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

Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF) patients, we demonstrate its ability to survey thousands of bacteria (or bacterial aggregates) over millimeter scales and quantify aggregation of individual species in polymicrobial communities. By analyzing aggregation patterns of four prominent CF pathogens, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus sp., and Achromobacter xylosoxidans, we demonstrate a spectrum of aggregation states: from mostly single cells (A. xylosoxidans), to medium-sized clusters (S. aureus), to a mixture of single cells and large aggregates (P. aeruginosa and Streptococcus sp.). Furthermore, MiPACT-HCR revealed an intimate interaction between Streptococcus sp. and specific host cells. Lastly, by comparing standard rRNA fluorescence in situ hybridization signals to those from HCR, we found that different populations of S. aureus and A. xylosoxidans grow slowly overall yet exhibit growth rate heterogeneity over hundreds of microns. These results demonstrate the utility of MiPACT-HCR to directly capture the spatial organization and metabolic activity of bacteria in complex systems, such as human sputum.

Cystic fibrosis-related diabetes affects up to half of cystic fibrosis patients and is associated with increased mortality and more frequent pulmonary exacerbations. However, it is unclear to what degree good glycemic control might mitigate these risks and clinical outcomes have not previously been studied in relation to glucose from the lower airways, the site of infection and CF disease progression. We initially hypothesized that diabetic cystic fibrosis patients with glycosylated hemoglobin (HbA1c) > 6.5% have worse pulmonary function, longer and more frequent exacerbations and also higher sputum glucose levels than patients with HbA1c ≤ 6.5% or cystic fibrosis patients without diabetes. To test this, we analyzed spontaneously expectorated sputum samples from 88 cystic fibrosis patients. The median sputum glucose concentration was 0.70 mM (mean, 4.75 mM; range, 0-64.6 mM). Sputum glucose was not correlated with age, sex, body mass index, diabetes diagnosis, glycemic control, exacerbation frequency or length, or pulmonary function. Surprisingly, sputum glucose was highest in subjects with normal glucose tolerance, suggesting the dynamics of glycemic control, sputum glucose and pulmonary infections are more complex than previously thought. Two-year mean HbA1c was positively correlated with the length of exacerbation admission (p < 0.01), and negatively correlated with measures of pulmonary function (p < 0.01). While total number of hospitalizations for exacerbations were not significantly different, subjects with an HbA1c > 6.5% were hospitalized on average 6 days longer than those with HbA1c ≤ 6.5% (p < 0.01). Current clinical care guidelines for cystic fibrosis-related diabetes target HbA1c ≤ 7% to limit long-term microvascular damage, but more stringent glycemic control (HbA1c ≤ 6.5%) may further reduce the short-term pulmonary complications.