High-throughput 16S rRNA Gene Amplicon Research Articles

Origin and modern microbial ecology of secondary mineral deposits in Lehman Caves, Great Basin National Park, NV, USA.

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ34SV-CDT between +9.7‰ and +26.1‰, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO2 phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.

Multiple approaches in the study of ancient human microbiome

Abstract Research related to the analysis of microbial communities in ancient human samples began two decades ago. Since then, new technologies as well as new bioinformatic tools have been developed for the manipulation of massive data. All this together, with the theoretical knowledge generated from microbial ecology has expanded our understanding of the human microbiome. Despite the challenge of studying ancient samples and their microbiome, some approaches applied to the contemporary human microbiome have been successfully applied to ancient samples. Most studies in ancient samples have focused on taxonomic profiling at the community level with metagenomic or high-throughput 16S rRNA gene amplicon sequencing data. However, other approaches have emerged in order to get deeper understanding of the ancient human microbiome. Therefore, the aim of this review is to discuss all the approaches and methodologies successfully applied to ancient samples and ancient microbiome as well as the information that can be generated using them, in order to understand the evolution of human microbiome interactions throughout history.

Distribution of bacterial community structures and spread of antibiotic resistome at industrially polluted sites of Mini River, Vadodara, Gujarat, India.

The influence of anthropogenic pollution on the distribution of bacterial diversity, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) was mapped at various geo-tagged sites of Mini River, Vadodara, Gujarat, India. The high-throughput 16S rRNA gene amplicon sequencing analysis revealed a higher relative abundance of Planctomycetota at the polluted sites, compared to the pristine site. Moreover, the relative abundance of Actinobacteriota increased, whereas Chloroflexi decreased in the water samples of polluted sites than the pristine site. The annotation of functional genes in the metagenome samples of Mini River sites indicated the presence of genes involved in the defence mechanisms against bacitracin, aminoglycosides, cephalosporins, chloramphenicol, streptogramin, streptomycin, methicillin, and colicin. The analysis of antibiotic resistome at the polluted sites of Mini River revealed the abundance of sulfonamide, beta-lactam, and aminoglycoside resistance. The presence of pathogens and ARB was significantly higher in water and sediment samples of polluted sites compared to the pristine site. The highest resistance of bacterial populations in the Mini River was recorded against sulfonamide (≥ 7.943 × 103CFU/mL) and ampicillin (≥ 8.128 × 103CFU/mL). The real-time PCR-based quantification of ARGs revealed the highest abundance of sulfonamide resistance genes sul1 and sul2 at the polluted sites of the Mini River. Additionally, the antimicrobial resistance genes aac(6')-Ib-Cr and blaTEM were also found abundantly at polluted sites of the Mini River. The findings provide insights into how anthropogenic pollution drives the ARG and ARB distribution in the riverine ecosystem, which may help with the development of antimicrobial resistance mitigation strategies.

Human gut microbiota fermentation of cooked eggplant, garlic, and onion supports distinct microbial communities.

Heating and cooking vegetables not only enhances their palatability but also modifies their chemical structure, which in turn might affect their fermentation by resident gut microbes. Three commonly consumed vegetables that are known to undergo chemical browning, also known as Maillard reaction, during cooking - eggplant, garlic, and onion - were each fried, grilled, or roasted. The cooked vegetables were then subjected to an in vitro digestion-fermentation process aimed to simulate the passage of food through the human oro-gastro-intestinal tract. In the last step, the undigested fractions of these foods were anaerobically fermented by the complex human gut microbiota. We assessed the structure of microbial communities maintained on each cooked vegetable by high-throughput 16S rRNA gene amplicon sequencing, measured the levels of furosine, a chemical marker of the Maillard browning reaction, by HPLC, and determined the antioxidant capacities in all samples with ABTS and FRAP methods. Overall, vegetable type had the largest, statistically significant, effect on the microbiota structure followed by the cooking method. Onion fermentation supported a more beneficial community including an expansion of Bifidobacterium members and inhibition of Enterobacteriaceae. Fermentation of cooked garlic promoted Faecalibacterium growth. Among cooking methods, roasting led to a much higher ratio of beneficial-to-detrimental microbes in comparison with grilling and frying, possibly due to the exclusion of any cooking oil in the cooking process.

