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Thesis
Peer Reviewed

Polymicrobial Infections in Cystic Fibrosis Lungs: The Need for Personalized Treatment

Lim, Yan Wei
Advisor(s): Rohwer, Forest L

UC San Diego Electronic Theses and Dissertations (2015)

Polymicrobial infection defines a combination of microorganisms, including viruses, bacteria, fungi, and sometimes parasites, which are simultaneously associated with an infected site. While the idea of polymicrobial infections is common nowadays, most clinical laboratories still focus on the culturing and identification of a single pathogen. The biological implications attributed to these singular microbial infections are deceptive, especially when considering the multiple complex interactions associated with polymicrobial infections. This dissertation used Cystic Fibrosis (CF) as a study system for polymicrobial infections in the lung. CF is a genetic disease caused by mutations in the Cystic Fibrosis Transmembrane Regulator (CFTR) gene. CF affects multiple organs across the body, but pulmonary infection remains the main cause of morbidity and mortality. Studying CF lungs is challenging. Here a set of comprehensive methods was developed to simultaneously study the viral, microbial, and host genetics using metagenomics and metatranscriptomics approaches. Many of the bacteria found in the lungs of CF patients are of oral origin. Detailed genetic analyses showed that these bacteria adapt to the CF lungs by acquiring essential genes while losing non-essential ones. One example is Rothia mucilaginosa – an organism that is present in more than 80% of San Diego patients. De novo assembly of a near-complete R. mucilaginosa genome showed potential physiological adaptations through the acquisition of multiple genes. A survey of more than 20 CF patients in San Diego revealed that every patient harbors a unique microbial community, suggesting that CF patients require personalized medicine for the treatment of their lung infections. Despite these personalized differences in microbial taxonomical profiles across CF patients, the functional potential across these different microbial communities is highly similar. The conserved functional potential in CF microbes allows them to carry out aerobic and anaerobic respirations, as well as fermentation, highlighting the importance of anaerobes in the CF lungs. CF microbes regulate their metabolic activities in response to perturbations. In vitro community culturing of CF microbes showed that the anaerobes were sensitive to antibiotics commonly used in CF patients and their metabolic activities could be associated with the patient’s health. The role and importance of CF anaerobes and their survival mechanisms are illustrated in this study. In summary, this dissertation provides novel insights into polymicrobial infections in CF lungs and demonstrates the potential and advantages of coupling omics and clinical approaches for the study of other complex polymicrobial infections.

Cover page: Polymicrobial Infections in Cystic Fibrosis Lungs: The Need for Personalized Treatment

Article
Peer Reviewed

Ecological networking of cystic fibrosis lung infections

UC Irvine Previously Published Works (2016)

In the context of a polymicrobial infection, treating a specific pathogen poses challenges because of unknown consequences on other members of the community. The presence of ecological interactions between microbes can change their physiology and response to treatment. For example, in the cystic fibrosis lung polymicrobial infection, antimicrobial susceptibility testing on clinical isolates is often not predictive of antibiotic efficacy. Novel approaches are needed to identify the interrelationships within the microbial community to better predict treatment outcomes. Here we used an ecological networking approach on the cystic fibrosis lung microbiome characterized using 16S rRNA gene sequencing and metagenomics. This analysis showed that the community is separated into three interaction groups: Gram-positive anaerobes, Pseudomonas aeruginosa, and Staphylococcus aureus. The P. aeruginosa and S. aureus groups both anti-correlate with the anaerobic group, indicating a functional antagonism. When patients are clinically stable, these major groupings were also stable, however, during exacerbation, these communities fragment. Co-occurrence networking of functional modules annotated from metagenomics data supports that the underlying taxonomic structure is driven by differences in the core metabolism of the groups. Topological analysis of the functional network identified the non-mevalonate pathway of isoprenoid biosynthesis as a keystone for the microbial community, which can be targeted with the antibiotic fosmidomycin. This study uses ecological theory to identify novel treatment approaches against a polymicrobial disease with more predictable outcomes.

Cover page: Ecological networking of cystic fibrosis lung infections

Article
Peer Reviewed

Metabolomics of pulmonary exacerbations reveals the personalized nature of cystic fibrosis disease

UC Irvine Previously Published Works (2016)

