scholarly journals Toolbox Approaches Using Molecular Markers and 16S rRNA Gene Amplicon Data Sets for Identification of Fecal Pollution in Surface Water

2015 ◽  
Vol 81 (20) ◽  
pp. 7067-7077 ◽  
Author(s):  
W. Ahmed ◽  
C. Staley ◽  
M. J. Sadowsky ◽  
P. Gyawali ◽  
J. P. S. Sidhu ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
High Throughput Sequencing ◽  
Community Analysis ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Potential Sources

ABSTRACTIn this study, host-associated molecular markers and bacterial 16S rRNA gene community analysis using high-throughput sequencing were used to identify the sources of fecal pollution in environmental waters in Brisbane, Australia. A total of 92 fecal and composite wastewater samples were collected from different host groups (cat, cattle, dog, horse, human, and kangaroo), and 18 water samples were collected from six sites (BR1 to BR6) along the Brisbane River in Queensland, Australia. Bacterial communities in the fecal, wastewater, and river water samples were sequenced. Water samples were also tested for the presence of bird-associated (GFD), cattle-associated (CowM3), horse-associated, and human-associated (HF183) molecular markers, to provide multiple lines of evidence regarding the possible presence of fecal pollution associated with specific hosts. Among the 18 water samples tested, 83%, 33%, 17%, and 17% were real-time PCR positive for the GFD, HF183, CowM3, and horse markers, respectively. Among the potential sources of fecal pollution in water samples from the river, DNA sequencing tended to show relatively small contributions from wastewater treatment plants (up to 13% of sequence reads). Contributions from other animal sources were rarely detected and were very small (<3% of sequence reads). Source contributions determined via sequence analysis versus detection of molecular markers showed variable agreement. A lack of relationships among fecal indicator bacteria, host-associated molecular markers, and 16S rRNA gene community analysis data was also observed. Nonetheless, we show that bacterial community and host-associated molecular marker analyses can be combined to identify potential sources of fecal pollution in an urban river. This study is a proof of concept, and based on the results, we recommend using bacterial community analysis (where possible) along with PCR detection or quantification of host-associated molecular markers to provide information on the sources of fecal pollution in waterways.

scholarly journals Restructuring of the Aquatic Bacterial Community by Hydric Dynamics Associated with Superstorm Sandy

2016 ◽  
Vol 82 (12) ◽  
pp. 3525-3536 ◽  
Author(s):  
Nikea Ulrich ◽  
Abigail Rosenberger ◽  
Colin Brislawn ◽  
Justin Wright ◽  
Collin Kessler ◽  
...  
Keyword(s):  
Community Structure ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Bacterial Community Structure ◽  
High Throughput Sequencing ◽  
Storm Event ◽  
Rrna Gene ◽  
Content Type

ABSTRACTBacterial community composition and longitudinal fluctuations were monitored in a riverine system during and after Superstorm Sandy to better characterize inter- and intracommunity responses associated with the disturbance associated with a 100-year storm event. High-throughput sequencing of the 16S rRNA gene was used to assess microbial community structure within water samples from Muddy Creek Run, a second-order stream in Huntingdon, PA, at 12 different time points during the storm event (29 October to 3 November 2012) and under seasonally matched baseline conditions. High-throughput sequencing of the 16S rRNA gene was used to track changes in bacterial community structure and divergence during and after Superstorm Sandy. Bacterial community dynamics were correlated to measured physicochemical parameters and fecal indicator bacteria (FIB) concentrations. Bioinformatics analyses of 2.1 million 16S rRNA gene sequences revealed a significant increase in bacterial diversity in samples taken during peak discharge of the storm. Beta-diversity analyses revealed longitudinal shifts in the bacterial community structure. Successional changes were observed, in whichBetaproteobacteriaandGammaproteobacteriadecreased in 16S rRNA gene relative abundance, while the relative abundance of members of theFirmicutesincreased. Furthermore, 16S rRNA gene sequences matching pathogenic bacteria, including strains ofLegionella,Campylobacter,Arcobacter, andHelicobacter, as well as bacteria of fecal origin (e.g.,Bacteroides), exhibited an increase in abundance after peak discharge of the storm. This study revealed a significant restructuring of in-stream bacterial community structure associated with hydric dynamics of a storm event.IMPORTANCEIn order to better understand the microbial risks associated with freshwater environments during a storm event, a more comprehensive understanding of the variations in aquatic bacterial diversity is warranted. This study investigated the bacterial communities during and after Superstorm Sandy to provide fine time point resolution of dynamic changes in bacterial composition. This study adds to the current literature by revealing the variation in bacterial community structure during the course of a storm. This study employed high-throughput DNA sequencing, which generated a deep analysis of inter- and intracommunity responses during a significant storm event. This study has highlighted the utility of applying high-throughput sequencing for water quality monitoring purposes, as this approach enabled a more comprehensive investigation of the bacterial community structure. Altogether, these data suggest a drastic restructuring of the stream bacterial community during a storm event and highlight the potential of high-throughput sequencing approaches for assessing the microbiological quality of our environment.