Differentiated responses of the phyllosphere bacterial community of the yellowhorn tree to precipitation and temperature regimes across Northern China.

As an ephemeral and oligotrophic environment, the phyllosphere harbors many highly diverse microorganisms. Importantly, it is known that their colonization of plant leaf surfaces is considerably influenced by a few abiotic factors related to climatic conditions. Yet how the dynamics of phyllosphere bacterial community assembly are shaped by detailed climatological elements, such as various bioclimatic variables, remains poorly understood. Using high-throughput 16S rRNA gene amplicon sequencing technology, we analyzed the bacterial communities inhabiting the leaf surfaces of an oilseed tree, yellowhorn (Xanthoceras sorbifolium), grown at four sites (Yinchuan, Otogqianqi, Tongliao, and Zhangwu) whose climatic status differs in northern China. We found that the yellowhorn phyllosphere's bacterial community was generally dominated by four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Nevertheless, bacterial community composition differed significantly among the four sampled site regions, indicating the possible impact of climatological factors upon the phyllosphere microbiome. Interestingly, we also noted that the α-diversities of phyllosphere microbiota showed strong positive or negative correlation with 13 bioclimatic factors (including 7 precipitation factors and 6 temperature factors). Furthermore, the relative abundances of 55 amplicon sequence variants (ASVs), including three ASVs representing two keystone taxa (the genera Curtobacterium and Streptomyces), exhibited significant yet contrary responses to the precipitation and temperature climatic variables. That pattern was consistent with all ASVs' trends of possessing opposite correlations to those two parameter classes. In addition, the total number of links and nodes, which conveys community network complexity, increased with rising values of most temperature variables. Besides that, remarkably positive relevance was found between average clustering coefficient and most precipitation variables. Altogether, these results suggest the yellowhorn phyllosphere bacterial community is capable of responding to variation in rainfall and temperature regimes in distinctive ways.

Optimization of high-throughput 16S rRNA gene amplicon sequencing: an assessment of PCR pooling, mastermix use and contamination.

16S rRNA gene sequencing is widely used to characterize human and environmental microbiomes. Sequencing at scale facilitates better powered studies but is limited by cost and time. We identified two areas in our 16S rRNA gene library preparation protocol where modifications could provide efficiency gains, including (1) pooling of multiple PCR amplifications per sample to reduce PCR drift and (2) manual preparation of mastermix to reduce liquid handling. Using nasal samples from healthy human participants and a serially diluted mock microbial community, we compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. One hundred and fifty-eight 16S rRNA gene sequencing libraries were prepared, including a replicate experiment. Comparing PCR pooling strategies, we found no significant difference in high-quality read counts and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate on principal coordinate analysis (PCoA) and non-metric dimensional scaling (NMDS) analysis. Choice of mastermix had no significant impact on high-quality read and alpha diversity, and beta diversity by Bray-Curtis index clustered by replicate in PCoA and NMDS analysis. Importantly, we observed contamination and variability of rare species (<0.01 %) across replicate experiments; the majority of contaminants were accounted for by removal of species present at <0.1 %, or were linked to reagents (including a primer stock). We demonstrate no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol.

Residual chlorine persistently changes antibiotic resistance gene composition and increases the risk of antibiotic resistance in sewer systems

During the COVID-19 pandemic, excessive amounts of disinfectants and their transformation products entered sewer systems worldwide, which was an extremely rare occurrence before. The stress of residual chlorine and disinfection by-products is not only likely to promote the spread of antibiotic resistance genes (ARGs), but also leads to the enrichment of chlorine-resistant bacteria that may also be resistant to antibiotics. Therefore, the potential impact of such discharge on ARG composition should be studied and the health risks should be assessed. Thus, this study combined high-throughput 16S rRNA gene amplicon sequencing and metagenomic analysis with long-term batch tests that involved two stages of stress and recovery to comprehensively evaluate the impact of residual chlorine on the microbial community and ARG compositions in sewer systems. The tests demonstrated that the disturbance of the microbial community structure by residual chlorine was reversible, but the change in ARG composition was persistent. This study found that vertical propagation and horizontal gene transfer jointly drove ARG composition succession in the biofilm, while the driving force was mainly horizontal gene transfer in the sediment. In this process, the biocide resistance gene (BRG) subtype chtR played an important role in promoting co-selection with ARGs through plasmids and integrative and conjugative elements. Moreover, it was further shown that the addition of sodium hypochlorite increased the risk of ARGs to human health, even after discontinuation of dosing, signifying that the impact was persistent. In general, this study strengthens the co-selection theory of ARGs and BRGs, and calls for improved disinfection strategies and more environmentally friendly disinfectants.