Background. Cystic fibrosis (CF) is a genetic disease that results in chronic infections of the lungs. CF patients experience intermittent pulmonary exacerbations (CFPE) that are associated with poor clinical outcomes. CFPE involves an increase in disease symptoms requiring more aggressive therapy. Methods. Longitudinal sputum samples were collected from 11 patients (n = 44 samples) to assess the effect of exacerbations on the sputum metabolome using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The data was analyzed with MS/MS molecular networking and multivariate statistics. Results. The individual patient source had a larger influence on the metabolome of sputum than the clinical state (exacerbation, treatment, post-treatment, or stable). Of the 4,369 metabolites detected, 12% were unique to CFPE samples; however, the only known metabolites significantly elevated at exacerbation across the dataset were platelet activating factor (PAF) and a related monacylglycerophosphocholine lipid. Due to the personalized nature of the sputum metabolome, a single patient was followed for 4.2 years (capturing four separate exacerbation events) as a case study for the detection of personalized biomarkers with metabolomics. PAF and related lipids were significantly elevated during CFPEs of this patient and ceramide was elevated during CFPE treatment. Correlating the abundance of bacterial 16S rRNA gene amplicons to metabolomics data from the same samples during a CFPE demonstrated that antibiotics were positively correlated to Stenotrophomonas and Pseudomonas, while ceramides and other lipids were correlated with Streptococcus, Rothia, and anaerobes. Conclusions. This study identified PAF and other inflammatory lipids as potential biomarkers of CFPE, but overall, the metabolome of CF sputum was patient specific, supporting a personalized approach to molecular detection of CFPE onset.

Cover page: Metabolomics of pulmonary exacerbations reveals the personalized nature of cystic fibrosis disease

Article
Peer Reviewed

Biogeochemical Forces Shape the Composition and Physiology of Polymicrobial Communities in the Cystic Fibrosis Lung

UC Irvine Previously Published Works (2014)

The cystic fibrosis (CF) lung contains thick mucus colonized by opportunistic pathogens which adapt to the CF lung environment over decades. The difficulty associated with sampling airways has impeded a thorough examination of the biochemical microhabitats these pathogens are exposed to. An indirect approach is to study the responses of microbial communities to these microhabitats, facilitated by high-throughput sequencing of microbial DNA and RNA from sputum samples. Microbial metagenomes and metatranscriptomes were sequenced from multiple CF patients, and the reads were assigned taxonomy and function through sequence homology to NCBI and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database hierarchies. For a comparison, saliva microbial metagenomes from the Human Microbiome Project (HMP) were also analyzed. These analyses identified that functions encoded and expressed by CF microbes were significantly enriched for amino acid catabolism, folate biosynthesis, and lipoic acid biosynthesis. The data indicate that the community uses oxidative phosphorylation as a major energy source but that terminal electron acceptors were diverse. Nitrate reduction was the most abundant anaerobic respiratory pathway, and genes for nitrate reductase were largely assigned to Pseudomonas and Rothia. Although many reductive pathways of the nitrogen cycle were present, the cycle was incomplete, because the oxidative pathways were absent. Due to the abundant amino acid catabolism and incomplete nitrogen cycle, the CF microbial community appears to accumulate ammonia. This finding was verified experimentally using a CF bronchiole culture model system. The data also revealed abundant sensing and transport of iron, ammonium, zinc, and other metals along with a low-oxygen environment. This study reveals the core biochemistry and physiology of the CF microbiome. IMPORTANCE The cystic fibrosis (CF) microbial community is complex and adapts to the environmental conditions of the lung over the lifetime of a CF patient. This analysis illustrates the core functions of the CF microbial community in the context of CF lung biochemistry. There are many studies of the metabolism and physiology of individual microbes within the CF lung, but none that collectively analyze data from the whole microbiome. Understanding the core metabolism of microbes that inhabit the CF lung can provide new targets for novel therapies. The fundamental processes that CF pathogens rely on for survival may represent an Achilles heel for this pathogenic community. Novel therapies that are designed to disrupt understudied survival strategies of the CF microbial community may succeed against otherwise untreatable or antibiotic-resistant microbes.

Cover page: Biogeochemical Forces Shape the Composition and Physiology of Polymicrobial Communities in the Cystic Fibrosis Lung

Article
Peer Reviewed

Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

UC Irvine Previously Published Works (2014)

The airways of cystic fibrosis (CF) patients are chronically colonized by patient-specific polymicrobial communities. The conditions and nutrients available in CF lungs affect the physiology and composition of the colonizing microbes. Recent work in bioreactors has shown that the fermentation product 2,3-butanediol mediates cross-feeding between some fermenting bacteria and Pseudomonas aeruginosa, and that this mechanism increases bacterial current production. To examine bacterial fermentation in the respiratory tract, breath gas metabolites were measured and several metagenomes were sequenced from CF and non-CF volunteers. 2,3-butanedione was produced in nearly all respiratory tracts. Elevated levels in one patient decreased during antibiotic treatment, and breath concentrations varied between CF patients at the same time point. Some patients had high enough levels of 2,3-butanedione to irreversibly damage lung tissue. Antibiotic therapy likely dictates the activities of 2,3-butanedione-producing microbes, which suggests a need for further study with larger sample size. Sputum microbiomes were dominated by P. aeruginosa, Streptococcus spp. and Rothia mucilaginosa, and revealed the potential for 2,3-butanedione biosynthesis. Genes encoding 2,3-butanedione biosynthesis were disproportionately abundant in Streptococcus spp, whereas genes for consumption of butanedione pathway products were encoded by P. aeruginosa and R. mucilaginosa. We propose a model where low oxygen conditions in CF lung lead to fermentation and a decrease in pH, triggering 2,3-butanedione fermentation to avoid lethal acidification. We hypothesize that this may also increase phenazine production by P. aeruginosa, increasing reactive oxygen species and providing additional electron acceptors to CF microbes.

