Successful Treatment of Recurrent Clostridioides difficile Infection Using a Novel, Drinkable, Oral Formulation of Fecal Microbiota.

BACKGROUND: Fecal microbiota transplants can be administered orally in encapsulated form or require invasive procedures to administer liquid formulations. There is a need for an oral liquid formulation of fecal microbiota for patients who are unable to swallow capsules, especially if they require multiple, repeated administrations. AIMS: These studies were conducted to develop a protocol to manufacture an organoleptically acceptable powdered fecal microbiota formulation that can be suspended in a liquid carrier and used for fecal microbiota transplantation. METHODS: Several processing steps were investigated, including extra washes of microbiota prior to lyophilization and an addition of a flavoring agent. The viability of bacteria in the transplant formulation was tested using live/dead microscopy staining and engraftment into antibiotic-treated mice. After development of a clinical protocol for suspension of the powdered microbiota, the new formulation was tested in three elderly patients with recurrent Clostridioides difficile infections and who have difficulties in swallowing capsules. Changes in the microbial community structure in one of the patients were characterized using 16S rRNA gene profiling and engraftment analysis. RESULTS: The processing steps used to produce an organoleptically acceptable suspension of powdered fecal microbiota did not result in loss of its viability. The powder could be easily suspended in a liquid carrier. The use of the new formulation was associated with abrogation of the cycle of C. difficile infection recurrences in the three patients. CONCLUSION: We developed a novel organoleptically acceptable liquid formulation of fecal microbiota that is suitable for use in clinical trials for patients with difficulties in swallowing capsules.

Differential hydrogen sulfide production by a human cohort in response to animal- and plant-based diet interventions.

BACKGROUND: Hydrogen sulfide (H2S) is a toxic end-product of microbial fermentation produced in the colon that may play a role in the pathogenesis of several diseases, including ulcerative colitis and colon cancer. However, the effect of diet interventions on intestinal burden of H2S gas exposure remains poorly understood. OBJECTIVE: Determine the effect of short-term (1-week) plant- and animal-based eating patterns on ex vivo fecal H2S production in healthy human volunteers. METHODS: The study design was an open-label, cross-over diet study and diets were self-administered. Each participant consumed two interventional diets: 1) an animal-based, low fiber (i.e. western) diet and 2) a plant-based, high fiber diet, separated by a two-week washout period. Participants collected full stool samples at the end of each week, which were processed within 2 h of collection to capture H2S production. Microfluidic qPCR (MFQPCR) was used to simultaneously quantify multiple taxonomic and functional groups involved in sulfate reduction and the fecal microbiota was characterized through high-throughput DNA sequencing. RESULTS: Median H2S production was higher following the animal-based diet compared to the plant-based diet (p = 0.02; median difference 29 ppm/g, 95% CI 16-97). However, there was substantial individual variability and 2 of 11 individuals (18%) produced more H2S on the plant-based diet. Using the top and bottom quartiles of H2S percent change between animal- and plant-based diet weeks to define responders and non-responders, significant taxonomic differences were observed between the responder and non-responder cohorts. CONCLUSIONS: Here we report that substrate changes associated with a 1-week plant-based diet intervention resulted in lower ex vivo H2S production compared to a 1-week animal-based diet intervention in most healthy individuals. However, H2S responsiveness to diet was not uniform across the entire cohort, and potential H2S production enterotypes were characterized that may predict individualized H2S responsiveness to diet.

Corrigendum: Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

[This corrects the article DOI: 10.3389/fmicb.2021.706683.].

Gut Microbiota Associated With Different Sea Lamprey (Petromyzon marinus) Life Stages.

Sea lamprey (SL; Petromyzon marinus), one of the oldest living vertebrates, have a complex metamorphic life cycle. Following hatching, SL transition into a microphagous, sediment burrowing larval stage, and after 2-10+ years, the larvae undergo a dramatic metamorphosis, transforming into parasitic juveniles that feed on blood and bodily fluids of fishes; adult lamprey cease feeding, spawn, and die. Since gut microbiota are critical for the overall health of all animals, we examined the microbiota associated with SLs in each life history stage. We show that there were significant differences in the gut bacterial communities associated with the larval, parasitic juvenile, and adult life stages. The transition from larval to the parasitic juvenile stage was marked with a significant shift in bacterial community structure and reduction in alpha diversity. The most abundant SL-associated phyla were Proteobacteria, Fusobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Firmicutes, with their relative abundances varying among the stages. Moreover, while larval SL were enriched with unclassified Fusobacteriaceae, unclassified Verrucomicrobiales and Cetobacterium, members of the genera with fastidious nutritional requirements, such as Streptococcus, Haemophilus, Cutibacterium, Veillonella, and Massilia, were three to four orders of magnitude greater in juveniles than in larvae. In contrast, adult SLs were enriched with Aeromonas, Iodobacter, Shewanella, and Flavobacterium. Collectively, our findings show that bacterial communities in the SL gut are dramatically different among its life stages. Understanding how these communities change over time within and among SL life stages may shed more light on the role that these gut microbes play in host growth and fitness.