Different Responses of Bacteria and Archaea to Environmental Variables in Brines of the Mahai Potash Mine, Qinghai-Tibet Plateau.

Salt mines feature both autochthonous and allochthonous microbial communities introduced by industrialization. It is important to generate the information on the diversity of the microbial communities present in the salt mines and how they are shaped by the environment representing ecological diversification. Brine from Mahai potash mine (Qianghai, China), an extreme hypersaline environment, is used to produce potash salts for hundreds of millions of people. However, halophiles preserved in this niche during deposition are still unknown. In this study, using high-throughput 16S rRNA gene amplicon sequencing and estimation of physicochemical variables, we examined brine samples collected from locations with the gradient of industrial activity intensity and discrete hydrochemical compositions in the Mahai potash mine. Our findings revealed a highly diverse bacterial community, mainly composed of Pseudomonadota in the hypersaline brines from the industrial area, whereas in the natural brine collected from the upstream Mahai salt lake, most of the 16S rRNA gene reads were assigned to Bacteroidota. Halobacteria and halophilic methanogens dominated archaeal populations. Furthermore, we discovered that in the Mahai potash mining area, bacterial communities tended to respond to anthropogenic influences. In contrast, archaeal diversity and compositions were primarily shaped by the chemical properties of the hypersaline brines. Conspicuously, distinct methanogenic communities were discovered in sets of samples with varying ionic compositions, indicating their strong sensitivity to the brine hydrochemical alterations. Our findings provide the first taxonomic snapshot of microbial communities from the Mahai potash mine and reveal the different responses of bacteria and archaea to environmental variations in this high-altitude aquatic ecosystem.

Evaluation of different 16S rRNA gene hypervariable regions and reference databases for profiling engineered microbiota structure and functional guilds in a swine wastewater treatment plant.

High-throughput 16S rRNA gene amplicon sequencing technology is widely applied for environmental microbiota structure analysis to derive knowledge that informs microbiome-based surveillance and oriented bioengineering. However, it remains elusive how the selection of 16S rRNA gene hypervariable regions and reference databases affects microbiota diversity and structure profiling. This study systematically evaluated the fitness of different frequently used reference databases (i.e. SILVA 138 SSU, GTDB bact120_r207, Greengenes 13_5 and MiDAS 4.8) and primers of 16S rRNA gene in microbiota profiling of anaerobic digestion and activated sludge collected from a full-scale swine wastewater treatment plant (WWTP). The comparative results showed that MiDAS 4.8 achieved the highest levels of taxonomic diversity and species-level assignment rate. For whichever sample groups, microbiota richness captured by different primers decreased in the following order: V4 > V4-V5 > V3-V4 > V6-V8/V1-V3. Using primer-bias-free metagenomic data results as the judging standard, V4 region also best characterized microbiota structure and well represented typical functional guilds (e.g. methanogens, ammonium oxidizers and denitrifiers), while V6-V8 regions largely overestimated the archaeal methanogens (mainly Methanosarcina) by over 30 times. Therefore, MiDAS 4.8 database and V4 region are recommended for best simultaneous analysis of bacterial and archaeal community diversity and structure of the examined swine WWTP.

