicum complex, Mycobacterium triplex, Mycobacterium tuberculosis, Mycobacterium tusciae, Mycobacterium ulcerans, Mycobacterium wolinskyi, and Mycobacterium xenopi.

6. The method according to claim 1, wherein the therapeutically effective amount of robenidine or a therapeutically acceptable salt thereof is administered to the subject by oral, parenteral or topical administration.