Use of exogenous volatile organic compounds to detect Salmonella in milk

Abstract

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.

Detection of β-alanyl aminopeptidase as a biomarker for Pseudomonas aeruginosa in the sputum of patients with cystic fibrosis using exogenous volatile organic compound evolution

“…Our aim was to therefore develop a rapid test for the detection of Pseudomonas and Burkholderia from CF patient sputum samples, utilising balanyl aminopeptidase activity. We have previously identied that using targeted enzyme substrates, a unique exogenous volatile organic compound can be liberated and detected using either static headspace-solid phase micro-extraction-gas chromatography-mass spectrometry, SHS-SPME-GC-MS [23][24][25][26] or static headspace multicapillary column-gas chromatographyion mobility spectrometry, SHS-MCC-GC-IMS. 27 The use of exogenous volatile organic compounds (VOCs) to distinguish 7 Gram-positive from 15 Gram-negative bacteria using the enzyme substrates 2-amino-N-phenylpropanamide (to liberate the VOC aniline) and 2-amino-N-(4-methylphenyl)propanamide (to liberate the VOC p-toluidine) has been demonstrated.…”

“…25 In addition, exogenous VOC detection has been used to identify Salmonella in milk samples using the enzyme substrates 2-chlorophenyl octanoate, phenyl a-D-galactopyranoside and L-pyrrolidonyl uoroanilide which liberate the VOCs 2-chlorophenol, phenol and 3-uoroaniline, respectively. 26 In this case however, b-alanyl aminopeptidase enzymes work by catalysing the cleavage, via hydrolysis, of amino acids from the amino (N) terminus of protein or peptide substrates. 28 The presence of b-alanyl aminopeptidase activity is well documented in P. aeruginosa, 29,30 and has subsequently led to studies on the development of chromogenic and uorogenic substrates targeting b-alanyl aminopeptidase activity as a method for the detection of P. aeruginosa.…”

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

“…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]. We have recently been interested in detecting hydrolytic enzymatic activities in pathogenic microorganisms using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), a technique that is amenable to automation [5][6][7]. In our previous work, 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.…”

“…We have recently been interested in detecting hydrolytic enzymatic activities in pathogenic microorganisms using headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS), a technique that is amenable to automation [5][6][7]. In our previous work, 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. The focus of this paper is on selecting a more universal enzyme system, but still based on exogenous VOC evolution and detection, for rapid screening of microorganisms of clinical interest.…”

“…Following on from our previous 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. Nitrobenzene and 1-fluoro-2-nitrobenzene were chosen as the nitroreductase substrates, as they are readily available at low cost and high purity, in order to generate the VOCs aniline and 2-fluoroaniline, respectively. The selection of the exogenous VOCs is done to try and avoid false positives in the data generated; earlier research on the use of HS-SPME-GC-MS for the analysis of bacteria, in broth, indicated that VOCs with specific functionality, e.g., an amino group, and additionally a fluorine were unlikely to naturally occur [18].…”