In vitro volatile organic compound profiling using GC×GC-TOFMS to differentiate bacteria associated with lung infections: a proof-of-concept study.

Chronic pulmonary infections are the principal cause of morbidity and mortality in individuals with cystic fibrosis (CF). Due to the polymicrobial nature of these infections, the identification of the particular bacterial species responsible is an essential step in diagnosis and treatment. Current diagnostic procedures are time-consuming, and can also be expensive, invasive and unpleasant in the absence of spontaneously expectorated sputum. The development of a rapid, non-invasive methodology capable of diagnosing and monitoring early bacterial infection is desired. Future visions of real-time, in situ diagnosis via exhaled breath testing rely on the differentiation of bacteria based on their volatile metabolites. The objective of this proof-of-concept study was to investigate whether a range of CF-associated bacterial species (i.e. Pseudomonas aeruginosa, Burkholderia cenocepacia, Haemophilus influenzae, Stenotrophomonas maltophilia, Streptococcus pneumoniae and Streptococcus milleri) could be differentiated based on their in vitro volatile metabolomic profiles. Headspace samples were collected using solid phase microextraction (SPME), analyzed using comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) and evaluated using principal component analysis (PCA) in order to assess the multivariate structure of the data. Although it was not possible to effectively differentiate all six bacteria using this method, the results revealed that the presence of a particular pattern of VOCs (rather than a single VOC biomarker) is necessary for bacterial species identification. The particular pattern of VOCs was found to be dependent upon the bacterial growth phase (e.g. logarithmic versus stationary) and sample storage conditions (e.g. short-term versus long-term storage at -18 °C). Future studies of CF-associated bacteria and exhaled breath condensate will benefit from the approaches presented in this study and further facilitate the production of diagnostic tools for the early detection of bacterial lung infections.

Profiling the decomposition odour at the grave surface before and after probing.

Human remains detection (HRD) dogs are recognised as a valuable and non-invasive search method for remains concealed in many different environments, including clandestine graves. However, the search for buried remains can be a challenging task as minimal odour may be available at the grave surface for detection by the dogs. Handlers often use a soil probe during these searches in an attempt to increase the amount of odour available for detection, but soil probing is considered an invasive search technique. The aim of this study was to determine whether the soil probe assists with increasing the abundance of volatile organic compounds (VOCs) available at the grave surface. A proof-of-concept method was developed using porcine remains to collect VOCs within the grave without disturbing the burial environment, and to compare their abundance at the grave surface before and after probing. Detection and identification of the VOC profiles required the use of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) due to its superior sensitivity and selectivity for decomposition odour profiling. The abundance of decomposition VOCs was consistently higher within the grave environment compared to the grave surface, except when the grave surface had been disturbed, confirming the reduced availability of odour at the grave surface. Although probing appeared to increase the abundance of VOCs at the grave surface on many of the sampling days, there were no clear trends identified across the study and no direct relationships with the environmental variables measured. Typically, the decomposition VOCs that were most prevalent in the grave soil were the same VOCs detected at the grave surface, whereas the trace VOCs detected in these environments varied throughout the post-burial period. This study highlighted that probing the soil can assist with releasing decomposition VOCs but is likely correlated to environmental and burial variables which require further study. The use of a soil probe to assist HRD dogs should not be disregarded but should only follow the use of non-invasive methods if deemed appropriate.