scholarly journals Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

2007 ◽  
Vol 60 (2) ◽  
pp. 341-350 ◽  
Author(s):  
Rita Sipos ◽  
Anna J. Székely ◽  
Márton Palatinszky ◽  
Sára Révész ◽  
Károly Márialigeti ◽  
...  
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Annealing Temperature ◽  
Community Analysis ◽  
Rrna Gene ◽  
Cycle Number ◽  
Primer Mismatch

scholarly journals Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing

Microbiome ◽  
2020 ◽  
Vol 8 (1) ◽  
Author(s):  
Luyang Song ◽  
Kabin Xie
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput Sequencing ◽  
Bacterial Species ◽  
Amplicon Sequencing ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Plant Microbiota

Abstract Background High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing. Results We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling. Conclusion Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics.

scholarly journals Temporal Assessment of the Impact of Exposure to Cow Feces in Two Watersheds by Multiple Host-Specific PCR Assays

2008 ◽  
Vol 74 (22) ◽  
pp. 6839-6847 ◽  
Author(s):  
Yong-Jin Lee ◽  
Marirosa Molina ◽  
Jorge W. Santo Domingo ◽  
Jonathan D. Willis ◽  
Michael Cyterski ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Water Samples ◽  
Fecal Pollution ◽  
Source Tracking ◽  
Rrna Gene ◽  
Gene Markers ◽  
Specific Pcr ◽  
Pcr Assays ◽  
The Impact

ABSTRACT Exposure to feces in two watersheds with different management histories was assessed by tracking cattle feces bacterial populations using multiple host-specific PCR assays. In addition, environmental factors affecting the occurrence of these markers were identified. Each assay was performed using DNA extracts from water and sediment samples collected from a watershed directly impacted by cattle fecal pollution (WS1) and from a watershed impacted only through runoff (WS2). In WS1, the ruminant-specific Bacteroidales 16S rRNA gene marker CF128F was detected in 65% of the water samples, while the non-16S rRNA gene markers Bac1, Bac2, and Bac5 were found in 32 to 37% of the water samples. In contrast, all source-specific markers were detected in less than 6% of the water samples from WS2. Binary logistic regressions (BLRs) revealed that the occurrence of Bac32F and CF128F was significantly correlated with season as a temporal factor and watershed as a site factor. BLRs also indicated that the dynamics of fecal-source-tracking markers correlated with the density of a traditional fecal indicator (P < 0.001). Overall, our results suggest that a combination of 16S rRNA gene and non-16S rRNA gene markers provides a higher level of confidence for tracking unknown sources of fecal pollution in environmental samples. This study also provided practical insights for implementation of microbial source-tracking practices to determine sources of fecal pollution and the influence of environmental variables on the occurrence of source-specific markers.

scholarly journals The porcine corneal surface bacterial microbiome: A distinctive niche within the ocular surface

