Influence of bacterial and alveolar cell co-culture on microbial VOC production using HS-GC/MS.

Volatile organic compounds (VOCs) found in exhaled breath continue to garner interest as an alternative diagnostic tool in pulmonary infections yet, their clinical integration remains a challenge with difficulties in translating identified biomarkers. Alterations in bacterial metabolism secondary to host nutritional availability may explain this but is often inadequately modelled in vitro. The influence of more clinically relevant nutrients on VOC production for two common respiratory pathogens was investigated. VOCs from Staphylococcus aureus (S.aureus) and Pseudomonas aeruginosa (P.aeruginosa) cultured with and without human alveolar A549 epithelial cells were analyzed using headspace extraction coupled with gas chromatography-mass spectrometry. Untargeted and targeted analyses were performed, volatile molecules identified from published data, and the differences in VOC production evaluated. Principal component analysis (PCA) could differentiate alveolar cells from either S. aureus or P. aeruginosa when cultured in isolation based on PC1 (p = 0.0017 and 0.0498, respectively). However, this separation was lost for S. aureus (p = 0.31) but not for P. aeruginosa (p = 0.028) when they were cultured with alveolar cells. S. aureus cultured with alveolar cells led to higher concentrations of two candidate biomarkers, 3-methyl-1-butanol (p = 0.001) and 3-methylbutanal (p = 0.002) when compared to S. aureus, alone. P. aeruginosa metabolism resulted in less generation of pathogen-associated VOCs when co-cultured with alveolar cells compared to culturing in isolation. VOC biomarkers previously considered indicative of bacterial presence are influenced by the local nutritional environment and this should be considered when evaluating their biochemical origin.

Microbial Volatiles as Diagnostic Biomarkers of Bacterial Lung Infection in Mechanically Ventilated Patients.

BACKGROUND: Early and accurate recognition of respiratory pathogens is crucial to prevent increased risk of mortality in critically ill patients. Microbial-derived volatile organic compounds (mVOCs) in exhaled breath could be used as noninvasive biomarkers of infection to support clinical diagnosis. METHODS: In this study, we investigated the diagnostic potential of in vitro-confirmed mVOCs in the exhaled breath of patients under mechanical ventilation from the BreathDx study. Samples were analyzed by thermal desorption-gas chromatography-mass spectrometry. RESULTS: Pathogens from bronchoalveolar lavage (BAL) cultures were identified in 45 of 89 patients and Staphylococcus aureus was the most commonly identified pathogen (n = 15). Of 19 mVOCs detected in the in vitro culture headspace of 4 common respiratory pathogens (S. aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli), 14 were found in exhaled breath samples. Higher concentrations of 2 mVOCs were found in the exhaled breath of patients infected with S. aureus compared to those without (3-methylbutanal: P < .01, area under the receiver operating characteristic curve [AUROC] = 0.81-0.87; and 3-methylbutanoic acid: P = .01, AUROC = 0.79-0.80). In addition, bacteria identified from BAL cultures that are known to metabolize tryptophan (E. coli, Klebsiella oxytoca, and Haemophilus influenzae) were grouped and found to produce higher concentrations of indole compared to breath samples with culture-negative (P = .034) and other pathogen-positive (P = .049) samples. CONCLUSIONS: This study demonstrates the capability of using mVOCs to detect the presence of specific pathogen groups with potential to support clinical diagnosis. Although not all mVOCs were found in patient samples within this small pilot study, further targeted and qualitative investigation is warranted using multicenter clinical studies.

Molecular phenotyping approaches for the detection and monitoring of carbapenem-resistant Enterobacteriaceae by mass spectrometry.

Antimicrobial resistance is increasing in prevalence and there is a clear need for the development of rapid detection methods in clinical diagnostics. This review explores -omics studies utilising mass spectrometry to investigate the molecular phenotype associated with carbapenem resistance. Whilst the specific mechanisms of carbapenem resistance are well characterised, the resistant phenotype is poorly understood. Understanding how the acquisition of resistance affects cellular physiology and cell metabolism through molecular phenotyping is a necessary step towards detecting resistance by diagnostic means. In addition, this article examines the potential of mass spectrometry for the identification of resistance biomarkers through molecular profiling of bacteria. Developments in mass spectrometry platforms are expanding the biomarker-based diagnostic landscape. Targeted measures, such as high-resolution mass spectrometry coupled with chromatographic separation show considerable promise for the identification of molecular signatures and the development of a rapid diagnostic assay for the detection of carbapenem resistance.

