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Agrochemicals often contaminate freshwater bodies, affecting microbial communities that underlie aquatic food webs. For example, the herbicide glyphosate has the potential to indirectly select for antibiotic-resistant bacteria. Such cross-selection could occur if the same genes (encoding efflux pumps, for example) confer resistance to both glyphosate and antibiotics. To test for cross-resistance in natural aquatic bacterial communities, we added a glyphosate-based herbicide (GBH) to 1,000-liter mesocosms filled with water from a pristine lake. Over 57 days, we tracked changes in bacterial communities with shotgun metagenomic sequencing and annotated metagenome-assembled genomes (MAGs) for the presence of known antibiotic resistance genes (ARGs), plasmids, and resistance mutations in the enzyme targeted by glyphosate (enolpyruvyl-shikimate-3-phosphate synthase; EPSPS). We found that high doses of GBH significantly increased ARG frequency and selected for multidrug efflux pumps in particular. The relative abundance of MAGs after a high dose of GBH was predictable based on the number of ARGs in their genomes (17% of variation explained) and, to a lesser extent, by resistance mutations in EPSPS. Together, these results indicate that GBHs can cross-select for antibiotic resistance in natural freshwater bacteria. IMPORTANCE Glyphosate-based herbicides (GBHs) such as Roundup formulations may have the unintended consequence of selecting for antibiotic resistance genes (ARGs), as demonstrated in previous experiments. However, the effects of GBHs on ARGs remain unknown in natural aquatic communities, which are often contaminated with pesticides from agricultural runoff. Moreover, the resistance provided by ARGs compared to canonical mutations in the glyphosate target enzyme, EPSPS, remains unclear. Here, we performed a freshwater mesocosm experiment showing that a GBH strongly selects for ARGs, particularly multidrug efflux pumps. These selective effects were evident after just a few days, and the ability of bacteria to survive and thrive after GBH stress was predictable by the number of ARGs in their genomes and, to a lesser extent, by mutations in EPSPS. Intensive GBH application may therefore have the unintended consequence of selecting for ARGs in natural freshwater communities.

Methylobacterium is a prevalent bacterial genus of the phyllosphere. Despite its ubiquity, little is known about the extent to which its diversity reflects neutral processes like migration and drift, versus environmental filtering of life history strategies and adaptations. In two temperate forests, we investigated how phylogenetic diversity within Methylobacterium is structured by biogeography, seasonality, and growth strategies. Using deep, culture-independent barcoded marker gene sequencing coupled with culture-based approaches, we uncovered a considerable diversity of Methylobacterium in the phyllosphere. We cultured different subsets of Methylobacterium lineages depending upon the temperature of isolation and growth (20°C or 30°C), suggesting long-term adaptation to temperature. To a lesser extent than temperature adaptation, Methylobacterium diversity was also structured across large (>100 km; between forests) and small (<1.2 km; within forests) geographical scales, among host tree species, and was dynamic over seasons. By measuring the growth of 79 isolates during different temperature treatments, we observed contrasting growth performances, with strong lineage- and season-dependent variations in growth strategies. Finally, we documented a progressive replacement of lineages with a high-yield growth strategy typical of cooperative, structured communities in favor of those characterized by rapid growth, resulting in convergence and homogenization of community structure at the end of the growing season. Together, our results show how Methylobacterium is phylogenetically structured into lineages with distinct growth strategies, which helps explain their differential abundance across regions, host tree species, and time. This work paves the way for further investigation of adaptive strategies and traits within a ubiquitous phyllosphere genus. IMPORTANCE Methylobacterium is a bacterial group tied to plants. Despite the ubiquity of methylobacteria and the importance to their hosts, little is known about the processes driving Methylobacterium community dynamics. By combining traditional culture-dependent and -independent (metabarcoding) approaches, we monitored Methylobacterium diversity in two temperate forests over a growing season. On the surface of tree leaves, we discovered remarkably diverse and dynamic Methylobacterium communities over short temporal (from June to October) and spatial (within 1.2 km) scales. Because we cultured different subsets of Methylobacterium diversity depending on the temperature of incubation, we suspected that these dynamics partly reflected climatic adaptation. By culturing strains under laboratory conditions mimicking seasonal variations, we found that diversity and environmental variations were indeed good predictors of Methylobacterium growth performances. Our findings suggest that Methylobacterium community dynamics at the surface of tree leaves results from the succession of strains with contrasting growth strategies in response to environmental variations.

