Rapid, inexpensive measurement of synthetic bacterial community composition by Sanger sequencing

AbstractSimple synthetic bacterial communities are powerful tools for studying microbial ecology and evolution, as they enable rapid iteration between controlled laboratory experiments and theoretical modeling. However, their utility is hampered by the lack of fast, inexpensive, and accurate methods for quantifying bacterial community composition. For instance, while next-generation amplicon sequencing can be very accurate, high costs (>$30 per sample) and turnaround times (>1 month) limit the nature and pace of experiments. Here, we introduce a new approach for quantifying composition in synthetic bacterial communities based on Sanger sequencing. First, for a given community, we PCR-amplify a universal marker gene (here, the 16S rRNA gene), which yields a mixture of amplicons. Second, we sequence this amplicon mixture in a single Sanger sequencing reaction, which produces a “mixed” electropherogram with contributions from each community member. We also sequence each community member’s marker gene individually to generate “individual” electropherograms. Third, we fit the mixed electropherogram as a linear combination of time-warped individual electropherograms, thereby allowing us to estimate the fractional amplicon abundance of each strain within the community. Importantly, our approach accounts for retention-time variability in electrophoretic signals, which is crucial for accurate compositional estimates. Using synthetic communities of marine bacterial isolates, we show that this approach yields accurate and reproducible abundance estimates for two-, four-, and seven-strain bacterial communities. Furthermore, this approach can provide results within one day and costs ~$5 USD per sample. We envision this approach will enable new insights in microbial ecology by increasing the number of samples that can be analyzed and enabling faster iteration between experiments and theory. We have implemented our method in a free and open-source R package called CASEU (“Compositional Analysis by Sanger Electropherogram Unmixing”), available at https://bitbucket.org/DattaManoshi/caseu.

Download Full-text

Impact of enzymatic digestion on bacterial community composition in CF airway samples

PeerJ ◽

10.7717/peerj.3362 ◽

2017 ◽

Vol 5 ◽

pp. e3362 ◽

Cited By ~ 1

Author(s):

Kayla M. Williamson ◽

Brandie D. Wagner ◽

Charles E. Robertson ◽

Emily J. Johnson ◽

Edith T. Zemanick ◽

...

Keyword(s):

Bacterial Community ◽

Dna Extraction ◽

Community Composition ◽

Bacterial Communities ◽

Beta Diversity ◽

Diversity Index ◽

Bacterial Community Composition ◽

Microbial Community Composition ◽

Enzymatic Digestion ◽

Rrna Gene

BackgroundPrevious studies have demonstrated the importance of DNA extraction methods for molecular detection ofStaphylococcus,an important bacterial group in cystic fibrosis (CF). We sought to evaluate the effect of enzymatic digestion (EnzD) prior to DNA extraction on bacterial communities identified in sputum and oropharyngeal swab (OP) samples from patients with CF.MethodsDNA from 81 samples (39 sputum and 42 OP) collected from 63 patients with CF was extracted in duplicate with and without EnzD. Bacterial communities were determined by rRNA gene sequencing, and measures of alpha and beta diversity were calculated. Principal Coordinate Analysis (PCoA) was used to assess differences at the community level and Wilcoxon Signed Rank tests were used to compare relative abundance (RA) of individual genera for paired samples with and without EnzD.ResultsShannon Diversity Index (alpha-diversity) decreased in sputum and OP samples with the use of EnzD. Larger shifts in community composition were observed for OP samples (beta-diversity, measured by Morisita-Horn), whereas less change in communities was observed for sputum samples. The use of EnzD with OP swabs resulted in significant increase in RA for the generaGemella(p < 0.01),Streptococcus(p < 0.01), andRothia(p < 0.01).Staphylococcus(p < 0.01) was the only genus with a significant increase in RA from sputum, whereas the following genera decreased in RA with EnzD:Veillonella(p < 0.01),Granulicatella(p < 0.01),Prevotella(p < 0.01), andGemella(p = 0.02). In OP samples, higher RA of Gram-positive taxa was associated with larger changes in microbial community composition.DiscussionWe show that the application of EnzD to CF airway samples, particularly OP swabs, results in differences in microbial communities detected by sequencing. Use of EnzD can result in large changes in bacterial community composition, and is particularly useful for detection ofStaphylococcusin CF OP samples. The enhanced identification ofStaphylococcus aureusis a strong indication to utilize EnzD in studies that use OP swabs to monitor CF airway communities.