Metabolic phenotyping of acquired ampicillin resistance using microbial volatiles from Escherichia coli cultures.

AIMS: This study sought to assess the volatile organic compound (VOC) profiles of ampicillin-resistant and -susceptible Escherichia coli to evaluate whether VOC analysis may be utilized to identify resistant phenotypes. METHODS AND RESULTS: An E. coli BL21 (DE3) strain and its pET16b plasmid transformed ampicillin-resistant counterpart were cultured for 6 h in drug-free, low- and high-concentrations of ampicillin. Headspace analysis was undertaken using thermal desorption-gas chromatography-mass spectrometry. Results revealed distinct VOC profiles with ampicillin-resistant bacteria distinguishable from their susceptible counterparts using as few as six compounds. A minimum of 30 compounds (fold change >2, p ≤ 0.05) were differentially expressed between the strains across all set-ups. Furthermore, three compounds (indole, acetoin and 3-methyl-1-butanol) were observed to be significantly more abundant (fold change >2, p ≤ 0.05) in the resistant strain compared to the susceptible strain both in the presence and in the absence of drug stress. CONCLUSIONS: Results indicate that E. coli with acquired ampicillin resistance exhibit an altered VOC profile compared to their susceptible counterpart both in the presence and in the absence of antibiotic stress. This suggests that there are fundamental differences between the metabolisms of ampicillin-resistant and -susceptible E. coli which may be detected by means of VOC analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that VOC profiles may be utilized to differentiate between resistant and susceptible bacteria using just six compounds. Consequently, the development of machine-learning models using VOC signatures shows considerable diagnostic applicability for the rapid and accurate detection of antimicrobial resistance.

Composition and diversity analysis of the lung microbiome in patients with suspected ventilator-associated pneumonia.

BACKGROUND: Ventilator-associated pneumonia (VAP) is associated with high morbidity and health care costs, yet diagnosis remains a challenge. Analysis of airway microbiota by amplicon sequencing provides a possible solution, as pneumonia is characterised by a disruption of the microbiome. However, studies evaluating the diagnostic capabilities of microbiome analysis are limited, with a lack of alignment on possible biomarkers. Using bronchoalveolar lavage fluid (BALF) from ventilated adult patients suspected of VAP, we aimed to explore how key characteristics of the microbiome differ between patients with positive and negative BALF cultures and whether any differences could have a clinically relevant role. METHODS: BALF from patients suspected of VAP was analysed using 16s rRNA sequencing in order to: (1) differentiate between patients with and without a positive culture; (2) determine if there was any association between microbiome diversity and local inflammatory response; and (3) correctly identify pathogens detected by conventional culture. RESULTS: Thirty-seven of 90 ICU patients with suspected VAP had positive cultures. Patients with a positive culture had significant microbiome dysbiosis with reduced alpha diversity. However, gross compositional variance was not strongly associated with culture positivity (AUROCC range 0.66-0.71). Patients with a positive culture had a significantly higher relative abundance of pathogenic bacteria compared to those without [0.45 (IQR 0.10-0.84), 0.02 (IQR 0.004-0.09), respectively], and an increased interleukin (IL)-1ß was associated with reduced species evenness (rs = - 0.33, p < 0.01) and increased pathogenic bacteria presence (rs = 0.28, p = 0.013). Untargeted 16s rRNA pathogen detection was limited by false positives, while the use of pathogen-specific relative abundance thresholds showed better diagnostic accuracy (AUROCC range 0.89-0.998). CONCLUSION: Patients with positive BALF culture had increased dysbiosis and genus dominance. An increased caspase-1-dependent IL-1b expression was associated with a reduced species evenness and increased pathogenic bacterial presence, providing a possible causal link between microbiome dysbiosis and lung injury development in VAP. However, measures of diversity were an unreliable predictor of culture positivity and 16s sequencing used agnostically could not usefully identify pathogens; this could be overcome if pathogen-specific relative abundance thresholds are used.