Beneath the seafloor, microbial life subsists in isolation from the surface world under persistent energy limitation. The nature and extent of genomic evolution in subseafloor microbes have been unknown. Here, we show that the genomes of Thalassospira bacterial populations cultured from million-year-old subseafloor sediments evolve in clonal populations by point mutation, with a relatively low rate of homologous recombination and elevated numbers of pseudogenes. Ratios of nonsynonymous to synonymous substitutions correlate with the accumulation of pseudogenes, consistent with a role for genetic drift in the subseafloor strains but not in type strains of Thalassospira isolated from the surface world. Consistent with this, pangenome analysis reveals that the subseafloor bacterial genomes have a significantly lower number of singleton genes than the type strains, indicating a reduction in recent gene acquisitions. Numerous insertion-deletion events and pseudogenes were present in a flagellar operon of the subseafloor bacteria, indicating that motility is nonessential in these million-year-old subseafloor sediments. This genomic evolution in subseafloor clonal populations coincided with a phenotypic difference: all subseafloor isolates have a lower rate of growth under laboratory conditions than the Thalassospira xiamenensis type strain. Our findings demonstrate that the long-term physical isolation of Thalassospira , in the absence of recombination, has resulted in clonal populations whereby reduced access to novel genetic material from neighbors has resulted in the fixation of new mutations that accumulate in genomes over millions of years. IMPORTANCE The nature and extent of genomic evolution in subseafloor microbial populations subsisting for millions of years below the seafloor are unknown. Subseafloor populations have ultralow metabolic rates that are hypothesized to restrict reproduction and, consequently, the spread of new traits. Our findings demonstrate that genomes of cultivated bacterial strains from the genus Thalassospira isolated from million-year-old abyssal sediment exhibit greatly reduced levels of homologous recombination, elevated numbers of pseudogenes, and genome-wide evidence of relaxed purifying selection. These substitutions and pseudogenes are fixed into the population, suggesting that the genome evolution of these bacteria has been dominated by genetic drift. Thus, reduced recombination, stemming from long-term physical isolation, resulted in small clonal populations of Thalassospira that have accumulated mutations in their genomes over millions of years.

Vibrio cholerae can cause a range of symptoms, from severe diarrhea to asymptomatic infection. Previous studies using whole-genome sequencing (WGS) of multiple bacterial isolates per patient showed that V. cholerae can evolve modest genetic diversity during symptomatic infection. To further explore the extent of V. cholerae within-host diversity, we applied culture-based WGS and metagenomics to a cohort of both symptomatic and asymptomatic cholera patients from Bangladesh. While metagenomics allowed us to detect more mutations in symptomatic patients, WGS of cultured isolates was necessary to detect V. cholerae diversity in asymptomatic carriers, likely due to their low V. cholerae load. Using both metagenomics and isolate WGS, we report three lines of evidence that V. cholerae hypermutators evolve within patients. First, we identified nonsynonymous mutations in V. cholerae DNA repair genes in 5 out of 11 patient metagenomes sequenced with sufficient coverage of the V. cholerae genome and in 1 of 3 patients with isolate genomes sequenced. Second, these mutations in DNA repair genes tended to be accompanied by an excess of intrahost single nucleotide variants (iSNVs). Third, these iSNVs were enriched in transversion mutations, a known hallmark of hypermutator phenotypes. While hypermutators appeared to generate mostly selectively neutral mutations, nonmutators showed signs of convergent mutation across multiple patients, suggesting V. cholerae adaptation within hosts. Our results highlight the power and limitations of metagenomics combined with isolate sequencing to characterize within-patient diversity in acute V. cholerae infections, while providing evidence for hypermutator phenotypes within cholera patients. IMPORTANCE Pathogen evolution within patients can impact phenotypes such as drug resistance and virulence, potentially affecting clinical outcomes. V. cholerae infection can result in life-threatening diarrheal disease or asymptomatic infection. Here, we describe whole-genome sequencing of V. cholerae isolates and culture-free metagenomic sequencing from stool of symptomatic cholera patients and asymptomatic carriers. Despite the typically short duration of cholera, we found evidence for adaptive mutations in the V. cholerae genome that occur independently and repeatedly within multiple symptomatic patients. We also identified V. cholerae hypermutator phenotypes within several patients, which appear to generate mainly neutral or deleterious mutations. Our work sets the stage for future studies of the role of hypermutators and within-patient evolution in explaining the variation from asymptomatic carriage to symptomatic cholera.

