Phage Cocktails Constrain the Growth of Enterococcus.

Phages that infect pathogenic bacteria present a valuable resource for treating antibiotic-resistant infections. We isolated and developed a collection of 19 Enterococcus phages, including myoviruses, siphoviruses, and a podovirus, that can infect both Enterococcus faecalis and Enterococcus faecium. Several of the Myoviridae phages that we found in southern California wastewater were from the Brockvirinae subfamily (formerly Spounavirinae) and had a broad host range across both E. faecium and E. faecalis. By searching the NCBI Sequence Read Archive, we showed that these phages are prevalent globally in human and animal microbiomes. Enterococcus is a regular member of healthy human gut microbial communities; however, it is also an opportunistic pathogen responsible for an increasing number of antibiotic-resistant infections. We tested the ability of each phage to clear Enterococcus host cultures and delay the emergence of phage-resistant Enterococcus. We found that some phages were ineffective at clearing Enterococcus cultures individually but were effective when combined into cocktails. Quantitative PCR was used to track phage abundance in cocultures and revealed dynamics ranging from one dominant phage to an even distribution of phage growth. Genomic characterization showed that mutations in Enterococcus exopolysaccharide synthesis genes were consistently found in the presence of phage infection. This work will help to inform cocktail design for Enterococcus, which is an important target for phage therapy applications. IMPORTANCE Due to the rise in antibiotic resistance, Enterococcus infections are a major health crisis that requires the development of alternative therapies. Phage therapy offers an alternative to antibiotics and has shown promise in both in vitro and early clinical studies. Here, we established a collection of 19 Enterococcus phages and tested whether combining phages into cocktails could delay growth and the emergence of resistant mutants in comparison with individual phages. We showed that cocktails of two or three phages often prevented the growth of phage-resistant mutants, and we identified which phages were replicating the most in each cocktail. When resistant mutants emerged to single phages, they showed consistent accumulation of mutations in exopolysaccharide synthesis genes. These data serve to demonstrate that a cocktail approach can inform efforts to improve efficacy against Enterococcus isolates and reduce the emergence of resistance.

High-Fiber, Whole-Food Dietary Intervention Alters the Human Gut Microbiome but Not Fecal Short-Chain Fatty Acids.

Dietary shifts can have a direct impact on the gut microbiome by preferentially selecting for microbes capable of utilizing the various dietary nutrients. The intake of dietary fiber has decreased precipitously in the last century, while consumption of processed foods has increased. Fiber, or microbiota-accessible carbohydrates (MACs), persist in the digestive tract and can be metabolized by specific bacteria encoding fiber-degrading enzymes. The digestion of MACs results in the accumulation of short-chain fatty acids (SCFAs) and other metabolic by-products that are critical to human health. Here, we implemented a 2-week dietary fiber intervention aiming for 40 to 50 g of fiber per day within the context of a course-based undergraduate research experience (CURE) (n = 20). By coupling shotgun metagenomic sequencing and targeted gas chromatography-mass spectrometry (GC-MS), we found that the dietary intervention significantly altered the composition of individual gut microbiomes, accounting for 8.3% of the longitudinal variability within subjects. Notably, microbial taxa that increased in relative abundance as a result of the diet change included known MAC degraders (i.e., Bifidobacterium and Lactobacillus). We further assessed the genetic diversity within Bifidobacterium, assayed by amplification of the groEL gene. Concomitant with microbial composition changes, we show an increase in the abundance of genes involved in inositol degradation. Despite these changes in gut microbiome composition, we did not detect a consistent shift in SCFA abundance. Collectively, our results demonstrate that on a short-term timescale of 2 weeks, increased fiber intake can induce compositional changes of the gut microbiome, including an increase in MAC-degrading bacteria.IMPORTANCE A profound decrease in the consumption of dietary fiber in many parts of the world in the last century may be associated with the increasing prevalence of type II diabetes, colon cancer, and other health problems. A typical U.S. diet includes about ∼15 g of fiber per day, far less fiber than the daily recommended allowance. Changes in dietary fiber intake affect human health not only through the uptake of nutrients directly but also indirectly through changes in the microbial community and their associated metabolism. Here, we conducted a 2-week diet intervention in healthy young adults to investigate the impact of fiber consumption on the gut microbiome. Participants increased their average fiber consumption by 25 g/day on average for 2 weeks. The high-fiber diet intervention altered the gut microbiome of the study participants, including increases in known fiber-degrading microbes, such as Bifidobacterium and Lactobacillus.

Cystic Fibrosis-Associated Stenotrophomonas maltophilia Strain-Specific Adaptations and Responses to pH.

The airway fluids of cystic fibrosis (CF) patients contain local pH gradients and are more acidic than those of healthy individuals. pH is a critical factor that is often overlooked in studies seeking to recapitulate the infection microenvironment. We sought to determine the impact of pH on the physiology of a ubiqituous yet understudied microbe, Stenotrophomonas maltophilia Phylogenomics was first used to reconstruct evolutionary relationships between 74 strains of S. maltophilia (59 from CF patients). Neither the core genome (2,158 genes) nor the accessory genome (11,978 genes) distinguish the CF and non-CF isolates; however, strains from similar isolation sources grouped into the same subclades. We grew two human and six CF S. maltophilia isolates from different subclades at a range of pH values and observed impaired growth and altered antibiotic tolerances at pH 5. Transcriptomes revealed increased expression of both antibiotic resistance and DNA repair genes in acidic conditions. Although the gene expression profiles of S. maltophilia in lab cultures and CF sputum were distinct, we found that the same genes associated with low pH were also expressed during infection, and the higher pH cultures were more similar to sputum metatranscriptomes. Our findings suggest that S. maltophilia is not well adapted to acidity and may cope with low pH by expressing stress response genes and colonizing less acidic microenvironments. As a whole, our study underlines the impact of microenvironments on bacterial colonization and adaptation in CF infections.IMPORTANCE Understanding bacterial responses to physiological conditions is an important priority for combating opportunistic infections. The majority of CF patients succumb to inflammation and necrosis in the airways, arising from chronic infection due to ineffective mucociliary clearance. Steep pH gradients characterize the CF airways but are not often incorporated in standard microbiology culture conditions. Stenotrophomonas maltophilia is a prevalent CF opportunistic pathogen also found in many disparate environments, yet this bacterium's contribution to CF lung damage and its response to changing environmental factors remain largely understudied. Here, we show that pH impacts the physiology and antibiotic susceptibility of S. maltophilia, with implications for the development of relevant in vitro models and assessment of antibiotic sensitivity.