Glycoside derivatives of quinizarin, anthrarufin, and 1-hydroxyanthraquinone for use in microbial diagnostics.

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.

Pseudomonas aeruginosa is a dominant cause of respiratory infection in individuals with cystic fibrosis (CF), leading to significant morbidity and mortality. Detection of P. aeruginosa is conducted by culture of respiratory samples but this process may occasionally be compromised due to overgrowth by other bacteria and fungi. We aimed to evaluate a novel chromogenic medium, Pseudomonas aeruginosa chromogenic agar (PACA), for culture of P. aeruginosa from respiratory samples, from patients with CF. A total of 198 respiratory samples were cultured onto PACA and three other media: CHROMID®P. aeruginosa, CHROMagar™ Pseudomonas and MacConkey agar. P. aeruginosa was recovered from 66 samples (33%), using a combination of all media. After 72 h incubation, the sensitivity of the four chromogenic media was as follows: 91% for PACA and CHROMagar™ Pseudomonas, 85% for CHROMID®P. aeruginosa and 83% for MacConkey agar. For the three chromogenic media, the positive predictive value after 72 h was as follows: 95% for PACA, 56% for CHROMagar™ Pseudomonas and 86% for CHROMID®P. aeruginosa. PACA proved to be a highly effective culture medium for the isolation and specific detection of P. aeruginosa from respiratory samples.

Development of a rapid approach for universal microbial detection is required in the healthcare, food and environmental sectors to aid with medical intervention, food safety and environmental protection. This research investigates the use of enzymatic hydrolysis of a substrate by a microorganism to generate a volatile organic compound (VOC). One such enzyme activity that can be used in this context is nitroreductase as such activity is prevalent across a range of microorganisms. A study was developed to evaluate a panel of 51 microorganisms of clinical interest for their nitroreductase activity. Two enzyme substrates, nitrobenzene and 1-fluoro-2-nitrobenzene, were evaluated for this purpose with evolution, after incubation, of the VOCs aniline and 2-fluoroaniline, respectively. Detection of the VOCs was done using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) with obtained limits of quantitation (LOQ) of 0.17 and 0.03 µg/mL for aniline and 2-fluoroaniline, respectively. The results indicated that both enzyme substrates were reduced by the same 84.3% of microorganisms producing the corresponding volatile anilines which were detected using HS-SPME-GC-MS. It was found that nitroreductase activity could be detected after 6–8 h of incubation for the selected pathogenic bacteria investigated. This approach shows promise as a rapid universal microbial detection system.

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 β-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 β-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.

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.

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.

Six N-nitroaryl-2-amino-1,3-dichloropropane derivatives have been prepared and evaluated against 18 cancer cell lines and two non-cancerous cell lines. Analysis of cell viability data and IC50 values indicated that the presence of a trifluoromethyl group in the nitroaryl moiety is an important structural feature associated with the compounds' cytotoxicities.

A new rapid method for the detection of hydrogen sulfide from pathogenic bacteria is reported. The developed method, static headspace multi-capillary column - gas chromatography - ion mobility spectrometry (SHS-MCC-GC-IMS), has been applied to detect hydrogen sulfide evolution from 61 bacteria. The developed method has been compared against a standard triple sugar iron (TSI) agar approach, and a modified single sugar iron (SSI) agar approach. Hydrogen sulfide detection by SHS-MCC-GC-IMS using an initial inoculum of 1-1.5 x 10(5) CFU/mL can be achieved within 6 h, after incubation at 37 degrees C, with a limit of detection of 1.6 ng/mL. Data for the standard agar method against the new instrumental approach, and the modified agar method against the new instrumental approach, are compared. The specificity for the new method compared against the standard method and the modified agar approach across all 61 strains was 85.2% and 88.5% respectively, and 86.7% and 91.3% across the 23 Salmonella strains tested.

The preparation and evaluation of chromogenic substrates for detecting bacterial glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene (6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A β-d-riboside of DHN and a β-d-glucuronide derivative of 6,7-dibromo-DHN were particularly effective for the detection of S. aureus and E. coli respectively.

