Dynamics of Gut Microbiota Recovery after Antibiotic Exposure in Young and Old Mice (A Pilot Study).

Antibiotics have improved survival from previously deadly infectious diseases. Antibiotics alter the microbial composition of the gut microbiota, and these changes are associated with diminished innate immunity and decline in cognitive function in older adults. The composition of the human microbiota changes with age over the human lifespan. In this pilot study, we sought to identify if age is associated with differential recovery of the microbiota after antibiotic exposure. Using 16S rRNA gene sequencing, we compared recovery of the gut microbiota after the 10-day broad-spectrum antibiotic treatment in wild-type C57BL/six young and older mice. Immediately after antibiotic cessation, as expected, the number of ASVs, representing taxonomic richness, in both young and older mice significantly declined from the baseline. Mice were followed up to 6 months after cessation of the single 10-day antibiotic regimen. The Bray-Curtis index recovered within 20 days after antibiotic cessation in young mice, whereas in older mice the microbiota did not fully recover during the 6-months of follow-up. Bifidobacterium, Dubosiella, Lachnospiraceae_NK4A136_group became dominant in older mice, whereas in young mice, the bacteria were more evenly distributed, with only one dominant genus of Anaeroplasma. From 45 genera that became extinct after antibiotic treatment in young mice, 31 (68.9%) did not recover by the end of the study. In older mice, from 36 extinct genera, 27 (75%) did not recover. The majority of the genera that became extinct and never recovered belonged to Firmicutes phylum and Clostridiales family. In our study, age was a factor associated with the long-term recovery of the gut microbiota after the 10-day antibiotic treatment.

Dynamic shifts within volatile fatty acid-degrading microbial communities indicate process imbalance in anaerobic digesters.

Buildup of volatile fatty acids (VFAs) in anaerobic digesters (ADs) often results in acidification and process failure. Understanding the dynamics of microbial communities involved in VFA degradation under stable and overload conditions may help optimize anaerobic digestion processes. In this study, five triplicate mesophilic completely mixed AD sets were operated at different organic loading rates (OLRs; 1-6 g chemical oxygen demand [COD] LR-1day-1), and changes in the composition and abundance of VFA-degrading microbial communities were monitored using amplicon sequencing and taxon-specific quantitative PCRs, respectively. AD sets operated at OLRs of 1-4 g COD LR-1day-1 were functionally stable throughout the operational period (120 days) whereas process instability (characterized by VFA buildup, pH decline, and decreased methane production rate) occurred in digesters operated at ≥ 5 g COD LR-1day-1. Though microbial taxa involved in propionate (Syntrophobacter and Pelotomaculum) and butyrate (Syntrophomonas) degradation were detected across all ADs, their abundance decreased with increasing OLR. The overload conditions also inhibited the proliferation of the acetoclastic methanogen, Methanosaeta, and caused a microbial community shift to acetate oxidizers (Tepidanaerobacter acetatoxydans) and hydrogenotrophic methanogens (Methanoculleus). This study's results highlight the importance of operating ADs with conditions that promote the maintenance of microbial communities involved in VFA degradation.

Influence of Library Composition on SourceTracker Predictions for Community-Based Microbial Source Tracking.

Community-based microbial source tracking (MST) utilizes high-throughput DNA sequencing to profile and compare the microbial communities in different fecal sources and environmental samples. SourceTracker, a program that compares a library of OTUs from fecal sources (i.e., sources) to those in environmental samples (i.e., sinks) in order to determine sources of fecal contamination, is an emerging tool for community-based MST studies. In this study, we investigated the ability of SourceTracker to determine sources of known fecal contamination in spiked, in situ mesocosms containing different source contributors. We also evaluated how SourceTracker results were impacted by accounting for autochthonous taxa present in the sink environment. While SourceTracker was able to predict most sources present in the in situ mesocosms, fecal source library composition substantially influenced the program's ability to predict source contributions. Moreover, prediction results were most reliable when the library contained only known sources, autochthonous taxa were accounted for and when source profiles had low intragroup variability. Although SourceTracker struggled to differentiate between sources with similar bacterial community structures, it was able to consistently identify abundant and expected sources, suggesting that the SourceTracker program can be a useful tool for community-based MST studies.

Spatial and temporal characterization of epiphytic microbial communities associated with Eurasian watermilfoil: a highly invasive macrophyte in North America.

