Detection of Microbial Nitroreductase Activity by Monitoring Exogenous Volatile Organic Compound Production Using HS-SPME-GC-MS

Ryan Thompson,Stephen P Stanforth,John R Dean,John D Perry

doi:10.3390/separations7040064

Abstract

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.

Highlights

The design and application of enzyme substrates, which facilitate the detection of specific enzymatic activities in pathogenic microorganisms have been widely exploited in diagnostic microbiology by many sectors of the economy including the health-care sector, the food industry and the environmental sector [1,2,3,4]
Following on from our previous volatile organic compound (VOC)-based microbial detection studies using esterase and aminopeptidase substrates [5,6,7], we report in this paper our studies relating to the detection of microbial nitroreductase activity within a panel of clinically important pathogenic microorganisms using HS-SPME-GC-MS
Nitroreductase activity was detected by 84.3% of the panel of 51 selected microorganisms when using nitrobenzene or 1-fluoro-2-nitrobenzene as enzyme substrates

Summary

Introduction

The design and application of enzyme substrates, which facilitate the detection of specific enzymatic activities in pathogenic microorganisms have been widely exploited in diagnostic microbiology by many sectors of the economy including the health-care sector (e.g., hospitals), the food industry (e.g., food quality control) and the environmental sector (e.g., monitoring of water contamination) [1,2,3,4]. The focus has been on identifying specific bacteria (e.g., Salmonella [5] and Pseudomonas aeruginosa [7]) and the ability to differentiate Gram-positive from Gram-negative bacteria [6] using the exogenous VOCs detected from esterase [5] and aminopeptidase [6,7] activities, i.e., phenols and anilines, respectively. Our research has been using enzyme substrates to liberate exogenous VOCS that can be analysed using HS-SPME-GC-MS; this approach would seem ideal for the detection of bacterial nitroreductase activity. The detection of enzyme activity in microorganisms is done using off-the-shelf or synthesized enzyme substrates that cleave, in the presence of the specific enzyme, liberating either a chromophore or fluorophore [4]. Microbial nitroreductase activity was first identified in Escherichia coli in 1957 [9] and its presence was later reported across a wide range of microorganisms [10] including

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