Novel substrates for the improved detection and identification of pathogenic bacteria

Many diseases are caused by pathogenic bacteria. A key example of this is sepsis, which is mostly caused by staphylococci and Gram-negative bacteria. In addition, the highly resistant ESKAPE pathogens are responsible for the majority of hospital acquired infections. In order to treat bacterial infections effectively, and to avoid promoting bacterial resistance against antibacterial drugs, the correct agents must be used, for which in turn the detection and identification of pathogenic strains is essential. This research aims to develop selective chromogenic culture media, by introducing new antibacterial agents for the improved selectivity and new chromogenic substrates for selective visualisation of certain bacterial strains. The intention of the major part of this work was to inhibit the growth of commensal bacteria in clinical samples, as they mask the growth of the infection-causing bacteria. New and known compounds were prepared for 3 evaluation as alanine racemase inhibitors. The compounds were tested on a range of clinical pathogenic and non-pathogenic bacterial strains. The molecules developed were based on the amino acid alanine and utilised bioisosteres and other replacements for the carboxylic acid moiety. Key compounds targeted included alanylmethanesulfonamide 27-L, 1-aminoethyl5-oxo-1,2,4-oxadiazole 33-L and 1-aminoethyltetrazole 32a-L. Each compound was tested initially as the alanyl-X dipeptide form. While most of the alanine bioisosteres were known structures, their novel peptide derivatives required synthetic development using both solution and solid phase techniques. The solid phase synthesis of several C-terminal 1aminoethyltetrazole peptides was successfully established by using 2-chlorotrityl chloride resin. The investigation of the antimicrobial activity of the synthesised compounds identified several clinically applicable selective inhibitors. These compounds were shown to provide differentiation between Salmonella and Escherichia coli, or enterococci and streptococci. This work also gave a useful comparison between the different alanine bioisosteres, and showed the importance of di- and oligopeptide permease systems in order to reach sufficient bacterial activity. The microbiological activity of 1- aminoethyltetrazole peptide derivatives was studied in more detail, due to their potential in clinical applications for the diagnosis of food poisoning. In other work, also directed towards the rapid and selective detection and identification of pathogenic bacteria in a clinical environment, new chromogenic substrates were prepared. Each of these compounds contained a chromogen with a phenoxazin-3-one scaffold linked to an amino acid residue. The purpose of the amino acid is to act as a unit recognised and cleaved by specific hydrolytic bacterial enzymes. Upon liberation, electronic differences between the conjugated and free forms of the chromogen resulted in the development of distinct colour changes, which provide the basis of 4 bacterial detection and identification. Synthetic methods have been developed for the efficient and economical production of this series of substrates. After preparation, these compounds were tested against a panel of clinically relevant bacteria. The aim of these substrates was to present an alternative substrate for (N-β-alanyl)-7-amino-1-pentylphenoxazine-3-one 86a, which is applied commercially in chromID® Pseudomonas aeruginosa chromogenic medium designed for the clinical detection of P.aeruginosa. The new substrates are designed to fully explore the chemical space of phenoxazinonebased chromogenic substrates, and to decrease the colour, as substrate 86a causes significant background colour in culture media. The future application of these substrates in chromogenic media resides in their potential to advance the identification of specific pathogenic bacteria and to thus facilitate the treatment of bacterial infections