Exhaled volatile organic compounds as markers for medication use in asthma.

INTRODUCTION: Asthma is a heterogeneous condition, characterised by chronic inflammation of the airways, typically managed with inhaled bronchodilators and corticosteroids. In the case of uncontrolled asthma, oral corticosteroids (OCSs) are often prescribed. Good adherence and inhalation technique are associated with improved outcomes; however, it is difficult to monitor appropriate drug intake and effectiveness in individual patients. Exhaled breath contains thousands of volatile organic compounds (VOCs) that reflect changes in the body's chemistry and may be useful for monitoring drug pharmacokinetics/pharmacodynamics. We aimed to investigate the association of exhaled VOCs in severe asthma patients from the U-BIOPRED cohort (by gas chromatography coupled with time-of-flight mass spectrometry) with urinary levels of salbutamol and OCSs (by liquid chromatography coupled with high-resolution mass spectrometry). METHODS: Samples were collected at baseline and after 12-18 months of follow-up. Statistical analysis was based on univariate and multivariate modelling, followed by area under the receiver operating characteristic curve (AUC) calculation. Results were verified through longitudinal replication and independent validation. RESULTS: Data were available for 78 patients (baseline n=48, replication n=30 and validation n=30). Baseline AUC values were 82.1% (95% CI 70.4-93.9%) for salbutamol and 78.8% (95% CI 65.8-91.8%) for OCS. These outcomes could be adequately replicated and validated. Additional regression analysis between qualified exhaled VOCs and urinary concentrations of salbutamol and prednisone showed statistically significant correlations (p<0.01). CONCLUSION: We have linked exhaled VOCs to urinary detection of salbutamol and OCSs. This merits further development of breathomics into a point-of-care tool for therapeutic drug monitoring.

Methodological considerations for large-scale breath analysis studies: lessons from the U-BIOPRED severe asthma project.

Methods for breath sampling and analysis require robust quality assessment to minimise the risk of false discoveries. Planning large-scale multi-site breath metabolite profiling studies also requires careful consideration of systematic and random variation as a result of sampling and analysis techniques. In this study we use breath sample data from the recent U-BIOPRED cohort to evaluate and discuss some important methodological considerations such as batch variation and correction, variation between sites, storage and transportation, as well as inter-instrument analytical differences. Based on this we provide a summary of recommended best practices for new large scale multi-site studies.

Development of an adaptable headspace sampling method for metabolic profiling of the fungal volatome.

Pulmonary aspergillosis can cause serious complications in people with a suppressed immune system. Volatile metabolites emitted by Aspergillus spp. have shown promise for early detection of pathogenicity. However, volatile profiles require further research, as effective headspace analysis methods are required for extended chemical coverage of the volatome; in terms of both very volatile and semi-volatile compounds. In this study, we describe a novel adaptable sampling method in which fungal headspace samples can be sampled continuously throughout a defined time period using both active (pumped) and passive (diffusive) methods, with the capability for samples to be stored for later off-line analysis. For this method we utilise thermal desorption-gas chromatography-mass spectrometry to generate volatile metabolic profiles using Aspergillus fumigatus as the model organism. Several known fungal-specific volatiles associated with secondary metabolite biosynthesis (including α-pinene, camphene, limonene, and several sesquiterpenes) were identified. A comparison between the wild-type A. fumigatus with a phosphopantetheinyl transferase null mutant strain (ΔpptA) that is compromised in secondary metabolite synthesis, revealed reduced production of sesquiterpenes. We also showed the lack of terpene compounds production during the early growth phase, whilst pyrazines were identified in both early and late growth phases. We have demonstrated that the fungal volatome is dynamic and it is therefore critically necessary to sample the headspace across several time periods using a combination of active and passive sampling techniques to analyse and understand this dynamism.

Headspace volatile organic compounds from bacteria implicated in ventilator-associated pneumonia analysed by TD-GC/MS.

