The substrates c e all produced highly fluorescent colonies
The substrates 18c–e all produced highly fluorescent colonies with the panel of Gram-negative microorganisms (Table 3). Thus, the benzothiazole derivative 18d gave intensely yellow fluorescent colonies with all of the Gram-negative bacteria. This substrate was inhibitory towards the Gram-positive bacteria and no growth was apparent and hence no fluorescent colonies were produced. The yeast species, C. albicans, grew moderately well and produced blue-fluorescent colonies. The substrates 18c and 18e both gave similar results to substrate 18d, except that the colonies of Gram-negative bacteria were associated with an intense green fluorescence (substrate 18c, data not shown) and a strong blue fluorescence (substrate 18e). The observed fluorescence with substrates 18c–e was restricted to the microorganism colonies and this is an advantage over our previously described substrates 10 in which noticeable ceftiofur of the fluorophore into the surrounding media was apparent. Additionally, some diffusion of the fluorophore is also observed with the aminocoumarin-derived substrate 8 in agar media. In accord with expectation, the di-l-alanyl substrate 22 was less inhibitory towards most of the selection of Gram-positive microorganisms compared to the mono-l-alanyl substrate 18e and consequently blue fluorescent colonies were produced with growing Gram-positive microorganisms and also with both yeasts. It is interesting to note that a chromogenic l-alanylaminopeptidase substrate based on a 9-(4-aminophenyl)-10-methylacridinium core was non-inhibitory to five of a panel of ten Gram-positive microorganisms and did not undergo hydrolysis even when microorganism growth occurred. The di-l-alanyl-analogue was even less inhibitory, allowing growth of nine of the same panel of Gram-positive microorganisms. This contrasts with our work described here and elsewhere, in which the l-alanyl fluorogenic substrates were often inhibitory to most Gram-positive microorganisms. This difference in detection profile between these two sets of fluorogenic and chromogenic substrates might be attributed to a variety of factors, including for example, the greater sensitivity of fluorogenic substrates, the higher degree of toxicity of the fluorogenic substrates to Gram-positive microorganisms, the degree of permeation of the substrates into the cell and structural differences between the substrates; the chromogenic substrates are quaternised heterocycles whereas the fluorogenic substrates are not.
Conclusions In conclusion, the substrates 18c–18e all produced highly fluorescent colonies with the panel of Gram-negative microorganisms. This has been attributed to the combination of two synergistic effects; the wide distribution of l-alanylaminopeptidase in Gram-negative microorganisms and the inhibitory effect of these substrates against Gram-positive microorganisms. Some Gram-positive microorganisms gave fluorescent colonies with substrates population were not inhibitory to their growth (substrates 18a and 22).