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.

Fluorogenic l-alanylaminopeptidase substrates derived from 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins and their potential applications in diagnostic microbiology.

Six novel fluorogenic enzyme substrates for detecting l-alanylaminopeptidase activity in microorganisms have been prepared and evaluated in Columbia agar media. The substrates are l-alanyl derivatives of 6-amino-2-hetarylquinolines and 7-amino-3-hetarylcoumarins. Both the quinoline and coumarin series of substrates produced fluorescence in the presence of Gram-negative microorganisms. In contrast, fluorescence generation in the presence of the Gram-positive microorganisms and yeasts was limited or absent.

Detection of l-alanylaminopeptidase activity in microorganisms using chromogenic self-immolative enzyme substrates.

Three potential chromogenic enzymatic probes, each possessing a self-immolative spacer unit, were synthesised for the purpose of detecting l-alanylaminopeptidase activity in microorganisms. An Alizarin-based probe was the most effective, allowing several species to generate strongly coloured colonies in the presence of metal ions.

The Synthesis of L-Alanyl and ß-Alanyl Derivatives of 2-Aminoacridone and Their Application in the Detection of Clinically-Important Microorganisms.

In clinical microbiology the speed with which pathogenic microorganisms may be detected has a direct impact on patient health. One important strategy used in the laboratory is the growth of cultures in the presence of an enzymatic substrate which, once transformed by the appropriate microbial enzyme, generates a detectable colour or fluorescence output. Such substrates have previously been prepared by our group and others and are available as commercial diagnostic kits, however they all suffer from some degree of diffusion when used in a solid growth medium. This diffusion complicates the detection and differentiation of species in polymicrobial cultures and so we sought to improve on our previous work. In this work we have prepared and evaluated a series of novel fluorogenic enzyme substrates based on N-substituted-2-aminoacridones. All of the prepared substrates were found to be suitable for the detection and differentiation of certain microorganisms, however those based on the 2-amino-10-benzylacridone core in particular showed no apparent diffusion when incorporated into solid growth media. On transformation these substrates generated brightly fluorescent colonies that are clearly contrasted with the background medium due to the difference in emission wavelength (λem 445-450 nm for the substrate, λem 550 nm for the product). Here we have shown that our L-alanyl aminopeptidase substrate, 2-(N-L-alanylamino)-10-benzylacridone, is particularly suited to the detection of Gram-negative bacteria, and our ß-alanyl aminopeptidase substrate, 2-(N- ß-alanylamino)-10-benzylacridone, to the detection of Pseudomonas aeruginosa and Serratia marcescens when grown on solid media incorporating these substrates. The resulting fluorophore shows no apparent diffusion from the colonies of interest, and the enhanced sensitivity offered by fluorescent emission may allow for the detection of these organisms as microcolonies using automated fluorescence microscopy.

Detection of l-alanylaminopeptidase activity in microorganisms using fluorogenic self-immolative enzyme substrates.

A series of fluorogenic enzymatic substrates that incorporate a self-immolative spacer were synthesised for the purpose of identifying l-alanylaminopeptidase activity in microorganisms in agar media. These substrates resulted in the generation of fluorescent microorganism colonies with Gram-negative microorganisms.

Synthesis and Evaluation of Novel 7- and 8-Aminophenoxazinones for the Detection of ß-Alanine Aminopeptidase Activity and the Reliable Identification of Pseudomonas aeruginosa in Clinical Samples.

A series of novel 8-aminophenoxazin-3-one and 7-aminophenoxazin-3-one chromogens and their corresponding ß-alanine derivatives were synthesized and evaluated for their ability to detect ß-alanyl aminopeptidase activity in bacteria known to hydrolyze ß-alanine derivatized substrates. The results provided insight into the structural requirements for effective visualization of enzymatic activity and the mechanism of formation of phenoxazinon-3-ones. 8-Aminophenoxazin-3-one substrates 23c, 23d, and 23e were prepared in good to high overall yield and were selective for ß-alanyl aminopeptidase activity in bacteria, producing a lighter agar background coloration facilitating visualization of colored colonies, with variable localization to the colonies, but had lower sensitivities for the detection of Pseudomonas aeruginosa in comparison to the analogous 7-aminophenoxazin-3-one substrates. The synthetic methodology employed here allows the preparation of a range of substrates for evaluation and the establishment of structure-activity relationships. For example, the 2-pentyl substituted aminophenoxazin-3-one 22b performed with analogous sensitivity to the corresponding 1-pentyl-7-aminophenoxazin-3-one substrate 1 used commercially, highlighting that the position of the pentyl substituent can be varied while maintaining detection sensitivity.

2-(Nitroaryl)benzothiazole and benzoxazole derivatives as fluorogenic substrates for the detection of nitroreductase activity in clinically important microorganisms.

