Severe and persistent bacterial lung infections characterize cystic fibrosis (CF). While several studies have documented the microbial diversity within CF lung mucus, we know much less about the inorganic chemistry that constrains microbial metabolic processes and their distribution. We hypothesized that sputum is chemically heterogeneous both within and between patients. To test this, we measured microprofiles of oxygen and sulfide concentrations as well as pH and oxidation-reduction potentials in 48 sputum samples from 22 pediatric patients with CF. Inorganic ions were measured in 20 samples from 12 patients. In all cases, oxygen was depleted within the first few millimeters below the sputum-air interface. Apart from this steep oxycline, anoxia dominated the sputum environment. Different sputum samples exhibited a broad range of redox conditions, with either oxidizing (16 mV to 355 mV) or reducing (−300 to −107 mV) potentials. The majority of reduced samples contained hydrogen sulfide and had a low pH (2.9 to 6.5). Sulfide concentrations increased at a rate of 0.30 µM H2S/min. Nitrous oxide was detected in only one sample that also contained sulfide. Microenvironmental variability was observed both within a single patient over time and between patients. Modeling oxygen dynamics within CF mucus plugs indicates that anoxic zones vary as a function of bacterial load and mucus thickness and can occupy a significant portion of the mucus volume. Thus, aerobic respiration accounts only partially for pathogen survival in CF sputum, motivating research to identify mechanisms of survival under conditions that span fluctuating redox states, including sulfidic environments.

Effective treatment for chronic infections is undermined by a significant gap in understanding of the physiological state of pathogens at the site of infection. Chronic pulmonary infections are responsible for the morbidity and mortality of millions of immunocompromised individuals worldwide, yet drugs that are successful in laboratory culture are far less effective against pathogen populations persisting in vivo. Laboratory models, upon which preclinical development of new drugs is based, can only replicate host conditions when we understand the metabolic state of the pathogens and the degree of heterogeneity within the population. In this study, we measured the anabolic activity of the pathogen Staphylococcus aureus directly in the sputum of pediatric patients with cystic fibrosis (CF), by combining the high sensitivity of isotope ratio mass spectrometry with a heavy water labeling approach to capture the full range of in situ growth rates. Our results reveal S. aureus generation times with a median of 2.1 d, with extensive growth rate heterogeneity at the single-cell level. These growth rates are far below the detection limit of previous estimates of CF pathogen growth rates, and the rates are slowest in acutely sick patients undergoing pulmonary exacerbations; nevertheless, they are accessible to experimental replication within laboratory models. Treatment regimens that include specific antibiotics (vancomycin, piperacillin/tazobactam, tobramycin) further appear to correlate with slow growth of S. aureus on average, but follow-up longitudinal studies must be performed to determine whether this effect holds for individual patients.slow growth | infectious disease | metabolic heterogeneity | cystic fibrosis | hydrogen isotope labeling

To measure single cell microbial activity and substrate utilization patterns in environmental systems, we employ a new technique using stable isotope labeling of microbial populations with heavy water (a passive tracer) and 15N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single cell isotopic compositions to the corresponding bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single cell microbial activities ranging from generational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % 2H). Applying this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 hours, 1 day and 2 week generation times), we observe the greatest anabolic activity diversity in the slowest growing populations. By using heavy water to constrain cellular growth activity, we can further infer the relative contributions of ammonium vs. amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle individual cell activities even in nutritionally complex environments.