Influence of seasonality on the aerosol microbiome of the Amazon rainforest.

The Amazon rainforest is the world's largest tropical forest, and this biome may be a significant contributor to primary biological aerosol (PBA) emissions on a global scale. These aerosols also play a pivotal role in modulating ecosystem dynamics, dispersing biological material over geographic barriers and influencing climate through radiation absorption, light scattering, or acting as cloud condensation nuclei. Despite their importance, there are limited studies investigating the effect of environmental variables on the bioaerosol composition in the Amazon rainforest. Here we present a 16S rRNA gene-based amplicon sequencing approach to investigate the bacterial microbiome in aerosols of the Amazon rainforest during distinct seasons and at different heights above the ground. Our data revealed that seasonal changes in temperature, relative humidity, and precipitation are the primary drivers of compositional changes in the Amazon rainforest aerosol microbiome. Interestingly, no significant differences were observed in the bacterial community composition of aerosols collected at ground and canopy levels. The core airborne bacterial families present in Amazon aerosol were Enterobacteriaceae, Beijerinckiaceae, Polyangiaceae, Bacillaceae and Ktedonobacteraceae. By correlating the bacterial taxa identified in the aerosol with literature data, we speculate that the phyllosphere may be one possible source of airborne bacteria in the Amazon rainforest. Results of this study indicate that the aerosol microbiota of the Amazon Rainforest are fairly diverse and principally impacted by seasonal changes in temperature and humidity.

Methanogen Abundance Thresholds Capable of Differentiating In Vitro Methane Production in Human Stool Samples.

BACKGROUND: Intestinal methane (CH4) gas production has been associated with a number of clinical conditions and may have important metabolic and physiological effects. AIMS: In this study, taxonomic and functional gene analyses and in vitro CH4 gas measurements were used to determine if molecular markers can potentially serve as clinical tests for colonic CH4 production. METHODS: We performed a cross-sectional study involving full stool samples collected from 33 healthy individuals. In vitro CH4 gas measurements were obtained after 2-h incubation of stool samples and used to characterize samples as CH4 positive (CH4+) and CH4 negative (CH4-; n = 10 and 23, respectively). Next, we characterized the fecal microbiota through high-throughput DNA sequencing with a particular emphasis on archaeal phylum Euryarchaeota. Finally, qPCR analyses, targeting the mcrA gene, were done to determine the ability to differentiate CH4+ versus CH4- samples and to delineate major methanogen species associated with CH4 production. RESULTS: Methanobrevibacter was found to be the most abundant methane producer and its relative abundance provides a clear distinction between CH4+ versus CH4- samples. Its sequencing-based relative abundance detection threshold for CH4 production was calculated to be 0.097%. The qPCR-based detection threshold separating CH4+ versus CH4- samples, based on mcrA gene copies, was 5.2 × 105 copies/g. CONCLUSION: Given the decreased time-burden placed on patients, a qPCR-based test on a fecal sample can become a valuable tool in clinical assessment of CH4 producing status.

Influence of Environmental Stressors on the Microbiota of Zebra Mussels (Dreissena polymorpha).

Host-associated microbiota play a critical role in host fitness by providing nutrition, enhancing digestion capabilities, and by providing protection from pathogens. Here, we investigated the effects of two environmental stressors, temperature, and salinity, on the microbiota associated with zebra mussels (ZMs), a highly invasive bivalve in North America. To examine this in detail, lake-collected ZMs were acclimated to laboratory conditions, and subjected to temperature and salinity stress conditions. The impact of these stressors on the diversity, composition, and dynamics of ZM-associated microbiota were assessed by using amplicon- and shotgun-based sequencing, and qPCR-based approaches. Elevated temperature was found to be the primary driver of ZM mortality, although salinity alone also increased its likelihood. Stressor-induced ZM mortality, which ranged between 53 and 100%, was concomitant with significant increases in the relative abundance of several genera of putative opportunistic pathogens including Aeromonas. These genera were only present in low relative abundance in ZMs obtained from the control tank with 0% mortality. Shotgun sequencing and qPCR analyses indicated that the relative and absolute abundances of pathogenic Aeromonas species (particularly A. veronii) were significantly greater in temperature-induced dead ZMs. Taken together, our results show that environmental stress, especially elevated temperature (> 25 °C), is associated with the rapid mortality of ZMs as well as the proliferation of putative opportunistic bacterial pathogens.

