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  • plx4720 The dihydroxynaphthalene derived substrates a a and

    2020-01-21

    The 2,3-dihydroxynaphthalene-derived substrates 18a, 20a and 22a (Table 2) allowed only moderate growth of all members of the panel of microorganisms suggesting that these substrates were inhibitory to some extent. Strong growth of the Gram-negative microorganisms and moderate growth of the Gram-positive microorganisms and the yeasts were seen with the glucuronide substrate 24a. In contrast, strong growth of all 20 microorganisms was observed in the presence of the ribofuranoside substrate 26a. Purple coloured colonies were produced with this series of substrates when appropriate enzymatic activity was present. As with the catechol-derived substrates, plx4720 of colour into the surrounding medium occurred and hence these substrates would be better suited for use in liquid media. For substrate 18a, coloration was generated only by known producers of β-glucosidase including enterococci and Listeria monocytogenes, suggesting this substrate could be exploited for detection of these species. Similarly, for substrate 20a activity was only observed for known producers of β-galactosidase. Substrate 22a is expected to detect β-hexosaminidase activity and several bacteria demonstrated hydrolysis of this substrate including known producers of this enzyme e.g. Serratia marcescens. Disappointingly, no coloration was generated by Candida albicans within the 18 h incubation period and this species is known to produce β-hexosaminidase. The glucuronide substrate 24a was only hydrolysed by E. coli, as expected, producing strongly coloured colonies. Substrate 26a effectively detected β-ribosidase activity in a number of Gram-negative species and activity was consistent with that previously reported for other β-ribosidase substrates.12, 37 Among Gram-positive bacteria, reactivity was restricted to S. aureus strains and there was clear differentiation from other Gram-positive species, suggesting that this may be a useful substrate for detection of S. aureus. Fig. 2 illustrates the differentiation of S. aureus from other Gram-positive microorganisms in liquid media. The usefulness of β-ribosidase detection for the identification of S. aureus has previously been observed. In order to produce non-diffusible chelates that might be suited for use in solid (agar) media, the series of 6,7-dibromo-2,3-dihydroxynaphthalene substrates 18b, 20b, 24b and 26b were prepared (Table 3) with the expectation that the incorporation of the bromine substituents would reduce the solubility of the resulting chelates in this medium, thus limiting their diffusion out of bacterial colonies. Microorganism growth was generally moderate in the presence of substrates 18b and 20b and strong in the presence of substrate 24b whereas substrate 26b was inhibitory to Gram-positive bacteria. In agreement with expectation, the brown colours generated by enzymatic hydrolysis of these substrates were largely localised within the boundaries of the microorganism colonies and diffusion of colour into the surrounding medium was minimal. A β-d-ribofuranoside derived from 1,4-dibromo-2,3-dihydroxynaphthalene was also prepared (i.e. the isomer of compound 26b) but this substrate produced a strong background coloration in agar media and no discernible enzyme activity could be observed against the background. This suggests that the location of bromine atoms adjacent to hydroxy-groups would not produce practical substrates as noted in the introduction. Substrate 18b did not show potential as a suitable substrate for detection of β-glucosidase producing pathogens as weak reactions were observed for enterococci and Listeria monocytogenes. Some unexpected (albeit weak) positive reactions were also observed for other species such as S. aureus. β-Galactosidase is most often targeted as a useful marker for coliforms and moderate to strong positive reactions were obtained as expected for E. coli, S. marcescens and C. freundii with substrate 20b. However, E. cloacae (a β-galactosidase producing coliform) did not produce coloration. It is likely that activity of this and other species could be improved by inclusion of an inducer of β-galactosidase such as isopropyl-β-d-thiogalactoside (IPTG). As anticipated, the glucuronide substrate 24b was only hydrolysed by E. coli producing strongly coloured colonies and illustrative agar plates are depicted in Fig. 3. In addition to the 20 microorganism plate which clearly shows a positive response for E. coli, four single microorganism plates which included two E. coli strains (expected to give positive responses) and S. marcescens and S. typhimurium (both expected to produce negative responses) were prepared. These four plates also showed a clear difference between positive and negative results and a good contrast between the colour of the colonies and the background. Growth inhibition was even more pronounced with substrate 26b with inhibition observed for E. coli, P. rettgeri and all of the eight Gram-positive bacteria tested. This precluded its potential application for the detection of S. aureus.