The detection of pathogenic bacteria via fluorogenic enzyme substrate probes

There is a strong demand for the methods for the rapid detection of pathogenic bacteria due to the rise in antimicrobial resistance and the reduction in the efficacy of antimicrobial agents. The present work explores the use of fluorogenic media, through the preparation of benzofurazan / coumarin derived enzyme substrates, exploration of their fluorescence characteristics and evaluation of their effectiveness in the detection of pathogenic bacteria. The synthesised enzyme substrates target specific bacterial aminopeptidases, resulting in fluorescent bacteria colonies upon hydrolysis. \nInitially, 5 coumarin and 4 benzofurazan analogues were synthesised; the coumarins were coupled to β-alanine, L-leucine, L-pyroglutamate and L-proline via a self-immolative linker and a benzofurazan was coupled directly to β-alanine and L-pyroglutamate creating a total of 12 different aminopeptidase specific enzyme substrates. Excitation / emission wavelengths and fluorescence intensities of these synthesised enzyme substrates were compared to the corresponding fluorophores, demonstrating significant differences and illustrating their potential suitability for use in the detection of pathogenic bacteria. \nThe synthesised enzyme substrates were incorporated into a variety of agar media, and their sensitivity and specificity for the detection of 20 pathogenic bacterial strains examined, including the growth of coliform bacteria in the presence of meropenem. Microbiological evaluation showed the successful uptake and fluorescence generation by bacterial colonies with the following outcomes: \n(1)\tβ-alanyl aminopeptidase substrates showed specificity for P. aeruginosa and S. marcescens colonies, \n(2)\tL-leucyl aminopeptidase substrates were hydrolysed by all 20 pathogenic bacterial strains, \n(3)\tL-pyroglutamyl aminopeptidase substrates showed specificity for K. pneumoniae, S. pyogenes, C. freundii, E. cloacae, S. marcescens, E. faecium and E. faecalis colonies, \n(4)\tL-pyroglutamyl aminopeptidase substrates were effective for the differentiation of K. pneumoniae, C. freundii, S. marcescens and E. cloacae coliform bacterial colonies in MacConkey solid agar media; and for observing the growth of meropenem resistant K. pneumoniae in both solid and liquid media, \n(5)\tsubstrates incorporating 6,8-dibromo-3-methyl-7-hydroxycoumarin demonstrated poor stability in agar media, and \nsubstrates incorporating 8-iodo-4-methyl-7-hydroxycoumarin and benzofurazans demonstrated no fluorescence following incubation with bacterial colonies.