Sequence-enabled community-based microbial source tracking in surface waters using machine learning classification: A review.

The development of Microbial Source Tracking (MST) technologies was borne out of necessity. This was largely due to the: 1) inadequacies of the fecal indicator bacterial paradigm, 2) fact that many fecal bacteria can survive and often grow in the environment, 3) inability of traditional microbiological methods to attribute source, 4) lack of correspondence between numbers of fecal indicator bacteria in waterways and many human pathogens, and 5) source allocation requirements and load determinations needed for total maximum daily loads. The MST tools have changed over time, evolving from culture-dependent to culture-independent molecular analyses. More recently, MST tools based on microbial community analyses, mainly DNA sequencing-based approaches, have been developed in an attempt to overcome some of these issues. These approaches generate large data sets and require the use of sophisticated machine learning algorithms to allocate potential host sources to contaminated waterways. In this review we discuss the origins and needs for community-based MST methods, as well as elaborate on the Bayesian algorithm-based program SourceTracker, which is increasingly being used for the determination of sources of fecal contamination of waterways.

Indigenous soil bacteria and the hyperaccumulator Pteris vittata mediate phytoremediation of soil contaminated with arsenic species.

Arsenic (As) is a pollutant of major concern worldwide, posing as a threat to both human health and the environment. Phytoremediation has been proposed as a viable mechanism to remediate As-contaminated soil environments. Pot experiments were performed to evaluate the phytoextraction efficiency of As by Pteris vittata, a known As hyperaccumulating fern, from soil amended with different concentrations of arsenate [As(V)] and arsenite [As(III)], the more common, inorganic As forms in soil. The greatest accumulation of As (13.3 ± 0.36 g/kg Dwt) was found in fronds of plants grown in soil spiked with 1.0 g As(V)/kg. The maximum As-bioaccumulation factor (27.3 ± 1.9) was achieved by plants grown in soil amended with 0.05 g As(V)/kg. A total of 864 bacterial cultures were isolated and examined for their ability to enhance phytoremediation of As-contaminated soils. Traits examined included tolerance to As (III and V), production of siderophores, and/or ability to solubilize calcium phosphate and indole acetic acid (IAA) production. A culture-based survey shows greater numbers of viable and As-resistant bacteria were found in the rhizosphere of As-grown plants compared to bulk and unplanted soils. The percentage of bacteria resistant to As(V) was greater (P < 0.0001) than those resistant to As(III) in culture medium containing 0.5, 1, 1.5, and 2 g As/L. Higher (P < 0.0001) percentages of siderophore producing (77%) and phosphate solubilizing (61%) bacteria were observed among cultures isolated from unplanted soil. About 5% (44 of 864) of the isolates were highly resistant to both As (III) and As (V) (2 g/L), and were examined for their As-transformation ability and IAA production. A great proportion of the isolates produced IAA (82%) and promoted As (V)-reduction (95%) or As(III)-oxidation (73%), and 71% exhibited dual capacity for both As(V) reduction and As(III) oxidation. Phylogenetic analysis indicated that 67, 23, and 10% of these isolates belonged to Proteobacteria, Actinobacteria, and Firmicutes, respectively. Analysis of the 16S rRNA gene sequences confirmed that these isolates were closely related to 12 genera and 25 species of bacteria and were dominated by members of the genus Pseudomonas (39%). These results show that these isolates could potentially be developed as inocula for enhancing plant uptake during large scale phytoremediation of As-impacted soils.

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.

Water and sediment act as reservoirs for microbial taxa associated with invasive dreissenid mussels.

Zebra mussels (Dreissena polymorpha) are invasive, filter-feeding, bivalves that have disrupted the ecology of thousands of freshwater biomes across North America. Due to their efficient filter-feeding activity, zebra mussels (ZMs) and other bivalves are extensively used to detect chemical contamination in waterways. In this study, we evaluated whether water and sediment serve as major sources of ZM tissue-associated microbiota, and whether ZMs serve as a reservoir for potentially pathogenic microbes in aquatic systems. High-throughput DNA sequencing of 16S rRNA gene was done to characterize the microbial community structure in 472 environmental samples, comprising ZMs, sediment, and the water column, collected from 15 lakes during the summer and fall months. Sequence analyses, done using the SourceTracker program, predicted that water and sediment contributed up to 91 and 86%, respectively, to the structure of microbiota within ZMs, and that mussels from the same site showed nearly identical source microbiota profiles. The relatively high local source contribution suggests that the microbiota in ZM tissue has the potential to reflect biological contamination and this phenomenon can be used to monitor microbial water quality. A preferential enrichment of several taxa was also observed in ZM tissues, including potential pathogenic groups such as Aeromonas, Enterobacteriaceae, and Pseudomonas. Taken together, our results contribute to an improved understanding of ZMs as a sentinel species in aquatic habitats and its potential impact to water quality management.