Diet is a major determinant of community composition in the human gut microbiome, and "traditional" diets have been associated with distinct and highly diverse communities, compared to Western diets. However, most traditional diets studied have been those of agrarians and hunter-gatherers consuming fiber-rich diets. In contrast, the Inuit of the Canadian Arctic have been consuming a traditional diet low in carbohydrates and rich in animal fats and protein for thousands of years. We hypothesized that the Inuit diet and lifestyle would be associated with a distinct microbiome. We used deep sequencing of the 16S rRNA gene to compare the gut microbiomes of Montrealers with a Western diet to those of the Inuit consuming a range of traditional and Western diets. At the overall microbial community level, the gut microbiomes of Montrealers and Inuit were indistinguishable and contained similar levels of microbial diversity. However, we observed significant differences in the relative abundances of certain microbial taxa down to the subgenus level using oligotyping. For example, Prevotella spp., which have been previously associated with high-fiber diets, were enriched in Montrealers and among the Inuit consuming a Western diet. The gut microbiomes of Inuit consuming a traditional diet also had significantly less genetic diversity within the Prevotella genus, suggesting that a low-fiber diet might not only select against Prevotella but also reduce its diversity. Other microbes, such as Akkermansia , were associated with geography as well as diet, suggesting limited dispersal to the Arctic. Our report provides a snapshot of the Inuit microbiome as Western-like in overall community structure but distinct in the relative abundances and diversity of certain genera and strains. IMPORTANCE Non-Western populations have been shown to have distinct gut microbial communities shaped by traditional diets. The hitherto-uncharacterized microbiome of the Inuit may help us to better understand health risks specific to this population such as diabetes and obesity, which increase in prevalence as many Inuit transition to a Western diet. Here we show that even Inuit consuming a mostly traditional diet have a broadly Western-like microbiome. This suggests that similarities between the Inuit diet and the Western diet (low fiber, high fat) may lead to a convergence of community structures and diversity. However, certain species and strains of microbes have significantly different levels of abundance and diversity in the Inuit, possibly driven by differences in diet. Furthermore, the Inuit diet provides an exception to the correlation between traditional diets and high microbial diversity, potentially due to their transitioning diet. Knowledge of the Inuit microbiome may provide future resources for interventions and conservation of Inuit heritage.

Pharmacodynamic data on ciprofloxacin indicate that a target area under the concentration-time curve from 0 to 24 h (AUC(0-24))/MIC ratio of >or=125 is necessary to achieve optimal bactericidal activity for the treatment of gram-negative pneumonia. The purpose of this prospective study was to (i) develop a pharmacokinetic (PK) model to be utilized for therapeutic drug monitoring (TDM) of ciprofloxacin and (ii) evaluate current ciprofloxacin dosing regimens for pneumonias in cystic fibrosis (CF) patients. Twelve adult CF patients received a single 400-mg dose of IV ciprofloxacin. Six blood samples were obtained over a 12-h interval. Serum drug concentrations were determined by high-pressure liquid chromotography and were fitted to one- and two-compartment models by using NPEM2. Ciprofloxacin MIC data for Pseudomonas aeruginosa were obtained from 1,213 CF patients enrolled in a large clinical trial. A Monte Carlo simulation was performed to estimate the fractional attainment of an AUC(0-24)/MIC ratio of >or=125. A two-compartment model best describes the serum drug concentration data. The mean fitted PK parameter values are volume of distribution in the central compartment, 0.29 liter/kg; volume of distribution at steady state, 1.1 liters/kg; total clearance, 0.34 liter/h/kg; distributional clearance, 0.89 liter/h/kg; half-life at alpha phase, 0.16 h; and half-life at beta phase, 2.9 h. The overall fractional attainment of achieving an AUC(0-24)/MIC ratio of >or=125 against P. aeruginosa isolates with ciprofloxacin (400 mg every 12 h [q12h] and 8 qh) were 10 and 30%, respectively. A clinical breakpoint MIC of <0.5 microg/ml for susceptibility is suggested, based on an examination of the fractional attainment of the AUC(0-24)/MIC target at each MIC. The recommended doses of 400 mg q8h or q12h may be inadequate to treat an acute pulmonary exacerbation when given alone. The poor and variable AUC(0-24)/MIC ratios support the use of TDM to monitor and adjust the dosage to optimize the efficacy of ciprofloxacin therapy in these patients.

Once-daily administration of aminoglycosides is routinely used in many institutions. However, comparative efficacy data for patients with cystic fibrosis (CF) are lacking. The purpose of the present study was to compare the predicted pharmacodynamic activity of tobramycin at 10 mg/kg of body weight/day administered every 24 h (q24h), q12h, and q8h. Pharmacokinetic (PK) data were derived from analysis of data on the drug concentration in sera from 60 adult CF patients. Individual maximum a posteriori probability Bayesian PK parameter values were used to construct serum concentration-versus-time curves and to determine various indices (peak concentration/MIC ratio [peak/MIC], area under the concentration-time curve/MIC ratio [AUC/MIC], and time that the concentration was less than the MIC [T