Bacterial Communities Are Less Diverse in a Strepsipteran Endoparasitoid than in Its Fruit Fly Hosts and Dominated by Wolbachia

Microbiomes play vital roles in insect fitness and health and can be influenced by interactions between insects and their parasites. Many studies investigate the microbiome of free-living insects, whereas microbiomes of endoparasitoids and their interactions with parasitised insects are less explored. Due to their development in the constrained environment within a host, endoparasitoids are expected to have less diverse yet distinct microbiomes. We used high-throughput 16S rRNA gene amplicon sequencing to characterise the bacterial communities of Dipterophagus daci (Strepsiptera) and seven of its tephritid fruit fly host species. Bacterial communities of D. daci were less diverse and contained fewer taxa relative to the bacterial communities of the tephritid hosts. The strepsipteran’s microbiome was dominated by Pseudomonadota (formerly Proteobacteria) (> 96%), mainly attributed to the presence of Wolbachia, with few other bacterial community members, indicative of an overall less diverse microbiome in D. daci. In contrast, a dominance of Wolbachia was not found in flies parasitised by early stages of D. daci nor unparasitised flies. Yet, early stages of D. daci parasitisation resulted in structural changes in the bacterial communities of parasitised flies. Furthermore, parasitisation with early stages of D. daci with Wolbachia was associated with a change in the relative abundance of some bacterial taxa relative to parasitisation with early stages of D. daci lacking Wolbachia. Our study is a first comprehensive characterisation of bacterial communities in a Strepsiptera species together with the more diverse bacterial communities of its hosts and reveals effects of concealed stages of parasitisation on host bacterial communities.

Comparing the Efficacy of MALDI-TOF MS and Sequencing-Based Identification Techniques (Sanger and NGS) to Monitor the Microbial Community of Irrigation Water.

In order to intensify and guarantee the agricultural productivity and thereby to be able to feed the world's rapidly growing population, irrigation has become very important. In parallel, the limited water resources lead to an increase in usage of poorly characterized sources of water, which is directly linked to a higher prevalence of foodborne diseases. Therefore, analyzing the microorganisms or even the complete microbiome of irrigation water used for food production can prevent the growing numbers of such cases. In this study, we compared the efficacy of MALDI-TOF Mass spectrometry (MALDI TOF MS) identification to 16S rRNA gene Sanger sequencing of waterborne microorganisms. Furthermore, we analyzed the whole microbial community of irrigation water using high-throughput 16S rRNA gene amplicon sequencing. The identification results of MALDI-TOF MS and 16S rRNA gene Sanger sequencing were almost identical at species level (66.7%; 64.3%). Based on the applied cultivation techniques, Acinetobacter spp., Enterobacter spp., Pseudomonas spp., and Brevundimonas spp. were the most abundant cultivable genera. In addition, the uncultivable part of the microbiome was dominated by Proteobacteria followed by Actinobacteria, Bacteroidota, Patescibacteria, and Verrucomicrobiota. Our findings indicate that MALDI-TOF MS offers a fast, reliable identification method and can act as an alternative to 16S rRNA gene Sanger sequencing of isolates. Moreover, the results suggest that MALDI-TOF MS paired with 16S rRNA gene amplicon sequencing have the potential to support the routine monitoring of the microbiological quality of irrigation water.

Effects of continuous cropping Jiashi muskmelon on rhizosphere microbial community.

The continuous cropping of crops can result in the deterioration of the soil environment and cause a decline in plant health and yield, which complicates agricultural production. However, the effects of continuous melon cropping on rhizospheric microbial communities remain poorly understood. In this study, high-throughput absolute quantification 16S rRNA gene amplicon sequencing was employed to analyze the bacterial community structure of greenhouse rhizosphere soil from Jiashi muskmelon replanted for 0, 1, 2, and 3 years (CK, 1a, 2a, and 3a, respectively). The results showed that long- term continuous cropping caused significant changes in soil physicochemical properties. The bacterial absolute abundances increased, but the bacterial community richness and diversity were significantly lost (p < 0.05). The composition of bacterial community was more similar after 2 and 3 years of continuous cropping. The longer the continuous cropping years were, the greater the shift in the bacterial diversity and abundance. Analysis of potential functional components revealed that different bacterial groups were enriched in different continuous cropping years. The significant reduction of the taxa associated with nitrate reduction may be responsible for the loss of soil nitrogen in continuous cropping soil. In summary, continuous cropping had a significant impact on the bacterial community structure of Jiashi muskmelon rhizospheric soil, and these results will provide a reference for soil management and scientific fertilization of melon and other crops under a continuous cropping regime.

