A chromatographic approach to distinguish Gram-positive from Gram-negative bacteria using exogenous volatile organic compound metabolites

• Published in: Journal of Chromatography A Vol. 1501; pp. 79 - 88
• Main Authors: Ramírez-Guízar, Susana, Sykes, Hannah, Perry, John D., Schwalbe, Edward C., Stanforth, Stephen P., Perez-Perez, Ma. Cristina I., Dean, John R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 09.06.2017
• Subjects: Discriminant Analysis; Enzyme substrate; Gas Chromatography-Mass Spectrometry - methods; Gram-Negative Bacteria - chemistry; Gram-Negative Bacteria - metabolism; Gram-Positive Bacteria - chemistry; Gram-Positive Bacteria - metabolism; L-alanine aminopeptidase; Solid Phase Microextraction - methods; Static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS); Volatile organic compounds; Volatile Organic Compounds - chemistry; Volatile Organic Compounds - isolation & purification; Volatile Organic Compounds - metabolism; Volatile organic compounds; Static headspace multicapillary column gas chromatography ion mobility spectrometry (SHS-MCC-GC-IMS); Enzyme substrate; L-alanine aminopeptidase
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2017.04.015

•Differentiation of Gram-positive and Gram-negative bacteria.•Exogenous volatile organic compound metabolites.•Novel enzyme substrates for VOC.•Static headspace multicapillary column gas chromatography ion mobility spectrometry.•Headspace solid phase microextraction gas chromatography mass spectrometry. 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 0.02μg/mL and 0.01μg/mL for p-toluidine. In addition, it was also possible to determine indole interference-free by HS-SPME-GC–MS at an LOD of 0.01μg/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.