Ventilator-associated pneumonia (VAP) is a healthcare-acquired infection arising from the invasion of the lower respiratory tract by opportunistic pathogens in ventilated patients. The current method of diagnosis requires the culture of an airway sample such as bronchoalveolar lavage, which is invasive to obtain and may take up to seven days to identify a causal pathogen, or indeed rule out infection. While awaiting results, patients are administered empirical antibiotics; risks of this approach include lack of effect on the causal pathogen, contribution to the development of antibiotic resistance and downstream effects such as increased length of intensive care stay, cost, morbidity and mortality. Specific biomarkers which could identify causal pathogens in a timely manner are needed as they would allow judicious use of the most appropriate antimicrobial therapy. Volatile organic compound (VOC) analysis in exhaled breath is proposed as an alternative due to its non-invasive nature and its potential to provide rapid diagnosis at the patient's bedside. VOCs in exhaled breath originate from exogenous, endogenous, as well as microbial sources. To identify potential markers, VAP-associated pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus were cultured in both artificial sputum medium and nutrient broth, and their headspaces were sampled and analysed for VOCs. Previously reported volatile markers were identified in this study, including indole and 1-undecene, alongside compounds that are novel to this investigation, cyclopentanone and 1-hexanol. We further investigated media components (substrates) to identify those that are essential for indole and cyclopentanone production, with potential implications for understanding microbial metabolism in the lung.

Exhaled breath analysis: a review of 'breath-taking' methods for off-line analysis.

BACKGROUND: The potential of exhaled breath sampling and analysis has long attracted interest in the areas of medical diagnosis and disease monitoring. This interest is attributed to its non-invasive nature, access to an unlimited sample supply (i.e., breath), and the potential to facilitate a rapid at patient diagnosis. However, progress from laboratory setting to routine clinical practice has been slow. Different methodologies of breath sampling, and the consequent difficulty in comparing and combining data, are considered to be a major contributor to this. To fulfil the potential of breath analysis within clinical and pre-clinical medicine, standardisation of some approaches to breath sampling and analysis will be beneficial. OBJECTIVES: The aim of this review is to investigate the heterogeneity of breath sampling methods by performing an in depth bibliometric search to identify the current state of art in the area. In addition, the review will discuss and critique various breath sampling methods for off-line breath analysis. METHODS: Literature search was carried out in databases MEDLINE, BIOSIS, EMBASE, INSPEC, COMPENDEX, PQSCITECH, and SCISEARCH using the STN platform which delivers peer-reviewed articles. Keywords searched for include breath, sampling, collection, pre-concentration, volatile. Forward and reverse search was then performed on initially included articles. The breath collection methodologies of all included articles was subsequently reviewed. RESULTS: Sampling methods differs between research groups, for example regarding the portion of breath being targeted. Definition of late expiratory breath varies between studies. CONCLUSIONS: Breath analysis is an interdisciplinary field of study using clinical, analytical chemistry, data processing, and metabolomics expertise. A move towards standardisation in breath sampling is currently being promoted within the breath research community with a view to harmonising analysis and thereby increasing robustness and inter-laboratory comparisons.

Exhaled Volatile Organic Compounds of Infection: A Systematic Review.

With heightened global concern of microbial drug resistance, advanced methods for early and accurate diagnosis of infection are urgently needed. Analysis of exhaled breath volatile organic compounds (VOCs) toward detecting microbial infection potentially allows a highly informative and noninvasive alternative to current genomics and culture-based methods. We performed a systematic review of research literature reporting human and animal exhaled breath VOCs related to microbial infections. In this Review, we find that a wide range of breath sampling and analysis methods are used by researchers, which significantly affects interstudy method comparability. Studies either perform targeted analysis of known VOCs relating to an infection, or non-targeted analysis to obtain a global profile of volatile metabolites. In general, the field of breath analysis is still relatively immature, and there is much to be understood about the metabolic production of breath VOCs, particularly in a host where both commensal microflora as well as pathogenic microorganisms may be manifested in the airways. We anticipate that measures to standardize high throughput sampling and analysis, together with an increase in large scale collaborative international trials, will bring routine breath VOC analysis to improve diagnosis of infection closer to reality.