Download Full-text

Effects of geographic location and water quality on bacterial communities in full-scale biofilters across North America

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz210 ◽

2020 ◽

Vol 96 (2) ◽

Cited By ~ 1

Author(s):

Ben Ma ◽

Timothy M LaPara ◽

Ashley N. Evans ◽

Raymond M Hozalski

Keyword(s):

Water Quality ◽

North America ◽

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Bacterial Communities ◽

Bacterial Community Composition ◽

Geographic Location ◽

Rrna Gene

ABSTRACT Spatial patterns of bacterial community composition often follow a distance–decay relationship in which community dissimilarity increases with geographic distance. Such a relationship has been commonly observed in natural environments, but less so in engineered environments. In this study, bacterial abundance and community composition in filter media samples (n = 57) from full-scale rapid biofilters at 14 water treatment facilities across North America were determined using quantitative polymerase chain reaction and Illumina HiSeq high-throughput sequencing targeting the 16S rRNA gene, respectively. Bacteria were abundant on the filter media (108.8±0.3 to 1010.7±0.2 16S rRNA gene copies/cm3 bed volume) and the bacterial communities were highly diverse (Shannon index: 5.3 ± 0.1 to 8.4 ± 0.0). Significant inter-filter variations in bacterial community composition were observed, with weighted UniFrac dissimilarity values following a weak but highly significant distance–decay relationship (z = 0.0057 ± 0.0006; P = 1.8 × 10−22). Approximately 50% of the variance in bacterial community composition was explained by the water quality parameters measured at the time of media sample collection (i.e. pH, temperature and dissolved organic carbon concentration). Overall, this study suggested that the microbiomes of biofilters are primarily shaped by geographic location and local water quality conditions but the influence of these factors on the microbiomes is tempered by filter design and operating conditions.

Download Full-text

The bacterial community composition and its environmental drivers in the rivers around eutrophic Chaohu Lake, China

BMC Microbiology ◽

10.1186/s12866-021-02252-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Keqiang Shao ◽

Xin Yao ◽

Zhaoshi Wu ◽

Xingyu Jiang ◽

Yang Hu ◽

...

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Bacterial Community Composition ◽

Cyanobacterial Blooms ◽

Environmental Drivers ◽

Chaohu Lake ◽

Rrna Gene ◽

Functional Profiles ◽

16 S Rrna

Abstract Background Bacterial community play a key role in environmental and ecological processes in river ecosystems. Rivers are used as receiving body for treated and untreated urban wastewaters that brings high loads of sewage and excrement bacteria. However, little is known about the bacterial community structure and functional files in the rivers around the eutrophic Chaohu Lake, the fifth largest freshwater lake in China, has been subjected to severe eutrophication and cyanobacterial blooms over the past few decades. Therefore, understanding the taxonomic and functional compositions of bacterial communities in the river will contribute to understanding aquatic microbial ecology. The main aims were to (1) examine the structure of bacterial communities and functional profiles in this system; (2) find the environmental factors of bacterial community variations. Results We studied 88 sites at rivers in the Chaohu Lake basin, and determined bacterial communities using Illumina Miseq sequencing of the 16 S rRNA gene, and predicted functional profiles using PICRUSt2. A total of 3,390,497 bacterial 16 S rRNA gene sequences were obtained, representing 17 phyla, and 424 genera; The dominant phyla present in all samples were Bacteroidetes (1.4-82.50 %), followed by Proteobacteria (12.6–97.30 %), Actinobacteria (0.1–17.20 %). Flavobacterium was the most numerous genera, and accounted for 0.12–80.34 % of assigned 16 S reads, followed by Acinetobacter (0.33–49.28 %). Other dominant bacterial genera including Massilia (0.06–25.40 %), Psychrobacter (0-36.23 %), Chryseobacterium (0.01–22.86 %), Brevundimonas (0.01–12.82 %), Pseudomonas (0-59.73 %), Duganella (0.08–23.37 %), Unidentified Micrococcaceae (0-8.49 %). The functional profiles of the bacterial populations indicated an relation with many human diseases, including infectious diseases. Overall results, using the β diversity measures, coupled with heatmap and RDA showed that there were spatial variations in the bacterial community composition at river sites, and Chemical oxygen demand (CODMn) and (NH4+ )were the dominant environmental drivers affecting the bacterial community variance. Conclusions The high proportion of the opportunistic pathogens (Acinetobacter, Massilia, Brevundimonas) indicated that the discharge of sewage without adequate treatment into the rivers around Chaohu Lake. We propose that these bacteria could be more effective bioindicators for long-term sewage monitoring in eutrophic lakes.

