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

BackgroundVentilator-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.MethodsBALF 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.ResultsThirty-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)-1β was associated with reduced species evenness (rs = - 0.33, p s = 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).ConclusionPatients 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

Microbial volatiles as diagnostic biomarkers of bacterial lung infection in mechanically ventilated patients.

BackgroundEarly 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 non-invasive biomarkers of infection to support clinical diagnosis.MethodsIn this study, we investigated the diagnostic potential of in vitro confirmed mVOCs in the exhaled breath of patients under mechanically ventilation from the BreathDx study. Samples were analysed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS).ResultsPathogens from bronchoalveolar lavage (BAL) cultures were identified in 45/89 patients and S. aureus was the most commonly identified pathogen (n = 15). Out of 19 mVOCs detected in the in vitro culture headspace of four common respiratory pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli), 14 were found in exhaled breath samples. Higher concentrations of two mVOCs were found in the exhaled breath of patients infected with S. aureus compared to those without (3-methylbutanal p ConclusionsThis 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 multi-centre clinical studies

TD/GC–MS analysis of volatile markers emitted from mono- and co-cultures of Enterobacter cloacae and Pseudomonas aeruginosa in artificial sputum

Introduction: Infections such as ventilator-associated pneumonia (VAP) can be caused by one or more pathogens. Current methods for identifying these pathogenic microbes often require invasive sampling, and can be time consuming, due to the requirement for prolonged cultural enrichment along with selective and differential plating steps. This results in delays in diagnosis which in such critically ill patients can have potentially life-threatening consequences. Therefore, a non-invasive and timely diagnostic method is required. Detection of microbial volatile organic compounds (VOCs) in exhaled breath is proposed as an alternative method for identifying these pathogens and may distinguish between mono- and poly-microbial infections. Objectives: To investigate volatile metabolites that discriminate between bacterial mono- and co-cultures. Methods: VAP-associated pathogens Enterobacter cloacae and Pseudomonas aeruginosa were cultured individually and together in artificial sputum medium for 24 h and their headspace was analysed for potential discriminatory VOCs by thermal desorption gas chromatography–mass spectrometry. Results: Of the 70 VOCs putatively identified, 23 were found to significantly increase during bacterial culture (i.e. likely to be released during metabolism) and 13 decreased (i.e. likely consumed during metabolism). The other VOCs showed no transformation (similar concentrations observed as in the medium). Bacteria-specific VOCs including 2-methyl-1-propanol, 2-phenylethanol, and 3-methyl-1-butanol were observed in the headspace of axenic cultures of E. cloacae, and methyl 2-ethylhexanoate in the headspace of P. aeruginosa cultures which is novel to this investigation. Previously reported VOCs 1-undecene and pyrrole were also detected. The metabolites 2-methylbutyl acetate and methyl 2-methylbutyrate, which are reported to exhibit antimicrobial activity, were elevated in co-culture only. Conclusion: The observed VOCs were able to differentiate axenic and co-cultures. Validation of these markers in exhaled breath specimens could prove useful for timely pathogen identification and infection type diagnosis

Exhaled breath metabolomics reveals a pathogen-specific response in a rat pneumonia model for two human pathogenic bacteria:a proof-of-concept study

Volatile organic compounds in breath can reflect host and pathogen metabolism and might be used to diagnose pneumonia. We hypothesized that rats with Streptococcus pneumoniae (SP) or Pseudomonas aeruginosa (PA) pneumonia can be discriminated from uninfected controls by thermal desorption-gas chromatography-mass-spectrometry (TD-GC-MS) and selected ion flow tube-mass spectrometry (SIFT-MS) of exhaled breath. Male adult rats (n = 50) received an intratracheal inoculation of 1) 200 µl saline, or 2) 1 × 107 colony-forming units of SP or 3) 1 × 107 CFU of PA. Twenty-four hours later the rats were anaesthetized, tracheotomized, and mechanically ventilated. Exhaled breath was analyzed via TD-GC-MS and SIFT-MS. Area under the receiver operating characteristic curves (AUROCCs) and correct classification rate (CCRs) were calculated after leave-one-out cross-validation of sparse partial least squares-discriminant analysis. Analysis of GC-MS data showed an AUROCC (95% confidence interval) of 0.85 (0.73-0.96) and CCR of 94.6% for infected versus noninfected animals, AUROCC of 0.98 (0.94-1) and CCR of 99.9% for SP versus PA, 0.92 (0.83-1.00), CCR of 98.1% for SP versus controls and 0.97 (0.92-1.00), and CCR of 99.9% for PA versus controls. For these comparisons the SIFT-MS data showed AUROCCs of 0.54, 0.89, 0.63, and 0.79, respectively. Exhaled breath analysis discriminated between respiratory infection and no infection but with even better accuracy between specific pathogens. Future clinical studies should not only focus on the presence of respiratory infection but also on the discrimination between specific pathogens

Untargeted Molecular Analysis of Exhaled Breath as a Diagnostic Test for Ventilator-Associated Lower Respiratory Tract Infections (BreathDx)

Patients suspected of ventilator-associated lower respiratory tract infections (VA-LRTIs) commonly receive broad-spectrum antimicrobial therapy unnecessarily. We tested whether exhaled breath analysis can discriminate between patients suspected of VA-LRTI with confirmed infection, from patients with negative cultures. Breath from 108 patients suspected of VA-LRTI was analysed by gas chromatography-mass spectrometry. The breath test had a sensitivity of 98% at a specificity of 49%, confirmed with a second analytical method. The breath test had a negative predictive value of 96% and excluded pneumonia in half of the patients with negative cultures. Trial registration number: UKCRN ID number 19086, registered May 201