A microfluidic platform for the simultaneous quantification of methanogen populations in anaerobic digestion processes.

Methanogens are a diverse group of archaea that play a critical role in the global carbon cycle. The lack of appropriate molecular tools to simultaneously quantify numerous methanogenic taxa, however, has largely limited our ability to study these communities in a wide variety of habitats, such as anaerobic digesters (ADs). In this study, 34 probe-based quantitative PCR (qPCR) assays were designed to target all known methanogenic genera within the archaeal phylum Euryarchaeota. These qPCR assays were adapted to a high-throughput microfluidic platform, which allowed for the simultaneous detection and absolute quantification of numerous taxa in a single run. The resulting microfluidic qPCR (MFQPCR) platform was successfully used to decipher structure-function relationships among methanogenic communities in four laboratory-scale digesters exposed to a transient organic overload. Twelve of the 34 genera targeted in the MFQPCR were detected in the ADs, similar to results obtained using high-throughput sequencing. The MFQPCR platform and conventional qPCR assays also generated similar quantitative results. The MFQPCR tool developed here will help optimize AD technologies for efficient waste treatment and enhanced biogas production and can facilitate studies that will increase our understanding of methanogenic communities in other environments.

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.

Association between submerged aquatic vegetation and elevated levels of Escherichia coli and potential bacterial pathogens in freshwater lakes.

Fecal indicator bacteria such as Escherichia coli have been reported to persist and potentially grow in a wide variety of secondary habitats, such as water, beach sand, sediment, periphyton and some algae. However, little is known about their association with submerged macrophytes and how this may influence water quality. In this study, we examined the association of E. coli and potential bacterial pathogens with Eurasian watermilfoil (EWM), an invasive, submerged, macrophyte that has spread across thousands of lakes in North America. EWM samples were collected from 10 lakes in Minnesota, once a month, for six consecutive months from early summer to late fall. Microbiota associated with EWM were examined using membrane filtration, quantitative PCR targeting various bacterial pathogens and host-associated marker genes, and high-throughput DNA sequencing. E. coli densities were generally elevated on EWM samples, and peaked during warmer months. Moreover, our results showed that EWM could serve as a temporal source for transmission of microbiota to the water column. Several potential pathogenic groups, including Aeromonas, Enterobacteriaceae, and Clostridium were present in significantly greater relative abundance on EWM than in water, and waterfowl was predicted to be the major source of fecal contamination. These findings have water quality implications with respect to the potential for submerged macrophytes to harbor and disperse E. coli and other bacterial pathogens in a large number of waterbodies.

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.

Quantitative detection of syntrophic fatty acid-degrading bacterial communities in methanogenic environments.

In methanogenic habitats, volatile fatty acids (VFA), such as propionate and butyrate, are major intermediates in organic matter degradation. VFA are further metabolized to H(2), acetate and CO(2) by syntrophic fatty acid-degrading bacteria (SFAB) in association with methanogenic archaea. Despite their indispensable role in VFA degradation, little is known about SFAB abundance and their environmental distribution. To facilitate ecological studies, we developed four novel genus-specific quantitative PCR (qPCR) assays, with primer sets targeting known SFAB: Syntrophobacter, Smithella, Pelotomaculum and Syntrophomonas. Primer set specificity was confirmed using in silico and experimental (target controls, clone libraries and melt-curve analysis) approaches. These qPCR assays were applied to quantify SFAB in a variety of mesophilic methanogenic habitats, including a laboratory propionate enrichment culture, pilot- and full-scale anaerobic reactors, cow rumen, horse faeces, an experimental rice paddy soil, a bog stream and swamp sediments. The highest SFAB 16S rRNA gene copy numbers were found in the propionate enrichment culture and anaerobic reactors, followed by the bog stream and swamp sediment samples. In addition, it was observed that SFAB and methanogen abundance varied with reactor configuration and substrate identity. To our knowledge, this research represents the first comprehensive study to quantify SFAB in methanogenic habitats using qPCR-based methods. These molecular tools will help investigators better understand syntrophic microbial communities in engineered and natural environments.