Download Full-text

Bacterial community composition and dynamics spanning five years in freshwater bog lakes

10.1101/127035 ◽

2017 ◽

Cited By ~ 1

Author(s):

Alexandra M. Linz ◽

Benjamin C. Crary ◽

Ashley Shade ◽

Sarah Owens ◽

Jack A. Gilbert ◽

...

Keyword(s):

Time Series ◽

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Community Dynamics ◽

Bacterial Community Composition ◽

Rrna Gene ◽

Bacterial Community Dynamics ◽

Bog Lakes

AbstractBacteria play a key role in freshwater biogeochemical cycling, but long-term trends in freshwater bacterial community composition and dynamics are not yet well characterized. We used a multi-year time series of 16S rRNA gene amplicon sequencing data from eight bog lakes to census the freshwater bacterial community and observe annual and seasonal trends in abundance. Multiple sites and sampling events were necessary to begin to fully describe the bacterial communities. Each lake and layer contained a distinct bacterial community, with distinct levels of richness and indicator taxa that likely reflected the environmental conditions of each site. The community present in each year and site was also unique. Despite high interannual variability in community composition, we detected a core community of ubiquitous freshwater taxa. Although trends in abundance did not repeat annually, each freshwater lineage within the communities had a consistent lifestyle, defined by persistence, abundance, and variability. The results of our analysis emphasize the importance of long-term observations, as analyzing only a single year of data would not have allowed us to describe the dynamics and composition of these freshwater bacterial communities to the extent presented here.ImportanceLakes are excellent systems for investigating bacterial community dynamics because they have clear boundaries and strong environmental gradients. The results of our research demonstrate that bacterial community dynamics operate on multi-year timescales, a finding which likely applies to other ecosystems, with implications for study design and interpretation. Understanding the drivers and controls of bacterial communities on long time scales would improve both our knowledge of fundamental properties of bacterial communities, and our ability to predict community states. In this specific ecosystem, bog lakes play a disproportionately large role in global carbon cycling, and the information presented here may ultimately help refine carbon budgets for these lakes. Finally, all data and code in this study are publicly available. We hope that this will serve as a resource to anyone seeking to answer their own microbial ecology questions using a multi-year time series.

Download Full-text

Rhizobacterial communities of five co-occurring desert halophytes

PeerJ ◽

10.7717/peerj.5508 ◽

2018 ◽

Vol 6 ◽

pp. e5508 ◽

Cited By ~ 3

Author(s):

Yan Li ◽

Yan Kong ◽

Dexiong Teng ◽

Xueni Zhang ◽

Xuemin He ◽

...