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247392
Author(s):  
Marina L. Leis ◽  
Gabriela M. Madruga ◽  
Matheus O. Costa
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Ocular Surface ◽  
High Throughput Sequencing ◽  
Rrna Gene ◽  
Environmental Niche ◽  
Corneal Surface ◽  
Schirmer Tear Test

Purpose The ocular surface microbiome has been described as paucibacterial. Until now, studies investigating the bacterial community associated with the ocular surface through high-throughput sequencing have focused on the conjunctiva. Conjunctival samples are thought to reflect and be representative of the microbiome residing on the ocular surface, including the cornea. Here, we hypothesized that the bacterial community associated with the corneal surface was different from those of the inferonasal and superotemporal conjunctival fornices, and from the tear film. Methods Both eyes from 15 healthy piglets were sampled using swabs (inferonasal fornix, superotemporal fornix, and corneal surface, n = 30 each) and Schirmer tear test strips (STT, n = 30). Negative sampling controls (swabs and STT, n = 2 each) and extraction controls (n = 4) were included. Total DNA was extracted and high-throughput sequencing targeting the 16S rRNA gene was performed. Bioinformatic analyses included multiple contamination-controlling steps. Results Corneal surface samples had a significantly lower number of taxa detected (P<0.01) and were compositionally different from all other sample types (Bray-Curtis dissimilarity, P<0.04). It also harbored higher levels of Proteobacteria (P<0.05), specifically Brevundimonas spp. (4.1-fold) and Paracoccus spp. (3.4-fold) than other sample types. Negative control STT strip samples yielded the highest amount of 16S rRNA gene copies across all sample types (P<0.05). Conclusions Our data suggests that the corneal surface provides a distinct environmental niche within the ocular surface, leading to a bacterial community compositionally different from all other sample types.

scholarly journals Diversity and Structure of the Endophytic Bacterial Communities Associated With Three Terrestrial Orchid Species as Revealed by 16S rRNA Gene Metabarcoding

2020 ◽  
Vol 11 ◽  
Author(s):  
Pasquale Alibrandi ◽  
Sylvia Schnell ◽  
Silvia Perotto ◽  
Massimiliano Cardinale
Keyword(s):  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Host Plant ◽  
Bacterial Communities ◽  
Reproductive Organs ◽  
Distribution Model ◽  
Rrna Gene ◽  
Orchid Species ◽  
Core Microbiota

The endophytic microbiota can establish mutualistic or commensalistic interactions within the host plant tissues. We investigated the bacterial endophytic microbiota in three species of Mediterranean orchids (Neottia ovata, Serapias vomeracea, and Spiranthes spiralis) by metabarcoding of the 16S rRNA gene. We examined whether the different orchid species and organs, both underground and aboveground, influenced the endophytic bacterial communities. A total of 1,930 operational taxonomic units (OTUs) were obtained, mainly Proteobacteria and Actinobacteria, whose distribution model indicated that the plant organ was the main determinant of the bacterial community structure. The co-occurrence network was not modular, suggesting a relative homogeneity of the microbiota between both plant species and organs. Moreover, the decrease in species richness and diversity in the aerial vegetative organs may indicate a filtering effect by the host plant. We identified four hub OTUs, three of them already reported as plant-associated taxa (Pseudoxanthomonas, Rhizobium, and Mitsuaria), whereas Thermus was an unusual member of the plant microbiota. Core microbiota analysis revealed a selective and systemic ascent of bacterial communities from the vegetative to the reproductive organs. The core microbiota was also maintained in the S. spiralis seeds, suggesting a potential vertical transfer of the microbiota. Surprisingly, some S. spiralis seed samples displayed a very rich endophytic microbiota, with a large number of OTUs shared with the roots, a situation that may lead to a putative restoring process of the root-associated microbiota in the progeny. Our results indicate that the bacterial community has adapted to colonize the orchid organs selectively and systemically, suggesting an active involvement in the orchid holobiont.

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