Interpretación y reporte de resultados

A concerted effort by society to intervene with the emergence and spread of antibiotic resistance is paramount; failure to do so could cause a reversion back to the pre-World War II era when life-threatening diseases were untreatable. Emphasis should be targeted towards methods to conserve the efficacy and prolong the lifespan of existing antibiotics (relying on the response by healthcare organisations) and providing new tactics for combating resistance mechanisms (identifying novel targets and classes of antibiotics). With the decline of investment in antimicrobial design by the pharmaceutical industry and the prevalence of resistance mechanisms, antimicrobial agents are and should be considered a precious and finite source.

1.3.3.1. Healthcare policy

Healthcare organisations represent a key area for the control of antibiotic resistance development and dissemination. The prudent use of current drugs, prevention of unnecessary prescriptions and complete dismissal of antimicrobials from treatment programs could decrease the occurrence of resistance. Frequent surveillance of administrated drugs can correlate usage with the incidence of resistance. Thus, knowledge of local antibiotic susceptibilities is vital to ensure the appropriate therapy is prescribed (Masterton, 2008).

Healthcare environments provide a reservoir for antibiotic resistant bacteria. Various strategies have been implemented to tackle the spread of resistance. Statistics provided by the National Audit Office (2009) have shown that over a four year period (2004-2008), a predicted £57 million was spent on national initiatives to deal with healthcare associated infections.

A new campaign, supported by the Department of Health, has been promoted across Europe: the European Antibiotic Awareness Day (EAAD). This is a single day aiming to build on the public awareness of the antibiotic resistance threat, alerting the need for prudent and responsible use of antibiotics.

1.3.3.2. Development of new antibiotics and strategies

1.3.3.2.1.Traditional approaches to identify novel antibiotics

Over the past 60 years, natural products have been the focal point in the discovery and development of new antimicrobial agents. Early methods of identifying antibiotics from natural sources, such as soil bacteria, were hindered by the high volume screening strategies and the rediscovery of established classes of antibiotics: 22 % of soil actinomycetes produce streptothricin (Taylor and Wright, 2008). However, modern detection methods, including mass spectrometry, allow for high- resolution separation and analysis of small molecules from the environment (Davies, 2011). It has been predicted that merely 1-10 % of the metagenome has been sampled, thus a plethora of undiscovered molecules remain to be identified (Walsh, 2003).

Optimisation of established antibiotic structures is a common approach to derive new antimicrobial active compounds. Using existing scaffolds, modifications including attachment, removal or substitution of functional groups can provide many alternatives to a common structure with desirable pharmacological properties (von Nussbaum et al., 2006). These alterations are often used to replace and consequently enhance the efficacy of natural products and represent the semisynthetic group of antibiotics. Between 1983 and 1994, 75 % of compounds were variants of natural products (Newman et al., 2003), thus filling the void generated by the absence of new structural classes of antibiotics.

1.3.3.2.2. Bioinformatics and genomics

Following the first report of the complete bacterial genome of Haemophilus influenzae in 1995 (Fleischmann et al., 1995), bioinformatics and genomics have been valuable tools in antibacterial drug discovery. Genome sequencing has allowed the identification of gene clusters involved in the biosynthesis of small compounds. From this analysis, it has been revealed that individual species of actinomycetes are capable of producing more than 20 secondary metabolites, some of which may have antibacterial activity (Baltz, 2008).

Genomics can also be exploited to discover new antimicrobial targets (McDevitt and Rosenberg, 2001). Various studies, such as gene disruption, deletion and over- expression, have identified essential genes for bacterial viability within a genome. In

E. coli, it has been established that there are approximately 300 essential genes, which represent approximately 7 % of the genome (Gong et al., 2008; Taylor and Wright, 2008). Comparisons with eukaryotic genomes can identify bacterial genes that do not share substantial homology, and consequently could present novel antimicrobial targets (Taylor and Wright, 2008).

1.3.3.2.3. High throughput screening

High-throughput screening involves a library of thousands of potential inhibitors, which are screened against a drug target. The efficacy of this process at identifying antimicrobials is illustrated with the following example. GlaxoSmithKline utilised the high-throughput technique to screen 67 antibiotic targets with ~530,000 small molecules. The structure of only five candidates were pursued, none of which passed through clinical trials (Payne et al., 2007).

Conventional high-throughput screening methods use libraries containing natural products, often large complex molecules, which are less likely to successfully interact with a specified target. These libraries are now being replaced by molecules of 250 Da or less. These smaller and structurally simpler ‘fragments’ are more accessible for target binding (Waldrop, 2009).

1.3.3.2.4.Virtual screening

Virtual screening is a computational technique used to identify compounds of interest from chemical databases using two different approaches. The ligand-based method uses the structures of active ligands to retrieve similar compounds, which could interact with a target in an analogous manner. The receptor-based approach employs the 3D structure of a target to screen for compounds that bind in an equivalent or dissimilar way to known ligands. The increasing availability of 3D structures of drug targets makes virtual screening a valid technique to rapidly identify lead compounds (Agarwal and Fishwick, 2010).

1.3.3.2.5.Rational and de novo drug design

Rational drug design is a targeted approach based on significant functional and structural information of the drug target. The structural insights of protein-ligand interactions provided by NMR and X-ray crystallography enable structures (often substrate derivatives) to be generated and optimised rationally.

De novo computational drug design utilises the known 3D structural features of an antimicrobial target to iteratively generate specific inhibitors. Compounds are computationally constructed based on the steric and chemical properties of the target site. SPROUT is an example of such computational software that can be employed in de novo drug design (Gillet et al., 1993). The application of SPROUT to the 3D- structures of E. coli DdlB (Besong et al., 2005), E. faecium VanA (Sova et al., 2009) and E. coli MurD (Horton et al., 2003) has yielded specific inhibitor molecules to these important enzymes involved in peptidoglycan biosynthesis. Although this demonstrates the utility of de novo drug design in generation of inhibitory ligands against essential bacterial targets, it does not circumvent the problems of penetration of the inhibitor to its target. This is exemplified by the fact that none of these molecules possessed antimicrobial properties.

1.3.3.2.6. Inhibition of the resistance mechanisms

Following the characterisation of a resistance mechanism (such as those described in Section 1.3.2), a viable strategy is to design drugs for its inhibition. Although not antibiotics themselves, these drugs can be used in combination with an active antimicrobial. This approach can potentially restore and preserve the efficacy of therapies against which resistance has developed.

A prime example of this is the secondary metabolite clavulanic acid, a potent !- lactamase inhibitor, isolated from Streptomyces clavuligerus in 1977 (Reading and Cole, 1977). It has weak intrinsic antibacterial activity, but can be co-administered with !-lactam antibiotics such as amoxicillin (augmentin). Clavulanic acid has a higher affinity for the !-lactamase than amoxicillin and can irreversibly bind to the active site serine of the !-lactamase catalytic site, forming a stable acylated intermediate (Yang et al., 1999). This combination therapy allows the antibiotic to exert its effect without the detrimental interference of a resistance mechanism.