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Plant Species ◽

Bacterial Communities ◽

Bacterial Community Composition ◽

Community Diversity ◽

Species Identity ◽

Halostachys Caspica ◽

Lycium Ruthenicum ◽

Limonium Gmelinii

BackgroundRecently, researches have begun to investigate the microbial communities associated with halophytes. Both rhizobacterial community composition and the environmental drivers of community assembly have been addressed. However, few studies have explored the structure of rhizobacterial communities associated with halophytic plants that are co-occurring in arid, salinized areas.MethodsFive halophytes were selected for study: these co-occurred in saline soils in the Ebinur Lake Nature Reserve, located at the western margin of the Gurbantunggut Desert of Northwestern China. Halophyte-associated bacterial communities were sampled, and the bacterial 16S rDNA V3–V4 region amplified and sequenced using the Illumina Miseq platform. The bacterial community diversity and structure were compared between the rhizosphere and bulk soils, as well as among the rhizosphere samples. The effects of plant species identity and soil properties on the bacterial communities were also analyzed.ResultsSignificant differences were observed between the rhizosphere and bulk soil bacterial communities. Diversity was higher in the rhizosphere than in the bulk soils. Abundant taxonomic groups (from phylum to genus) in the rhizosphere were much more diverse than in bulk soils. Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Planctomycetes were the most abundant phyla in the rhizosphere, while Proteobacteria and Firmicutes were common in bulk soils. Overall, the bacterial community composition were not significantly differentiated between the bulk soils of the five plants, but community diversity and structure differed significantly in the rhizosphere. The diversity ofHalostachys caspica,Halocnemum strobilaceumandKalidium foliatumassociated bacterial communities was lower than that ofLimonium gmeliniiandLycium ruthenicumcommunities. Furthermore, the composition of the bacterial communities ofHalostachys caspicaandHalocnemum strobilaceumwas very different from those ofLimonium gmeliniiandLycium ruthenicum. The diversity and community structure were influenced by soil EC, pH and nutrient content (TOC, SOM, TON and AP); of these, the effects of EC on bacterial community composition were less important than those of soil nutrients.DiscussionHalophytic plant species played an important role in shaping associated rhizosphere bacterial communities. When salinity levels were constant, soil nutrients emerged as key factors structuring bacterial communities, while EC played only a minor role. Pairwise differences among the rhizobacterial communities associated with different plant species were not significant, despite some evidence of differentiation. Further studies involving more halophyte species, and individuals per species, are necessary to elucidate plant species identity effects on the rhizosphere for co-occurring halophytes.

Download Full-text

Correlation of Breed, Growth Performance, and Rumen Microbiota in Two Rustic Cattle Breeds Reared Under Different Conditions

Frontiers in Microbiology ◽

10.3389/fmicb.2021.652031 ◽

2021 ◽

Vol 12 ◽

Author(s):

Matteo Daghio ◽

Francesca Ciucci ◽

Arianna Buccioni ◽

Alice Cappucci ◽

Laura Casarosa ◽

...

Keyword(s):

Bacterial Community ◽

Growth Performance ◽

Community Composition ◽

Lipid Composition ◽

High Throughput Sequencing ◽

Bacterial Community Composition ◽

Daily Weight Gain ◽

Rrna Gene ◽

Rumen Microbiota ◽

Cattle Breeds

The use of rustic cattle is desirable to face challenges brought on by climate change. Maremmana (MA) and Aubrac (AU) are rustic cattle breeds that can be successfully used for sustainable production. In this study, correlations between two rearing systems (feedlot and grazing) and the rumen microbiota, the lipid composition of rumen liquor (RL), and the growth performance of MA and AU steers were investigated. Bacterial community composition was characterized by high-throughput sequencing of 16S rRNA gene amplicons, and the RL lipid composition was determined by measuring fatty acid (FA) and the dimethyl acetal profiles. The main factor influencing bacterial community composition was the cattle breed. Some bacterial groups were positively correlated to average daily weight gain for the two breeds (i.e., Rikenellaceae RC9 gut group, Fibrobacter and Succiniclasticum in the rumen of MA steers, and Succinivibrionaceae UCG-002 in the rumen of AU steers); despite this, animal performance appeared to be influenced by short chain FAs production pathways and by the presence of H2 sinks that divert the H2 to processes alternative to the methanogenesis.