Bacterial succession in an abandoned circum-neutral opencast coal mine in India

Context Abandoned mines provide an ideal opportunity to study the succession of microbial communities, which is crucial for the development and stability of the soil. Aims We examined the diversity and temporal succession of the microbial community in an abandoned coal mine. Methods We investigated soil geochemical parameters and microbial succession using high-throughput 16S rRNA gene amplicon sequences over a 4-year period in a derelict opencast circum-neutral coal mine in Tasra colliery, Jharkhand, India. Results Soil pH (4.46–6.13), organic carbon (0.86–4.53%), available nitrogen (0.001–0.003%) and total cultivable heterotrophic microbial load increased, and concentrations of chloride (24 865–15 067 ppm), sulfate (26 417–417 ppm) and heavy metals reduced over the period of 4 years. The 16S rRNA gene sequences identified Proteobacteria as the dominant phylum across samples except for the fourth year, where Actinobacteria was predominant. Chlorobi, WPS-2 and Spirochaetes were exclusively identified in the first year. Twelve phyla present in the first year were completely absent by the end. In the fourth year, an abundance of nitrogen-fixing members such as Burkholderiales, Rhizobiales, Roseiflexales and Actinomycetales was noted and semi-quantitative PCR suggested that nitrogenases were predominately vanadium dependent (relative to molybdenum). Gradual shifts from purportedly chemolithotrophic to heterotrophic metabolic strategies were also observed. Conclusion This work establishes that soil improvement in abandoned coal mines involves the dynamic interplay of physicochemical parameters and shifts in microbial communities and dominant metabolic guilds. This knowledge can be used to plan and monitor remediation measures in such sites.

Application of ecosystem-specific reference databases for increased taxonomic resolution in soil microbial profiling.

Intensive agriculture systems have paved the way for a growing human population. However, the abundant use of mineral fertilizers and pesticides may negatively impact nutrient cycles and biodiversity. One potential alternative is to harness beneficial relationships between plants and plant-associated rhizobacteria to increase nutrient-use efficiency and provide pathogen resistance. Plant-associated microbiota profiling can be achieved using high-throughput 16S rRNA gene amplicon sequencing. However, interrogation of these data is limited by confident taxonomic classifications at high taxonomic resolution (genus- or species level) with the commonly applied universal reference databases. High-throughput full-length 16S rRNA gene sequencing combined with automated taxonomy assignment (AutoTax) can be used to create amplicon sequence variant resolved ecosystems-specific reference databases that are superior to the traditional universal reference databases. This approach was used here to create a custom reference database for bacteria and archaea based on 987,353 full-length 16S rRNA genes from Askov and Cologne soils. We evaluated the performance of the database using short-read amplicon data and found that it resulted in the increased genus- and species-level classification compared to commonly use universal reference databases. The custom database was utilized to evaluate the ecosystem-specific primer bias and taxonomic resolution of amplicon primers targeting the V5-V7 region of the 16S rRNA gene commonly used within the plant microbiome field. Finally, we demonstrate the benefits of custom ecosystem-specific databases through the analysis of V5-V7 amplicon data to identify new plant-associated microbes for two legumes and two cereal species.

Triclosan Promotes Conjugative Transfer of Antibiotic Resistance Genes to Opportunistic Pathogens in Environmental Microbiome.

Although triclosan, as a widely used antiseptic chemical, is known to promote the transmission of antibiotic resistance to diverse hosts in pure culture, it is still unclear whether and how triclosan could affect the transmission of broad-host-range plasmids among complex microbial communities. Here, bacterial culturing, fluorescence-based cell sorting, and high-throughput 16S rRNA gene amplicon sequencing were combined to investigate contributions of triclosan on the transfer rate and range of an IncP-type plasmid from a proteobacterial donor to an activated sludge microbiome. Our results demonstrate that triclosan significantly enhances the conjugative transfer of the RP4 plasmid among activated sludge communities at environmentally relevant concentrations. High-throughput 16S rRNA gene sequencing on sorted transconjugants demonstrates that triclosan not only promoted the intergenera transfer but also the intragenera transfer of the RP4 plasmid among activated sludge communities. Moreover, triclosan mediated the transfer of the RP4 plasmid to opportunistic human pathogens, for example, Legionella spp. The mechanism of triclosan-mediated conjugative transfer is primarily associated with excessive oxidative stress, followed by increased membrane permeability and provoked SOS response. Our findings offer insights into the impacts of triclosan on the dissemination of antibiotic resistance in the aquatic environmental microbiome.

