Chromogenic enzyme substrates based on [2-(nitroaryl)ethenyl]pyridinium and quinolinium derivatives for the detection of nitroreductase activity in clinically important microorganisms.

A series of [2-(nitroaryl)ethenyl]pyridinium and quinolinium derivatives have been synthesised as potential indicators of microbial nitroreductase activity. When assessed against a selection of 20 clinically important pathogenic microorganisms, microbial colonies of various colours (yellow, green, red, brown, black) were produced and attributed to nitroreductase activity. Most substrates elicited colour responses with Gram-negative microorganisms. In contrast, the growth of several species of Gram-positive microorganisms and yeasts was often inhibited by the substrates and hence coloured responses were not seen.

Detection of ß-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution.

A novel, rapid and sensitive analytical method has been developed and applied to 105 sputum samples from patients with cystic fibrosis, including 5 samples from post-lung transplant patients. This new method is specifically targeted to measure ß-alanyl aminopeptidase activity which is characteristic of some important Gram-negative pathogens. Of relevance to this study are Pseudomonas aeruginosa and pathogens of the Burkholderia cepacia complex both of which are commonly associated with respiratory infections as well as increased morbidity and mortality in adult cystic fibrosis patients. The analytical method involves the addition of a novel enzyme substrate (i.e. 3-amino-N-(3-fluorophenyl)propanamide) that interacts with ß-alanyl aminopeptidase to generate an exogenous volatile organic compound 3-fluoroaniline (LOD 0.02 µg mL-1; LOQ 0.06 µg mL-1). 3-Fluoroaniline was determined at 20 times above its calculated limit of quantification in the sputum samples by HS-SPME-GC-MS and then the results compared with standard culture methods and bacterial identification using MALDI-TOF-MS. Detection of 3-fluoroaniline was possible after only 8 h incubation of the sputum samples with a 95% success rate; this increased to 100% at 24 h which was well within the typical routine timeframe of 48 h. To our knowledge, this is the first demonstration of detection of P. aeruginosa by use of a custom-designed substrate to liberate a detectable and unique VOC. The very high negative predictive value (100% in this study) means such an assay could be appropriate as a screening technique for patients who are not yet colonized by this pathogen.

Fluorogenic 7-azidocoumarin and 3/4-azidophthalimide derivatives as indicators of reductase activity in microorganisms.

A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.