Download Full-text

Significant Differences in the Gut Bacterial Communities of Hooded Crane (Grus monacha) in Different Seasons at a Stopover Site on the Flyway

Animals ◽

10.3390/ani10040701 ◽

2020 ◽

Vol 10 (4) ◽

pp. 701 ◽

Cited By ~ 1

Author(s):

Fengling Zhang ◽

Xingjia Xiang ◽

Yuanqiu Dong ◽

Shaofei Yan ◽

Yunwei Song ◽

...

Keyword(s):

Community Structure ◽

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Bacterial Community Structure ◽

Bacterial Community Composition ◽

Stopover Site ◽

Intestinal Bacterial Community ◽

Hooded Crane ◽

Grus Monacha

Intestinal bacterial communities form an integral component of the organism. Many factors influence gut bacterial community composition and diversity, including diet, environment and seasonality. During seasonal migration, birds use many habitats and food resources, which may influence their intestinal bacterial community structure. Hooded crane (Grus monacha) is a migrant waterbird that traverses long distances and occupies varied habitats. In this study, we investigated the diversity and differences in intestinal bacterial communities of hooded cranes over the migratory seasons. Fecal samples from hooded cranes were collected at a stopover site in two seasons (spring and fall) in Lindian, China, and at a wintering ground in Shengjin Lake, China. We analyzed bacterial communities from the fecal samples using high throughput sequencing (Illumina Mi-seq). Firmicutes, Proteobacteria, Tenericutes, Cyanobacteria, and Actinobacteria were the dominant phyla across all samples. The intestinal bacterial alpha-diversity of hooded cranes in winter was significantly higher than in fall and spring. The bacterial community composition significantly differed across the three seasons (ANOSIM, P = 0.001), suggesting that seasonal fluctuations may regulate the gut bacterial community composition of migratory birds. This study provides baseline information on the seasonal dynamics of intestinal bacterial community structure in migratory hooded cranes.

Download Full-text

Dynamics of the bacterial community in a channel catfish nursery pond with a cage–pond integration system

Canadian Journal of Microbiology ◽

10.1139/cjm-2018-0268 ◽

2018 ◽

Vol 64 (12) ◽

pp. 954-967 ◽

Cited By ~ 2

Author(s):

Liqiang Zhong ◽

Daming Li ◽

Minghua Wang ◽

Xiaohui Chen ◽

Wenji Bian ◽

...

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

16S Rrna Gene ◽

Community Composition ◽

Channel Catfish ◽

Bacterial Community Composition ◽

Rrna Gene ◽

Integration System ◽

Nursery Pond ◽

Site Variation

The changes in the bacterial community composition in a channel catfish nursery pond with a cage–pond integration system were investigated by sequencing of the 16S rRNA gene through Illumina MiSeq sequencing platforms. A total of 1 362 877 sequences and 1440 operational taxonomic units were obtained. Further analysis showed that the dominant phyla in the cage and pond groups were similar, including Actinobacteria, Cyanobacteria, Proteobacteria, and Bacteroidetes, although a significant difference was detected between them by ANOSIM (P < 0.05). Temporal changes and site variation were significantly related to the variation of the bacterial community. A comprehensive analysis of the diversity and evenness of the bacterial 16S rRNA gene, redundancy analysis (RDA), and partial Mantel test showed that the bacterial community composition in a cage–pond integration system was shaped more by temporal variation than by site variation. RDA also indicated that water temperature, total dissolved solids, and Secchi depth had the largest impact on bacterial populations.

Download Full-text

Multiple Stressors Determine Community Structure and Estimated Function of River Biofilm Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.00291-20 ◽

2020 ◽

Vol 86 (12) ◽

Cited By ~ 1

Author(s):

Ferran Romero ◽

Vicenç Acuña ◽

Sergi Sabater

Keyword(s):

