Trace incorporation of heavy water reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum

Abstract

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

Gel-Entrapped Staphylococcus aureus Bacteria as Models of Biofilm Infection Exhibit Growth in Dense Aggregates, Oxygen Limitation, Antibiotic Tolerance, and Heterogeneous Gene Expression

“…After a day of incubation, the specific growth rate was lowered to less than 10% of its maximum value, with estimates by two different approaches being 0.06 h Ϫ1 and 0.02 h Ϫ1 . These measurements recapitulate the observation of slow growth that has been reported in many conventional biofilm systems (23)(24)(25)(26) and has recently been demonstrated by ex vivo measurements of bacterial growth rates in sputum specimens from people with cystic fibrosis (27) and in a rat model of prosthetic joint infection (28). For example, the median growth rate of S. aureus in the sputum of people with cystic fibrosis was 0.014 h Ϫ1 (27), and a typical value in the rodent joint was 0.087 h Ϫ1 (28).…”

Measurement of bacterial replication rates in microbial communities

“…Measurements in such environments are very challenging using alternative approaches such as isotope tracing 26 . These studies typically target specific organisms, and such measurements have only recently been implemented in the human lung microbiome 26 . Large-scale comparisons using PTR are not possible due to a lack of complete reference genomes.…”

Global and local selection acting on the pathogen Stenotrophomonas maltophilia in the human lung

“…Mutations can sweep through the population8 or lead to diversification into distinct subpopulations that differ genotypically and phenotypically5679101112131415. Subpopulations within an individual can be spatially segregated across the structured environment of the human body1416171819, but it is unknown to what extent such spatial variations reflect local differences in selective pressures. Separating which genes are important for the local versus global adaptation of a pathogen is critical for predicting and possibly manipulating the survival strategy of the pathogen.…”