Cover page: Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate active pH neutral 2,3-butanedione fermentation

Article
Peer Reviewed

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.

UC Irvine Previously Published Works (2015)

Article
Peer Reviewed

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

UC Irvine Previously Published Works (2015)

There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.

Article
Peer Reviewed

Microbial, host and xenobiotic diversity in the cystic fibrosis sputum metabolome

UC Irvine Previously Published Works (2016)

Cystic fibrosis (CF) lungs are filled with thick mucus that obstructs airways and facilitates chronic infections. Pseudomonas aeruginosa is a significant pathogen of this disease that produces a variety of toxic small molecules. We used molecular networking-based metabolomics to investigate the chemistry of CF sputa and assess how the microbial molecules detected reflect the microbiome and clinical culture history of the patients. Metabolites detected included xenobiotics, P. aeruginosa specialized metabolites and host sphingolipids. The clinical culture and microbiome profiles did not correspond to the detection of P. aeruginosa metabolites in the same samples. The P. aeruginosa molecules that were detected in sputum did not match those from laboratory cultures. The pseudomonas quinolone signal (PQS) was readily detectable from cultured strains, but absent from sputum, even when its precursor molecules were present. The lack of PQS production in vivo is potentially due to the chemical nature of the CF lung environment, indicating that culture-based studies of this pathogen may not explain its behavior in the lung. The most differentially abundant molecules between CF and non-CF sputum were sphingolipids, including sphingomyelins, ceramides and lactosylceramide. As these highly abundant molecules contain the inflammatory mediator ceramide, they may have a significant role in CF hyperinflammation. This study demonstrates that the chemical makeup of CF sputum is a complex milieu of microbial, host and xenobiotic molecules. Detection of a bacterium by clinical culturing and 16S rRNA gene profiling do not necessarily reflect the active production of metabolites from that bacterium in a sputum sample.

Cover page: Microbial, host and xenobiotic diversity in the cystic fibrosis sputum metabolome

Article
Peer Reviewed

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation.

UC Irvine Previously Published Works (2015)

There is a poor understanding of how the physiology of polymicrobial communities in cystic fibrosis (CF) lungs contributes to pulmonary exacerbations and lung function decline. In this study, a microbial culture system based on the principles of the Winogradsky column (WinCF system) was developed to study the physiology of CF microbes. The system used glass capillary tubes filled with artificial sputum medium to mimic a clogged airway bronchiole. Chemical indicators were added to observe microbial physiology within the tubes. Characterization of sputum samples from seven patients showed variation in pH, respiration, biofilm formation and gas production, indicating that the physiology of CF microbial communities varied among patients. Incubation of homogenized tissues from an explant CF lung mirrored responses of a Pseudomonas aeruginosa pure culture, supporting evidence that end-stage lungs are dominated by this pathogen. Longitudinal sputum samples taken through two exacerbation events in a single patient showed that a two-unit drop in pH and a 30% increase in gas production occurred in the tubes prior to exacerbation, which was reversed with antibiotic treatment. Microbial community profiles obtained through amplification and sequencing of the 16S rRNA gene showed that fermentative anaerobes became more abundant during exacerbation and were then reduced during treatment where P. aeruginosa became the dominant bacterium. Results from the WinCF experiments support the model where two functionally different CF microbial communities exist, the persistent Climax Community and the acute Attack Community. Fermentative anaerobes are hypothesized to be the core members of the Attack Community and production of acidic and gaseous products from fermentation may drive developing exacerbations. Treatment targeting the Attack Community may better resolve exacerbations and resulting lung damage.

Article
Peer Reviewed

Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem.

UC San Diego Previously Published Works (2016)

Managing ecosystems to maintain biodiversity may be one approach to ensuring their dynamic stability, productivity, and delivery of vital services. The applicability of this approach to industrial ecosystems that harness the metabolic activities of microbes has been proposed but has never been tested at relevant scales. We used a tag-sequencing approach with bacterial small subunit rRNA (16S) genes and eukaryotic internal transcribed spacer 2 (ITS2) to measuring the taxonomic composition and diversity of bacteria and eukaryotes in an open pond managed for bioenergy production by microalgae over a year. Periods of high eukaryotic diversity were associated with high and more-stable biomass productivity. In addition, bacterial diversity and eukaryotic diversity were inversely correlated over time, possibly due to their opposite responses to temperature. The results indicate that maintaining diverse communities may be essential to engineering stable and productive bioenergy ecosystems using microorganisms.

Cover page: Diversity, Productivity, and Stability of an Industrial Microbial Ecosystem.