Antimicrobial peptides are key effector molecules of the innate immune system and integral components of the first line of defence against microbial infections of all eukaryotic organisms. A number of prokaryotic organisms also utilise antimicrobial peptides as means to compete against challenge from other microorganisms. Many antimicrobial peptides are characterised by cationic properties that facilitate interactions with the negatively charged phospholipids of the microbial membrane which then lead to microbial lysis and death following membrane permeabilisation. For example, it has been shown that antimicrobial peptide molecules can aggregate and form voltage dependent channels in the lipid bilayer resulting in the permeabilisation of both the inner and outer membrane of the microorganism (Lehrer, R. I., J. Clin. Investigation, 84:553 (1989)). The amphiphilic nature of these molecules may also facilitate the insertion of the hydrophobic residue into the lipid bilayer by electrostatic attraction while the polar residues project into and above the membrane.
Drug resistant microorganisms, especially bacteria, are becoming increasingly problematic as infection rates continue to rise and effective methods of control become more and more limited. Prolific use of antibiotics over the last 50 or so years, together with the indiscriminate prescribing of antibiotics and patient non-compliance with treatment regimes, has selected for microorganisms that have developed or acquired means of overcoming the effects of antibiotics. The transmission and control of drug-resistant organisms is becoming one of the most significant problems within healthcare.
All Gram positive genera, including Staphylococcus spp., Enterococcus spp., Listeria spp., Clostridium spp., Corynebacterium spp., Nocardia spp., Bacillus spp. and Streptococcus spp., including those that have developed or obtained varying levels of resistance to antibiotics such as methicillin (meticillin), are of particular interest as are the Gram negative genera Escherichia spp., Pseudomonas spp., Klebsiella spp. and Acetinobacter spp. Other Gram negative pathogens of interest include the Enterobacteriaceae (especially those producing either extended-spectrum β-lactamase (ESBL) or carbapenemase). Coagulase-negative Staphylococci, such as S. epidermidis, have also emerged as important drug-resistant nosocomial pathogens. The treatment options for infections contributed to or caused by methicillin or multi-drug resistant bacteria are now limited and there is an urgent need to discover new therapies which inhibit or kill such organisms. Other bacterial pathogens of particular interest include Mycobacterium spp., e.g. Mycobacterium tuberculosis; Enterobacter spp.; Campylobacter spp.; Salmonella spp.; Helicobacter spp., e.g. Helicobacter pylori; Neisseria spp., e.g. Neisseria gonorrhea, Neisseria meningitidis; Borrelia burgdorferi; Shigella spp., e.g. Shigella flexnerii; Haemophilus spp., e.g. Haemophilus influenzae; Chlamydia spp., e.g. Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci; Francisella tularensis; Yersinia spp., e.g. Yersinia pestis; Treponema spp.; Burkholderia spp.; e.g. Burkholderia mallei and B. pseudomallei. 
Pseudomonas aeruginosa is an opportunistic pathogen that causes, respiratory tract infections, urinary tract infections, dermatitis, soft tissue infections, bacteraemia and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed. Respiratory infections caused by Pseudomonas aeruginosa occur almost exclusively in individuals with a compromised lower respiratory tract or a compromised systemic defence mechanism (for example in patients with cystic fibrosis or chronic obstructive pulmonary disease). Primary pneumonia occurs in patients with chronic lung disease and congestive heart failure. Bacteraemic pneumonia commonly occurs in neutropenic cancer patients undergoing chemotherapy. Lower respiratory tract colonisation of cystic fibrosis patients by mucoid strains of Pseudomonas aeruginosa is common and difficult to treat. There is a need to develop an effective means of treating Pseudomonas aeruginosa infections.
Staphylococcus aureus is an opportunistic pathogen that is normally encountered on the skin and in the nose of many healthy people where it lives completely harmlessly. S. aureus can, however, cause problems when it is able to enter the body causing abscesses, boils, pimples, impetigo and wound infections, whether accidental or surgical. If the infection gets into the bloodstream and travel to different parts of the body it can cause blood poisoning (septicaemia), bone infection (osteomyelitis), heart valve infection (endocarditis) and lung infection (pneumonia). MRSA is a type of S. aureus that is resistant to many commonly prescribed antibiotics, including methicillin (˜40% of S. aureus infections in the UK are resistant to methicillin and other antibiotics), and is commonly referred to in the popular press as a “superbug”. MRSA is one of the most prevalent microbes involved with healthcare-associated infections. Infections are normally confined to hospitals, and in particular to vulnerable and/or debilitated patients, including patients in intensive care units, burns units and orthopaedic wards. MRSA is more difficult to treat because many antibiotics are ineffective, and those that are effective often need to be given at much higher doses, intravenously, over prolonged periods of time (several weeks) thereby highlighting the need to develop alternative antimicrobial therapies.