A series of carboxy-substituted 2-(nitroaryl)benzothiazole derivatives and carboxy-substituted 2-(nitroaryl)benzoxazole derivatives were prepared and evaluated as potential nitroreductase substrates for the purpose of detecting clinically important microorganisms. Several of the substrates produced highly fluorescent colonies with the majority of a panel of 10 Gram-negative bacteria and also with two of a panel of 8 Gram-positive bacteria.

Novel chromogenic aminopeptidase substrates for the detection and identification of clinically important microorganisms.

A series of amino acid derivatives 8-10, 42 and 43 have been prepared as chromogenic enzyme substrates in order to detect aminopeptidase activity in clinically important Gram-negative and Gram-positive bacteria. Enzymatic hydrolysis liberates the amino acid moiety and either a 4-aminophenol or a 4-dialkylaminoaniline derivative which undergoes oxidative coupling with 1-naphthol or a substituted 1-naphthol giving an indophenol dye. Substrates and 1-naphthols were incorporated into an agar-based culture medium and this allowed growth of intensely coloured bacterial colonies based on hydrolysis by specific enzymes. Red/pink coloured colonies were produced by the substrates 8-10 and blue coloured colonies were formed by the substrates 42 and 43. The L-alanyl aminopeptidase substrates 8 targeted L-alanyl aminopeptidase activity and gave coloured colonies with a range of Gram-negative bacteria. Substrates 9 targeted ß-alanyl aminopeptidase activity and generated coloured colonies with selected Gram-negative species including Pseudomonas aeruginosa. Three substrates for L-pyroglutamyl acid aminopeptidase (10a, 10c and 43) were hydrolysed by enterococci and Streptococcus pyogenes to generate coloured colonies. Two yeasts were also included in the study, but they did not produce coloured colonies with any of the substrates examined.

Synthesis of 2-arylbenzothiazole derivatives and their application in bacterial detection.

A series of 2-arylbenzothiazole derivatives have been prepared as fluorogenic enzyme substrates in order to detect aminopeptidase, esterase, phosphatase and ß-galactosidase activity in clinically important Gram-negative and Gram-positive bacteria. Substrates were incorporated into an agar-based culture medium and this allowed growth of intensely fluorescent bacterial colonies based on hydrolysis by specific enzymes. Substrate 20 targeted L-alanine aminopeptidase activity and was hydrolysed exclusively by a range of Gram-negative bacteria and inhibited the growth of a range of Gram-positive bacteria. Substrate 19a targeted ß-alanyl aminopeptidase activity and generated fluorescent colonies of selected Gram-negative species including Pseudomonas aeruginosa. Substrate 21b targeted C8-esterase activity and resulted in strongly fluorescent colonies of selected species known to harbour such enzyme activity (e.g., Salmonella and Pseudomonas). Most Gram-negative species produced colonies with an intense blue fluorescence due to hydrolysis of phosphatase substrates 24a-c and substrate 24c was also hydrolysed by strains of Staphylococcus aureus. Compounds 26b and 26c targeted ß-galactosidase activity and generated strongly fluorescent colonies with coliform bacteria that produced this enzyme (e.g., Escherichia coli).

Synthesis and evaluation of fluorogenic 2-amino-1,8-naphthyridine derivatives for the detection of bacteria.

Several novel fluorogenic N-aminoacylnaphthyridine substrates were synthesized in good yield and tested for their ability to detect pathogenic bacteria in agar-based cell culture. Simple 2-N-(ß-alanyl)amino-5,7-dialkylnaphthyridine substrates were selectively hydrolysed by ß-alanylaminopeptidase expressing bacteria, but were subject to diffusion in the agar medium. Diffusion was reduced in the 2-N-(ß-alanyl)amino-7-alkylnaphthyridine substrates with longer alkyl chains, but inhibition of growth was increased. 2-N-(ß-Alanyl)amino-7-octylnaphthyridine inhibited the growth of all species tested, except for strains resistant to colistin/polymyxin, providing a rationale for the development of substrates for the selective detection of drug resistant species in clinical samples.

In vitro activity of S-(3,4-dichlorobenzyl)isothiourea hydrochloride and novel structurally related compounds against multidrug-resistant bacteria, including Pseudomonas aeruginosa and Burkholderia cepacia complex.