Rapid, sensitive, and selective detection and identification of pathogenic bacteria is required in terms of food security. In this study, exogenous VOCs liberated by Salmonella strains have been identified and quantified via head space-solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) in milk samples. The specific enzymes targeted for detection and/or differentiation of Salmonella were C8 esterase, α-galactosidase and pyrrolidonyl peptidase using the following enzyme substrates: 2-chlorophenyl octanoate, phenyl α-d-galactopyranoside and L-pyrrollidonyl fluoroanilide, respectively. Detection of the exogenous VOCs, 2-chlorophenol, phenol and 3-fluoraniline was possible with typical limits of detection of 0.014, 0.045 and 0.005 μg/mL, respectively and correlation coefficients >0.99. The developed methodology was able to detect and identify Salmonella species within a 5 h incubation at 37 °C by the detection of the liberated VOCs. It was found that the milk samples tested were Salmonella free.

The Type B mesoionic heterocycle, 3,5-dipheny1-1,2-dithiolium-4-olate, reacts with phenylisocyanate producing a thioketone derivative rather than a pseudo-semi-conjugated heterocyclic mesomeric betaine. The structure of the thioketone product was confirmed by an X-ray crystallographic investigation. (C) 2017 Elsevier Ltd. All rights reserved.

Listeria monocytogenes is a Gram-positive bacterium and an opportunistic food-borne pathogen which poses significant risk to the immune-compromised and pregnant due to the increased likelihood of acquiring infection and potential transmission of infection to the unborn child. Conventional methods of analysis suffer from either long turn-around times or lack the ability to discriminate between Listeria spp. reliably. This paper investigates an alternative method of detecting Listeria spp. using two novel enzyme substrates that liberate exogenous volatile organic compounds in the presence of α-mannosidase and d-alanyl aminopeptidase. The discriminating capabilities of this approach for identifying L. monocytogenes from other species of Listeria are investigated. The liberated volatile organic compounds (VOCs) are detected using an automated analytical technique based on static headspace–multi-capillary column–gas chromatography–ion mobility spectrometry (SHS–MCC–GC–IMS). The results obtained by SHS–MCC–GC–IMS are compared with those obtained by the more conventional analytical technique of headspace–solid phase microextraction–gas chromatography–mass spectrometry (HS–SPME–GC–MS). The results found that it was possible to differentiate between L. monocytogenes and L. ivanovii, based on their VOC response from α-mannosidase activity.

This paper utilized L-alanine aminopeptidase activity as a useful approach to distinguish between Gram-negative and Gram-positive bacteria. This was done using two enzyme substrates, specifically 2-amino-N-phenylpropanamide and 2-amino-N-(4-methylphenyl)propanamide which liberated the volatile compounds aniline and p-toluidine, respectively. Two complementary analytical techniques have been used to identify and quantify the VOCs, specifically static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS) and headspace solid phase microextraction gas chromatography mass spectrometry (HS-SPME-GC MS). Superior limits of detection were obtained using HS-SPME-GC MS, typically by a factor of x6 such that the LOD for aniline was 0.02 mu g/mL and 0.01 mu g/mL for p-toluidine. In addition, it was also possible to determine indole interference-free by HS-SPME-GC MS at an LOD of 0.01 mu g/mL. The approach was applied to a range of selected bacteria: 15 Gram-negative and 7 Gram-positive bacteria. Use of pattern recognition, in the form of Principal Component Analysis, confirmed that it is possible to differentiate between Gram-positive and Gram-negative bacteria using the enzyme generated VOCs, aniline and p-toluidine. The exception was Stenotrophomonas maltophilia which showed negligible VOC concentrations for both aniline and p-toluidine, irrespective of the analytical techniques used and hence was not characteristic of the other Gram-negative bacteria investigated. The developed methodology has the potential to be applied for clinical and food applications. (C) 2017 Elsevier B.V. All rights reserved.

The evolution of volatile organic compounds (VOCs) provides an opportunistic approach for the detection of pathogenic bacteria. This approach can be enhanced by the application of VOC-labelled enzyme substrates that target specific enzyme activities of the bacteria under investigation. Detection of the VOCs provides a novel, specific and sensitive approach for the detection of pathogenic bacteria. This review highlights the importance of this approach alongside a range of alternative detection techniques for the identification of pathogenic bacteria.

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 β-alanyl aminopeptidase substrate, 2-(N- β-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.

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.

A series of N-nitroarylated-3-chloromethyl-1,2,3,4-tetrahydroisoquinoline derivatives, several of which also possessed a trifluoromethyl substituent, were prepared and assessed as potential nitroaromatic prodrugs. The enzymatic reduction of these compounds and their cytotoxicities were studied. The compounds were cytotoxic, but this is probably not related to their enzymatic reduction.

A novel, low-cost and simple method for the detection of pathogenic bacteria is proposed.

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.