Elevated Carbon Monoxide in the Exhaled Breath of Mice during a Systemic Bacterial Infection Alan G. Barbour 1 *, Charlotte M. Hirsch 2 , Arash Ghalyanchi Langeroudi 1 , Simone Meinardi 2 , Eric R. G. Lewis 1 , Azadeh Shojaee Estabragh 1 , Donald R. Blake 2 1 Departments of Medicine and Microbiology & Molecular Genetics, University of California Irvine, Irvine, California, United States of America, 2 Department of Chemistry and the Environmental Molecular Sciences Institute, University of California Irvine, Irvine, California, United States of America Abstract Blood is the specimen of choice for most laboratory tests for diagnosis and disease monitoring. Sampling exhaled breath is a noninvasive alternative to phlebotomy and has the potential for real-time monitoring at the bedside. Improved instrumentation has advanced breath analysis for several gaseous compounds from humans. However, application to small animal models of diseases and physiology has been limited. To extend breath analysis to mice, we crafted a means for collecting nose-only breath samples from groups and individual animals who were awake. Samples were subjected to gas chromatography and mass spectrometry procedures developed for highly sensitive analysis of trace volatile organic compounds (VOCs) in the atmosphere. We evaluated the system with experimental systemic infections of severe combined immunodeficiency Mus musculus with the bacterium Borrelia hermsii. Infected mice developed bacterial densities of ,10 7 per ml of blood by day 4 or 5 and in comparison to uninfected controls had hepatosplenomegaly and elevations of both inflammatory and anti-inflammatory cytokines. While 12 samples from individual infected mice on days 4 and 5 and 6 samples from uninfected mice did not significantly differ for 72 different VOCs, carbon monoxide (CO) was elevated in samples from infected mice, with a mean (95% confidence limits) effect size of 4.2 (2.8–5.6), when differences in CO 2 in the breath were taken into account. Normalized CO values declined to the uninfected range after one day of treatment with the antibiotic ceftriaxone. Strongly correlated with CO in the breath were levels of heme oxygenase-1 protein in serum and HMOX1 transcripts in whole blood. These results (i) provide further evidence of the informativeness of CO concentration in the exhaled breath during systemic infection and inflammation, and (ii) encourage evaluation of this noninvasive analytic approach in other various other rodent models of infection and for utility in clinical management. Citation: Barbour AG, Hirsch CM, Ghalyanchi Langeroudi A, Meinardi S, Lewis ERG, et al. (2013) Elevated Carbon Monoxide in the Exhaled Breath of Mice during a Systemic Bacterial Infection. PLoS ONE 8(7): e69802. doi:10.1371/journal.pone.0069802 Editor: Ben Adler, Monash University, Australia Received February 25, 2013; Accepted June 13, 2013; Published July 31, 2013 Copyright: ß 2013 Barbour et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant U54 AI065359 to the Pacific-Southwest Regional Center of Excellence for Biodefense and Emerging Infections. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: abarbour@uci.edu Introduction Body temperature, respiratory rate, and pulse rhythm were known to the ancients as informative parameters for diagnosing, staging, and monitoring disease. Subsequent advances in bedside monitoring were through technical enhancement of human capabilities and include the electrocardiogram, sphygmomanom- eter, and oximeter. A lengthening list of laboratory-based assays of substances and cells of the blood, urine, and other specimens provide additional information on the bodily status. However, assays of blood are seldom performed at the bedside, and it may be hours or days before results are known. When a patient’s status is rapidly changing, and supportive and therapeutic maneuvers are underway, such as in an intensive care unit, real-time data on the function of the cardiovascular, respiratory, and other systems is of critical importance for distinguishing salutary from counterpro- ductive actions. Ancient physicians also recognized the diagnostic utility of smelling the exhaled breath for tell-tale aromas, such as with hepatic or renal failure [1]. For some conditions unaided olfaction of the practitioner was sufficiently accurate for clinical decision making. These examples inspired efforts to extend human olfaction by means of instrumentation that provided better sensitivity and discrimination. While the potential value of breath analysis for disease-specific diagnosis, such as in diabetes [2], cystic fibrosis [3], or cancer [4], is undoubted, our interest instead is in compounds in the breath that might provide a unique dimension for the assessment and monitoring of patients with a variety of conditions. To this end we initiated studies of experimental animals, but this required developing means for the noninvasive characterization of exhaled breath of individual mice. While there have been several examples of non-invasive breath analysis of humans (reviewed in [5]), nose-only methods have not to our knowledge been extended to laboratory mice who are awake and not intubated. A challenge has been the size of Mus musculus, which is ,0.04% the mass of an adult human. In addition, for breath analysis to be feasible at the bedside, sampling durations should be in minutes if not seconds. Here, we report non-invasive assessment of the exhaled breath of mice using analytical methods of atmospheric chemistry for trace gases. This approach was evaluated with a bacterial disease, Borrelia hermsii infection, with blood-borne dissemination and systemic inflammation [6]. After a survey for a large number of PLOS ONE | www.plosone.org 1 July 2013 | Volume 8 | Issue 7 | e69802