Bacterial communities that inhabit the surface of aquatic plants are thought to play a critical role in relation to host fitness and function. However, little is known about their structure and dynamics in comparison with those of bacterioplankton. In this study, we performed a comprehensive spatial and temporal characterization of epibacterial communities associated with Eurasian watermilfoil (EWM; Myriophyllum spicatum), an invasive macrophyte, which has established itself in thousands of lakes across North America. EWM samples were collected from 10 lakes in Minnesota, once a month, for six consecutive months, along with surrounding water and sediment. High-throughput DNA sequencing analyses, performed on all samples (n = 522) using the Illumina platform, indicated that EWM-associated epibacterial communities were distinct from those found in water and sediment. EWM-specific microbiota was comprised of operational taxonomic units classified to the families Rhodobacteraceae, Comamonadaceae, Cyanobacteria Subsection I Family I, Aeromonadaceae, Planctomycetaceae, Sphingomonadaceae and Verrucomicrobiaceae. In addition, several identified taxa were overrepresented in EWM samples when compared to water and sediment. Amongst all the environmental factors examined, water temperature had the greatest influence on epibacterial community structure. Our findings suggest that EWM harbor specific, but temporally adapted, epibacterial communities that are potentially involved in host-microbe interactions.

Fecal pollution: new trends and challenges in microbial source tracking using next-generation sequencing.

In this minireview, we expand upon traditional microbial source tracking (MST) methods by discussing two recently developed, next-generation-sequencing (NGS)-based MST approaches to identify sources of fecal pollution in recreational waters. One method defines operational taxonomic units (OTUs) that are specific to a fecal source, e.g., humans and animals or shared among multiple fecal sources to determine the magnitude and likely source association of fecal pollution. The other method uses SourceTracker, a program using a Bayesian algorithm, to determine which OTUs have contributed to an environmental community based on the composition of microbial communities in multiple fecal sources. Contemporary NGS-based MST tools offer a promising avenue to rapidly characterize fecal source contributions for water monitoring and remediation efforts at a broader and more efficient scale than previous molecular MST methods. However, both NGS methods require optimized sequence processing methodologies (e.g. quality filtering and clustering algorithms) and are influenced by primer selection for amplicon sequencing. Therefore, care must be taken when extrapolating data or combining datasets. Furthermore, traditional limitations of library-dependent MST methods, including differential decay of source material in environmental waters and spatiotemporal variation in source communities, remain to be fully understood. Nevertheless, increasing use of these methods, as well as expanding fecal taxon libraries representative of source communities, will help improve the accuracy of these methods and provide promising tools for future MST investigations.

Application of SourceTracker for Accurate Identification of Fecal Pollution in Recreational Freshwater: A Double-Blinded Study.

The efficacy of SourceTracker software to attribute contamination from a variety of fecal sources spiked into ambient freshwater samples was investigated. Double-blinded samples spiked with ≤5 different sources (0.025-10% vol/vol) were evaluated against fecal taxon libraries characterized by next-generation amplicon sequencing. Three libraries, including an initial library (17 nonlocal sources), a blinded source library (5 local sources), and a composite library (local and nonlocal sources), were used with SourceTracker. SourceTracker's predictions of fecal compositions in samples were made, in part, based on distributions of taxa within abundant genera identified as discriminatory by discriminant analyses but also using a large percentage of low abundance taxa. The initial library showed poor ability to characterize blinded samples, but, using local sources, SourceTracker showed 91% accuracy (31/34) at identifying the presence of source contamination, with two false positives for sewage and one for horse. Furthermore, sink predictions of source contamination were positively correlated (Spearman's ρ ≥ 0.88, P < 0.001) with spiked source volumes. Using the composite library did not significantly affect sink predictions ( P > 0.79) compared to those made using the local sources alone. Results of this study indicate that geographically associated fecal samples are required for SourceTracker to assign host sources accurately.

A High-Throughput DNA-Sequencing Approach for Determining Sources of Fecal Bacteria in a Lake Superior Estuary.

Current microbial source-tracking (MST) methods, employed to determine sources of fecal contamination in waterways, use molecular markers targeting host-associated bacteria in animal or human feces. However, there is a lack of knowledge about fecal microbiome composition in several animals and imperfect marker specificity and sensitivity. To overcome these issues, a community-based MST method has been developed. Here, we describe a study done in the Lake Superior-Saint Louis River estuary using SourceTracker, a program that calculates the source contribution to an environment. High-throughput DNA sequencing of microbiota from a diverse collection of fecal samples obtained from 11 types of animals (wild, agricultural, and domesticated) and treated effluent (n = 233) was used to generate a fecal library to perform community-based MST. Analysis of 319 fecal and environmental samples revealed that the community compositions in water and fecal samples were significantly different, allowing for the determination of the presence of fecal inputs and identification of specific sources. SourceTracker results indicated that fecal bacterial inputs into the Lake Superior estuary were primarily attributed to wastewater effluent and, to a lesser extent, geese and gull wastes. These results suggest that a community-based MST method may be another useful tool for determining sources of aquatic fecal bacteria.