Bacterial Community ◽

Community Composition ◽

Bacterial Communities ◽

Pesticide Exposure ◽

Multiple Stressors ◽

Bacterial Community Composition ◽

Freshwater Ecosystems ◽

Low Flow ◽

Hydrological Stress ◽

The Individual

ABSTRACT Freshwater ecosystems are exposed to multiple stressors, but their individual and combined effects remain largely unexplored. Here, we investigated the response of stream biofilm bacterial communities to warming, hydrological stress, and pesticide exposure. We used 24 artificial streams on which epilithic (growing on coarse sediments) and epipsammic (growing on fine sediments) stream biofilms were maintained. Bacterial community composition and estimated function of biofilms exposed during 30 days to individual and combined stressors were assessed using 16S rRNA gene metabarcoding. Among the individual effects by stressors, hydrological stress (i.e., a simulated low-flow situation) was the most relevant, since it significantly altered 57% of the most abundant bacterial taxa (n = 28), followed by warming (21%) and pesticide exposure (11%). Regarding the combined effects, 16% of all stressor combinations resulted in significant interactions on bacterial community composition and estimated function. Antagonistic responses prevailed (57 to 89% of all significant interactions), followed by synergisms (11 to 43%), on specific bacterial taxa, indicating that multiple-stressor scenarios could lead to unexpected shifts in the community composition and associated functions of riverine bacterial communities. IMPORTANCE Freshwater ecosystems such as rivers are of crucial importance for human well-being. However, human activities result in many stressors (e.g., toxic chemicals, increased water temperatures, and hydrological alterations) cooccurring in rivers and streams worldwide. Among the many organisms inhabiting rivers and streams, bacteria are ecologically crucial; they are placed at the base of virtually all food webs and they recycle the organic matter needed for bigger organisms. Most of these bacteria are in close contact with river substratum, where they form the biofilms. There is an urgent need to evaluate the effects of these stressors on river biofilms, so we can anticipate future environmental problems. In this study, we experimentally exposed river biofilms to a pesticide mixture, an increase in water temperature and a simulated low-flow condition, in order to evaluate the individual and joint effects of these stressors on the bacterial community composition and estimated function.

Download Full-text

Variation of bacterial communities in Muztagh ice core from 1869 to 2000

10.5194/egusphere-egu2020-6328 ◽

2020 ◽

Author(s):

Yongqin Liu ◽

Tandong Yao ◽

Baiqing Xu

Keyword(s):

Bacterial Community ◽

Temporal Variation ◽

Community Composition ◽

Bacterial Communities ◽

Environmental Variables ◽

Climate Changes ◽

Bacterial Community Composition ◽

Ice Core ◽

The Past ◽

Stage 1

Many studies focusing on the physical and chemical indicators of the ice core reflected the climate changes. However, only few biological indicators indicated the past climate changes which are mainly focused in biomass rather than diversity. How the biodiversity response to the climate change during the past hundred years is still unknow. Glaciers in Mt. Muztagh Ata region are in&#64258;uenced by the year-round westerly circulation. We firstly disclosed annual variations of bacterial community compositions in ice core over the past 130 years from Muztagh Glacier, the western Tibetan Plateau. Temporal variation in bacterial abundance was strongly controlled by DOC, TN, &#948;18O, Ca2+, SO42&#8722;, NH4+ and NO3&#8722;. Proteobacteria, Actinobacteria and Firmicutes were the three most abundant bacterial phyla, accounting for 49.3%, 21.3% and 11.0% of the total community, respectively. The abundances of Firmicutes and Bacteroidetes pronouncedly increased over time throughout the entire ice core. UPGMA cluster analysis of the bacterial community composition separated the all ice core samples into two main clusters along the temporal variation. The first cluster consisted of samples from 1951 to 2000 and the second cluster contained main samples during the period of 1869-1950. The stage 1 and stage 2 bacterial community dissimilarities increased linearly with time on the basis of the Bray-Curtis distance, indicating a similar temporal&#8211;decay relationship between the stage 1 and stage 2 bacterial communities. Of all the environmental variables examined, only DOC and NH4+ exhibited very strong negative correlations with bacterial Chao1-richness. 18O was another important variable in shaping the ice core bacterial community composition and contributed 1.6% of the total variation. Moreover, DistLM analysis indicated that the environmental variables explained more variation in the stage 1 community (20.1%) than that of the stage 2 community (19.9%).

Download Full-text