The aim of this study was to establish the antimicrobial activities of S-(3,4-dichlorobenzyl)isothiourea hydrochloride (A22) and a series of structurally related compounds against multidrug-resistant (MDR) bacteria. The minimum inhibitory concentrations (MICs) of 21 compounds were determined against 18 strains of pathogenic bacteria in addition to Pseudomonas aeruginosa (n=19) and Burkholderia cepacia complex (BCC) (n=20) isolated from the sputa of cystic fibrosis patients. Selected compounds were tested against further isolates, including P. aeruginosa (n=100), BCC (n=12) and Stenotrophomonas maltophilia (n=19). The interaction of S-(4-chlorobenzyl)isothiourea hydrochloride (C2) in combination with conventional antimicrobials was examined against 10 P. aeruginosa strains. Selected compounds were also tested against Enterobacteriaceae producing NDM-1 carbapenemase (n=64) and meticillin-resistant Staphylococcus aureus (MRSA) (n=37). Of the 21 compounds, 14 showed antimicrobial activity that was generally more pronounced against Gram-negative bacteria. Against P. aeruginosa, the most active compound was C2 [MIC for 50% of the organisms (MIC(50))=32µg/mL]. This compound was also the most active against BCC, with all isolates inhibited by 64µg/mL. For all ten strains of P. aeruginosa subjected to combination testing with C2 and conventional antimicrobials, a bactericidal effect was achieved with at least one combination. C2 and A22 both showed strong activity [MIC for 90% of the organisms (MIC(90))=4µg/mL] against Enterobacteriaceae that produced NDM-1 carbapenemase. Finally, S-(4-chlorobenzyl)-N-(2,4-dichlorophenyl)isothiourea hydrochloride showed good activity (MIC(90)=8µg/mL) against MRSA. This work establishes the activity of isothiourea derivatives against a broad range of clinically important MDR bacteria.

2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for the detection of nitroreductase and aminopeptidase activity in clinically important bacteria.

A series of 2-(2-nitrophenyl)benzothiazole 7, 2-(2-nitrophenyl)benzoxazole 10 and 2-(2-nitrophenyl)benzimidazole 13 derivatives have been synthesised and assessed as indicators of nitroreductase activity across a range of clinically important Gram negative and Gram positive bacteria. The majority of Gram negative bacteria produced strongly fluorescent colonies with substrates 7 and 10 whereas fluorescence production in Gram positive bacteria was less widespread. The l-alanine 16 and 19 and ß-alanine 21 and 23 derivatives have been prepared from 2-(2-aminophenyl)benzothiazole 14 and 2-(2-aminophenyl)benzoxazole 17. These four compounds have been evaluated as indicators of aminopeptidase activity. The growth of Gram positive bacteria was generally inhibited by these substrates but fluorescent colonies were produced with the majority of Gram negative bacteria tested.

Enzymatic substrates in microbiology.

Enzymatic substrates are powerful tools in biochemistry. They are widely used in microbiology to study metabolic pathways, to monitor metabolism and to detect, enumerate and identify microorganisms. Synthetic enzymatic substrates have been customized for various microbial assays, to detect an expanding range of both new enzymatic activities and target microorganisms. Recent developments in synthetic enzymatic substrates with new spectral, chemical and biochemical properties allow improved detection, enumeration and identification of food-borne microorganisms, clinical pathogens and multi-resistant bacteria in various sample types. In the past 20 years, the range of synthetic enzymatic substrates used in microbiology has been markedly extended supporting the development of new multi-test systems (e.g., Microscan, Vitek 2, Phoenix) and chromogenic culture media. The use of such substrates enables an improvement in time to detection and specificity over conventional tests that employ natural substrates. In the era of intense developments in molecular biology, phenotypic tests involving enzymatic substrates remain useful to analyse both simple and complex samples. Such tests are applicable to diagnostic and research laboratories all over the world.

A novel chromogenic medium for isolation of Pseudomonas aeruginosa from the sputa of cystic fibrosis patients.

BACKGROUND: A novel chromogenic medium for isolation and identification of Pseudomonas aeruginosa from sputa of cystic fibrosis (CF) patients was evaluated and compared with standard laboratory methods. METHODS: One hundred sputum samples from distinct CF patients were cultured onto blood agar (BA), Pseudomonas CN selective agar (CN) and a Pseudomonas chromogenic medium (PS-ID). All Gram-negative morphological variants from each medium were subjected to antimicrobial susceptibility testing, and identification using a combination of biochemical and molecular methods. RESULTS: P. aeruginosa was isolated from 62 samples after 72 h incubation. Blood agar recovered P. aeruginosa from 56 samples (90.3%) compared with 59 samples (95.2%) using either CN or PS-ID. The positive predictive value of PS-ID (98.3%) was significantly higher than growth on CN (88.5%) for identification of P. aeruginosa (P<0.05). CONCLUSIONS: PS-ID is a promising medium allowing for the isolation and simultaneous identification of P. aeruginosa from sputa of CF patients.

Synthesis and evaluation of novel chromogenic peptidase substrates based on 9-(4'-aminophenyl)-10-methylacridinium salts as diagnostic tools in clinical bacteriology.

The synthesis and initial evaluation of novel chromogenic substrates with potential in the detection and differentiation of cultured bacterial colonies are described. The substrates were readily hydrolysed by specific aminopeptidase activity to release the chromogen, 9-(4'-aminophenyl)-10-methylacridinium salt, which provided a clear visual indication of the presence of the corresponding bacteria.

Synthesis and evaluation of novel chromogenic aminopeptidase substrates for microorganism detection and identification.

The amides 8a-e and 10a-c were prepared as chromogenic aminopeptidase substrates. A range of microorganisms were grown in the presence of these compounds and coloured colonies were produced in several cases after addition of acetic acid-thus giving potential methods for the detection of aminopeptidase activity and for microorganism identification.