For 70 years antibiotics have saved countless lives and enabled the development of modern medicine, but it is becoming clear that the success of antibiotics may have only been temporary and we now anticipate a long-term, generational and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. As the search for new conventional antibiotics has become less productive and there are no clear strategies to improve success, a broader approach to address bacterial infection is needed. This review of potential alternatives to antibiotics (A2As) was commissioned by the Wellcome Trust, jointly funded by the Department of Health, and involved scientists and physicians from academia and industry. For the purpose of this review, A2As were defined as non-compound approaches (that is, products other than classical antibacterial agents) that target bacteria or approaches that target the host. In addition, the review was limited to agents that had potential to be administered orally, by inhalation or by injection for treatment of systemic/invasive infection. Within these criteria, the review has identified 19 A2A approaches now being actively progressed. The feasibility and potential clinical impact of each approach was considered. The most advanced approaches (and the only ones likely to deliver new treatments by 2025) are antibodies, probiotics, and vaccines now in Phase II and Phase III trials. These new agents will target infections caused by P. aeruginosa, C. difficile and S. aureus. However, other than probiotics for C. difficile, this first wave will likely best serve as adjunctive or preventive therapies. This suggests that conventional antibiotics will still be needed. The economics of pathogen-specific therapies must improve to encourage innovation, and greater investment into A2As with broad-spectrum activity (e.g. antimicrobial-, host defense-and, anti-biofilm peptides) is needed. Increased funding, estimated at >£1.5 bn over 10 years is required to validate and then develop these A2As. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches at Clinical Phase II proof of concept. Such an approach could transform our understanding of A2As as effective new therapies and should provide the catalyst required for both active engagement and investment by the pharma/biotech industry. Only a sustained, concerted and coordinated international effort will provide the solutions needed for the next decade.

The DNA in dormant spores of Bacillus species is saturated with a group of nonspecific DNA-binding proteins, termed oa/13-type small, acid-soluble spore proteins (SASP). These proteins alter DNA structure in vivo and in vitro, providing spore resistance to UV light. In addition, heat treatments (e.g., 850C for 30 min) which give little killing of wild-type spores of B. subtilis kill >99%o of spores which lack most a/13-type SASP (termed a-1-spores). Similar large differences in survival of wild-type and a-13-spores were found at 90, 80, 65, 22, and 10'C. After heat treatment (850C for 30 min) or prolonged storage (220C for 6 months) that gave >99%o killing of at-13-spores, 10 to 20%o of the survivors contained auxotrophic or asporogenous mutations.However, at-A-spores heated for 30 min at 850C released no more dipicolinic acid than similarly heated wild-type spores (<20%o of the total dipicolinic acid) and triggered germination normally. In contrast, after a heat treatment (930C for 30 min) that gave .99%6 killing of wild-type spores, <1% of the survivors had acquired new obvious mutations, >85% of the spore's dipicolinic acid had been released, and <1% of the surviving spores could initiate spore germination. Analysis of DNA extracted from heated (850C, 30 min) and unheated wild-type spores and unheated cC 1 spores revealed very few single-strand breaks (< 1 per 20 kb) in the DNA. In contrast, the DNA from heated a-0-spores had more than 10 single-strand breaks per 20 kb.These data suggest that binding of at/,3-type SASP to spore DNA in vivo greatly reduces DNA damage caused by heating, increasing spore heat resistance and long-term survival. While the precise nature of the initial DNA damage after heating of 13-spores that results in the single-strand breaks is not clear, a likely possibility is DNA depurination. A role for oc/13-type SASP in protecting DNA against depurination (and thus promoting spore survival) was further suggested by the demonstration that these proteins reduce the rate of DNA depurination in vitro at least 20-fold. Britain.tures to those at lower temperatures suggests that spores should be able to survive for years at common environmental temperatures (5, 24). Indeed, there are reports of spore survival over thousands of years (although obviously no details on spore population survival) (34). If spores are to survive for these extended times, their DNA must somehow be protected against the accumulation of potentially lethal damage during these periods, as the absence of metabolism and ATP in dormant spores precludes DNA repair (29,30,32). As noted above, spore DNA is protected against damage by UV radiation through the binding of a/1-type SASP. Other possible types of DNA damage are oxidative damage to bases and base loss through depurination; either of these types of DNA damage can lead to a mutagenic or lethal change in the DNA (7,10).DNA depurination appears to be of special significance, as this process is water catalyzed at neutral pH, with the rate of depurination rising dramaticall...

Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues. alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT). While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes. UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct. Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed. These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation.

Wild-type spores of BaciUlus subtilis were resistant to eight cycles of freeze-drying, whereas about 901% of spores lacking the two major DNA-binding proteins (small, acid-soluble proteins a and I8) were killed by three to four cycles of freeze-dryings, with significant mutagenesis and DNA damage accompanying the killing. This role for a/p-type small, acid-soluble proteins in spore resistance to freeze-drying may be important in spore survival in the environment. Dormant spores of Bacillus species are much more resistant than their growing counterparts to a variety of treatments, including heat, UV radiation, and chemicals such as hydrogen peroxide (6-8, 13). While many factors are involved in spore resistance to heat and hydrogen peroxide (6, 11, 13), one common factor is the saturation of spore DNA with a group of a/,-type small, acid-soluble proteins (a/,B-type SASP) (11, 13). These proteins are made in the forespore late in sporulation in amounts sufficient to completely cover the spore chromosome but are degraded in the first minutes of spore germination (12, 13). B. subtilis spores lacking the two major a/n-type SASP (termed o-4spores) are significantly more heat and hydrogen peroxide sensitive than are wild-type spores (5, 11). Studies

Antibiotic resistance is a global problem, and with bacteria having developed resistance to all approved antibacterial agents there is a growing need for innovative solutions. Phico Therapeutics has developed a new class of antibacterial agent, a platform technology called SASPject. SASPject comprises modified, disabled bacterial viruses (bacteriophages) injecting a gene encoding an antibacterial protein, SASP, into target bacteria. SASP, or Small, Acid-soluble Spore Protein(s), inactivate bacterial DNA in a non-sequence-specific manner so their activity is unaffected by DNA mutations. Selected pathogens can be targeted, avoiding the normal flora. A Staphylococcus aureus-targeted SASPject, PT1.2, developed for the nasal decolonization of S. aureus, including methicillin-resistant (MRSA) strains, is expected to complete phase I in 2009. SASPject PT1.2 shows good in vitro activity against a wide range of diverse clinical S. aureus isolates, including MRSA strains. A systemic SASPject PT1.2, and SASPjects targeted against Clostridium difficile and multidrug-resistant Gram-negative organisms are in development. The SASPject technology could represent a new paradigm in antibacterial therapeutics.