Since microbial pathogens do not readily acquire resistance to cationic peptides, despite evolutionary pressure from millions of years of co-existence, they remain attractive therapeutic targets. In our co-pending applications, WO 2006/018652 and WO 2008/093058, we describe the identification of peptides that can be used to treat microbial infections, including bacterial infections.
A microbial biofilm is a community of microbial cells embedded in an extracellular matrix of polymeric substances and adherent to a biological or a non-biotic surface. A range of microorganisms (bacteria, fungi, and/or protozoa, with associated bacteriophages and other viruses) can be found in these biofilms. Biofilms are ubiquitous in nature and are commonly found in a wide range of environments. Biofilms are being increasingly recognised by the scientific and medical community as being implicated in many infections, and especially their contribution to the recalcitrance of infection treatment.
Biofilm formation is not limited solely to the ability of microbes to attach to a surface. Microbes growing in a biofilm are able to interact more between each other than with the actual physical substratum on which the biofilm initially developed. For example, this phenomenon favours conjugative gene transfer, which occurs at a greater rate between cells in biofilms than between planktonic cells. This represents an increased opportunity for horizontal gene transfer between bacteria, and is important because this can facilitate the transfer of antibiotic resistance or virulence determinant genes from resistant to susceptible microbes. Bacteria can communicate with one another by a system known as quorum sensing, through which signalling molecules are released into the environment and their concentration can be detected by the surrounding microbes. Quorum sensing enables bacteria to co-ordinate their behaviour, thus enhancing their ability to survive. Responses to quorum sensing include adaptation to availability of nutrients, defence against other microorganisms which may compete for the same nutrients and the avoidance of toxic compounds potentially dangerous for the bacteria. It is very important for pathogenic bacteria during infection of a host (e.g. humans, other animals or plants) to co-ordinate their virulence in order to escape the immune response of the host in order to be able to establish a successful infection.
Biofilm formation plays a key role in many infectious diseases, such as cystic fibrosis and periodontitis, in bloodstream and urinary tract infections and as a consequence of the presence of indwelling medical devices. The suggested mechanisms by which biofilm-associated microorganisms elicit diseases in their host include the following: (i) delayed penetration of the antimicrobial agent through the biofilm matrix, (ii) detachment of cells or cell aggregates from indwelling medical device biofilms, (iii) production of endotoxins, (iv) resistance to the host immune system, (v) provision of a niche for the generation of resistant organisms through horizontal gene transfer of antimicrobial resistance &/or virulence determinant genes, and (vi) altered growth rate (i.e. metabolic dormancy) (Donlan and Costerton, Clin Microbiol Rev 15: 167-193, 2002; Parsek and Singh, Annu Rev Microbiol 57: 677-701, 2003; Costerton J W, Resistance of biofilms to stress. In ‘The biofilm primer’. (Springer Berlin Heidelberg). pp. 56-64.2007).
Recent experimental evidence has indicated the existence within biofilms of a small sub-population of specialized non-metabolising persister cells (dormant cells). It is thought that these cells may be responsible for the high resistance/tolerance of biofilm to antimicrobial agents. Multi-drug-tolerant persister cells are present in both planktonic and biofilm populations and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to this sub-population. The protection offered by the polymeric matrix allows persister cells to evade elimination and serve as a source for re-population. There is evidence that persisters may be largely responsible for the multi-drug tolerance of microbial biofilms (LaFleur et al., Antimicrob Agents Chemother. 50: 3839-46, 2006; Lewis, Nature Reviews Microbiology 5, 48-56 2007).
There is a requirement, therefore, for further agents that can be used to treat microbial infections. In particular, there remains a pressing need for further antimicrobial actives that can be used in the treatment of bacterial infections such as those caused by Staphylococci, Streptococci, Acinetobacter spp., Klebsiella spp., E. coli and Pseudomonas spp. There is also an urgent requirement for better therapies for preventing biofilm formation and treating conditions associated with microbial biofilms.
The present inventors have identified peptides that, surprisingly, have improved antimicrobial activity over natural antimicrobial peptides, such as the defensins, cathelicidins, etc. The claimed compounds have potent antimicrobial properties, whilst exhibiting low toxicity in vitro and in vivo to animals and humans.