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

Abstract

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.

Sniffing animals as a diagnostic tool in infectious diseases

“…whether 1-propanol is related to the presence of E. coli), and no breath test has been shown to have sufficient diagnostic accuracy for pneumonia. However, recent proof-ofconcept studies performed as part of the BreathDx consortium [39] showed that exhaled breath metabolomics measured by TD-GC-MS reveal a pathogen-specific response in a rat pneumonia model for S. pneumoniae and P. aeruginosa [40], and in an artificial sputum model from monocultures and co-cultures of Enterobacter cloacae and P. aeruginosa [41].…”

carP, encoding a Ca2+-regulated putative phytase, is evolutionarily conserved in Pseudomonas aeruginosa and has potential as a biomarker

“…The early established and commonly used strategies for detecting P. aeruginosa in clinics mostly rely on using selective growth media, phenotypic characterization, or amplification and sequencing of 16S rRNA gene, oprL [22], algD [23] or toxA genes [24]. More recently proposed methods for identifying P. aeruginosa include detection of pyocyanin by a silver/gold chip [25], mass spectrometry (MS) of volatile compounds, such as 2-aminoacetophenone, in patient breath samples [26,27] and detection of specific host proteins produced in response to P. aeruginosa infections [28]. While these methodologies are proven useful, they require expensive equipment and processing.…”

“…More recently proposed methods for identifying P. aeruginosa include detection of pyocyanin by a silver/gold chip [25], mass spectrometry (MS) of volatile compounds, such as 2-aminoacetophenone, in patient breath samples [26, 27] and detection of specific host proteins produced in response to P. aeruginosa infections [28].…”

Challenges and Progress Toward Determining Pneumonia Etiology

“…Such an innovative and minimally invasive approach may be the detection of pathogen-specific antibodysecreting cells (Figure 1) [24], which allow differentiation between M. pneumoniae infection and carriage in children with CAP [14]. Other promising diagnostic approaches are exhaled breath analysis [25], novel biomarkers [26], new point-ofcare and antigen detection assays [27], multidimensional (molecular) assessment of the host response [28], and new analytical approaches [11]. Efforts to determine the microbial etiology and understand the complex pathophysiology of pneumonia are key to reducing antibiotic overuse and resistance.…”