Occurrence and driving mechanism of antibiotic resistance genes in marine recreational water around Qinhuangdao, China

Rapid urbanization and other human activities have exacerbated the global human health risks associated with antibiotic resistance genes (ARGs). Swimming in contaminated waters is one of important pathways by which humans can be exposed to ARGs. However, little is currently known about the overall levels of ARGs in recreational waters and the forces driving their occurrence. In this study, we analyzed the ARGs and bacterial communities in water samples taken from five recreational coastal waters (RCWs) and their adjacent estuaries in the Qinhuangdao area of Bohai Bay, China, using high-throughput quantitative PCR and 16S rRNA gene amplicon sequencing technique. The relative abundances of ARGs in RCWs varied greatly, and occurred at levels close to, or even higher than, those in the neighboring estuaries, ranging from 9.4×10-2 to 2.5 copies/16S rRNA gene. A total of 159 different ARGs able to express resistance to almost all antibiotics were detected in all of the water samples. β-lactamase resistance genes (blaTEM gene) were the most abundance of all ARGs. Patterns of ARG occurrence were significantly correlated with those of bacterial communities (Mantel test, P &amp;lt; 0.05). A partial redundancy analysis showed that differences in bacterial communities accounted for 50.3% of the total ARG variations, which was much higher than the sum of the contribution of mobile genetic elements (18.1%) and the other selected environmental factors (8.3%). Principal coordinate analysis based on Bray-Curtis distance revealed the similarities between ARG profiles in the RCWs and in their adjacent estuaries. To our knowledge, this is the first study to report the ARG profiles in RCWs using high throughput qPCR technology. These results implied that it is necessary to develop proper regulations and monitoring strategies of RCWs for reducing risks of ARGs on human health.

Metagenomic Insights into the Gut Microbiota of Eudrilus eugeniae (Kinberg) and Its Potential Roles in Agroecosystem.

Gut microbiomes, a consortium of microorganisms that live in the animal gut, are highly engineered microbial communities. It makes a major contribution to digestive health, metabolism management, and the development of a strong immune system in the host. The present study was taken up to answer the long-running question about the existence of truly indigenous microflora of the epigeic earthworm gut. This is due to the general difficulties of culturing many of the microorganisms found in soil or earthworms' gut. Keeping this fact in a view, the metagenomics approach using 16S rRNA marker gene incorporated with amplicon-based sequencing was used to explore microbiota of commercially overriding, diversely fed epigeic earthworm Eudrilus eugeniae (Kinberg) in three varied habitats viz., artificial soil (AS), organic agricultural farm soil (OAFS) and conventional agriculture farm soil (CAFS). There are predominant bacteria that belong to different phyla such as Proteobacteria (29.72-76.81%), Actinobacteria (11.06-34.42%), Firmicutes (6.02-19.81%), and Bacteroidetes (2.40-9.22%) present in the gut of E. eugeniae. The alpha diversity (Observed species, Chao1, ACE, Shannon, Simpson, and Fisher alpha) indices showed that OAFS had significantly higher alpha diversity than AS and CAFS groups. The core microbiota analysis showed that OAFS and AS groups had a relatively similar bacterial panel in comparison to the CAFS group. Various statistical tools i.e. MetagenomeSeq, LEfSe, and Random Forest analysis were performed and the findings demonstrated prevalence of the most significant bacterial genera; Aeromonas, Gaiella, and Burkholderia in CAFS group. Nonetheless, in AS and OAFS groups, the common existence of Anaerosporobacter and Aquihabitans were found respectively. Metagenomic functional prediction revealed that earthworms' gut microbial communities were actively involved in multiple organic and xenobiotics compound degradation-related pathways. This is the first research to use high-throughput 16S rRNA gene amplicon sequencing to show the gut microbiota of E. eugeniae in diverse agricultural systems. The findings suggest the configuration of the gut microbiota of earthworms and its potential role in the soil ecosystem depends on the microbial communities of the soil.

Spatial variability of bacterial community compositions in the Mariana Trench.

