Making It Last: Storage Time and Temperature Have Differential Impacts on Metabolite Profiles of Airway Samples from Cystic Fibrosis Patients.

Metabolites of human or microbial origin have the potential to be important biomarkers of the disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography-mass spectrometry (GC-MS) when CF sputum samples were stored at 4°C, -20°C, or -80°C with one or two freeze-thaw cycles. Daily measurements were taken for 1 week and then weekly for 4 weeks (4°C) and 8 weeks (-20°C). The metabolites in samples stored at -20°C maintained abundances similar to those found at-80°C over the course of 8 weeks (average change in Bray-Curtis distance, 0.06 ± 0.04) and were also stable after one or two freeze-thaw cycles. However, the metabolite profiles of samples stored at 4°C shifted after 1 day and continued to change over the course of 4 weeks (average change in Bray-Curtis distance, 0.31 ± 0.12). The abundances of several amino acids and other metabolites increased with time of storage at 4°C but remained constant at -20°C. Storage temperature was a significant factor driving the metabolite composition (permutational multivariate analysis of variance: r2 = 0.32 to 0.49, P < 0.001). CF sputum samples stored at -20°C at the time of sampling maintain a relatively stable untargeted GC-MS profile. Samples should be frozen on the day of collection, as more than 1 day at 4°C impacts the global composition of the metabolites in the sample. IMPORTANCE Metabolomics has great potential for uncovering biomarkers of the disease state in CF and many other contexts. However, sample storage timing and temperature may alter the abundance of clinically relevant metabolites. To assess whether existing samples are stable and to direct future study design, we conducted untargeted GC-MS metabolomic analysis of CF sputum samples after one or two freeze-thaw cycles and storage at 4°C and -20°C for 4 to 8 weeks. Overall, storage at -20°C and freeze-thaw cycles had little impact on metabolite profiles; however, storage at 4°C shifted metabolite abundances significantly. GC-MS profiling will aid in our understanding of the CF lung, but care should be taken in studies using sputum samples to ensure that samples are properly stored

Predictable Molecular Adaptation of Coevolving Enterococcus faecium and Lytic Phage EfV12-phi1

Bacteriophages are highly abundant in human microbiota where they coevolve with resident bacteria. Phage predation can drive the evolution of bacterial resistance, which can then drive reciprocal evolution in the phage to overcome that resistance. Such coevolutionary dynamics have not been extensively studied in human gut bacteria, and are of particular interest for both understanding and eventually manipulating the human gut microbiome. We performed experimental evolution of an Enterococcus faecium isolate from healthy human stool in the absence and presence of a single infecting Myoviridae bacteriophage, EfV12-phi1. Four replicates of E. faecium and phage were grown with twice daily serial transfers for 8 days. Genome sequencing revealed that E. faecium evolved resistance to phage through mutations in the yqwD2 gene involved in exopolysaccharide biogenesis and export, and the rpoC gene which encodes the RNA polymerase β’ subunit. In response to bacterial resistance, phage EfV12-phi1 evolved varying numbers of 1.8 kb tandem duplications within a putative tail fiber gene. Host range assays indicated that coevolution of this phage-host pair resulted in arms race dynamics in which bacterial resistance and phage infectivity increased over time. Tracking mutations from population sequencing of experimental coevolution can quickly illuminate phage entry points along with resistance strategies in both phage and host – critical information for using phage to manipulate microbial communities

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Global phylogeography and ancient evolution of the widespread human gut virus crAssphage

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world’s countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome

Evolution of bacteriophages infecting Enterococcus from the human microbiome

Enterococcus can be both a friend and a foe in the human microbiome. As an opportunistic pathogen, Enterococcus is normally benign. However, Enterococcus is responsible for many hospital-acquired infections and has shown rising rates of vancomycin resistance. Bacteriophages (phages) could be an alternative to antibiotics to target these antibiotic resistant bacteria, but the evolutionary and phenotypic outcomes of phage-bacteria interactions need to be investigated more thoroughly. We begin by investigating the gut microbiome of preterm infants that had been exposed to antibiotics to learn about the scenarios in which Enterococcus blooms occur in the gut (Chapter 1). We show that antibiotic exposed gut microbiomes are dominated by facultative anaerobes such as Enterococcus and Enterobacteriaceae. Further, we show that the bacterial composition is correlated to the overall metabolite profile. Metabolomics is a powerful tool for investigating microbial metabolism, and we contribute to the effort of developing standardized practices for metabolomics in the human microbiome by showing that freezing microbial samples required for long term storage (Chapter 2). Next, we investigated how phages could be used as therapeutics for treating Enterococcus blooms and infections (Chapters 3, 4, and 5). When Enterococcus is grown with its phages in vitro, it evolves resistance to phage infection by mutating exopolysaccharide synthesis genes. These mutations alter the exopolysaccharides on the surface of the bacterial cell to prevent binding of phage. Further, this mechanism of resistance appears to be a general mechanism leading to resistance against a diverse array of phages. This work demonstrates that experimental evolution is a powerful tool for characterizing interactions between bacteria and phages.Phage therapy is often administered as a cocktail of multiple phages, but there are no rules or best practices described for combining phages to be most effective. We show that in vitro, Enterococcus phage cocktails are more effective at preventing the growth of phage-resistant mutants, but the composition of the cocktail is important. Genetically diverse phage cocktails performed better than cocktails of related phages. My work demonstrates some of the outcomes of phage-Enterococcus interactions and will move us closer to applying phage therapy to treat Enterococcus infections

Data from: Making it last: storage time and temperature have differential impacts on metabolite profiles of airway samples from cystic fibrosis patients

Metabolites of human or microbial origin have the potential to be important biomarkers of disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography mass spectrometry (GC-MS) when CF sputum samples were stored at either 4°C, -20°C, or -80°C with one or two freeze-thaw cycles. Daily time points were taken for one week and then weekly for 4 weeks (4°C) and 8 weeks (-20°C). The metabolites in samples stored at -20°C maintained similar abundances compared to -80°C over the course of eight weeks (average change in Bray-Curtis distance: 0.06±0.04), and were also stable after one or two freeze-thaw cycles. However, metabolite profiles of samples stored at 4°C shifted after one day and continued to change over the course of four weeks (average change in Bray-Curtis distance: 0.31±0.12). Several amino acids and other metabolite abundances increased with time when stored at 4°C, but remained constant at -20°C. Storage temperature was a significant factor driving the metabolite composition (PERMANOVA R2 = 0.32 to 0.49, p= <0.001). CF sputum samples stored at -20°C at the time of sampling maintain a relatively stable untargeted GC-MS profile. Samples should be frozen on the day of collection, as more than one day at 4°C impacts the global composition of the metabolites in the sample