Hadal microorganisms play an important role in the biogeochemical processes in marine ecosystems and act as a valuable resource for industrial applications. This paper presents the bacterial community analysis of samples taken from the Challenger Deep within the Mariana Trench, which is the deepest site in the ocean. High-throughput 16S rRNA gene amplicon sequencing was used to reveal that the vertically sampled bacterial populations at eight stations varied at the surface to 10 km depth. The surface water samples harbored a distinct bacterial assemblage, while the mesopelagic and bathyal samples manifested different bacterial community composition, which was not consistent with previous studies. Gammaproteobacteria was the most abundant bacteria in the bathyal and hadal waters. The hadal bacterial community consisted mostly of Alteromonadales and Oceanospirillales. The former was widely spread in the water column, which might suggest habitat partitioning at the genus and operational taxonomic unit levels, while the latter might represent hadal-enriched hydrocarbon degraders. The present work complements the current knowledge and understanding of the bathyal and hadal bacterial communities of the Mariana Trench.

QT-AMP: Sequencing PCR amplicons from Quanti-Tray wells to analyze enterococci communities

Enterococcus is ubiquitous in human feces and has been adopted as a useful indicator of human fecal pollution in water. Although regular enterococci monitoring only examines their numbers, identifying human-specific Enterococcus species or genotypes could help discriminate human fecal contamination from other environmental sources. We documented a new approach to characterize enterococci using a high-throughput 16S rRNA gene amplicon sequencing platform from Quanti Trays after following the counting of the most probable numbers of enterococci. We named this method QT-AMP (Quanti-Tray-based amplicon sequencing). We tested surface water samples collected from three rivers in southwest Florida. We detected 11 Enterococcus species from 45 samples in 1.1 million sequence reads. The method detected three rare species and eight cosmopolitan species (Enterococcus faecalis, E. faecium, E. casseliflavus, E. hirae, E. mundtii, E. gallinarum, E. avium, and E. durans) which have been commonly documented in previous studies. The approximate detection level of QT-AMP was four orders of magnitude higher than regular 16S rRNA gene amplicon sequencing. The current Enterolert MPN method only provides quantitative information but now we can look into the relative abundance of Enterococci species composition by accompanying Illumina sequencing. This QT-AMP could be a useful tool to streamline the quantification and identification of enterococci and could be used in various water management projects and human health risk assessment.

Student-Driven Course-Based Undergraduate Research Experience (CUREs) Projects in Identifying Vaginal Microorganism Species Communities to Promote Scientific Literacy Skills.

ObjectivesWe aim to build a students' own engagement in original microbiological course-based undergraduate research experience (CUREs) model served two research and teaching scientific purposes including students' scientific literacy skills and instructors' role, which could further be applied as contribution to broader scientific knowledge and conduct novel research in their future research experience and careers.MethodsWe describe a student-driven CUREs model on the microorganism species in female vaginal using general bacterial culture techniques and high-throughput 16S rRNA gene amplicon sequencing to enable students to center experimental research method under the direction of instructors. A total of 8 undergraduate students and 5 instructors from Shanghai Jiao Tong University School of Medicine participated in the project. The CUREs were divided in four operating scopes: project planning, implementation, summarizing and feedback phases. Instructors help students to develop learning research goals.ResultsThis project helped students to gain “hard skills” experiences in scientific theoretical research process and technical practices. Students reached the conclusion that Lactobacillus species dominated the primary vaginal microbiota in reproductive-age women, 16S rRNA sequencing is a method widely applied for microbiology detection. CUREs also increased students' engagement in scientific experiments and promote 3 learning goals in “soft skills”: (1) Develop students' self-study and efficacy ability, expression capability and professional research communication skills; (2) Strengthen students' motivation and ownership in science research, overcoming failure, benefitting persistence and patience, building professional science identity, competence, and confidence in collaboration, implement spirit of rigorous and carefulness; (3) Obtain authorship, independent and logical thinking capability, summarizing ability and confidence enhancement. Instructors proposed guiding research question for the students and determine evidence in achieving pedagogical goals in CUREs.ConclusionsOur microbiological CUREs project served two scientific purposes: research and teaching, which increase students' engagement in promoting learning gains in scientific research skills, ownership, identity development, and spirit of motivation, self-efficacy, persistence, collaboration, communication, as well as opportunities to make relevant scientific discoveries. These abilities equipped them with essential foundation for the subsequent collaborative experiments and future scientific study.