High-Throughput Sequencing for Examining Salmonella Prevalence and Pathogen—Microbiota Relationships in Barn Swallows

Studies in both humans and model organisms suggest that the microbiome may play a significant role in host health, including digestion and immune function. Microbiota can offer protection from exogenous pathogens through colonization resistance, but microbial dysbiosis in the gastrointestinal tract can decrease resistance and is associated with pathogenesis. Little is known about the effects of potential pathogens, such as Salmonella, on the microbiome in wildlife, which are known to play an important role in disease transmission to humans. Culturing techniques have traditionally been used to detect pathogens, but recent studies have utilized high throughput sequencing of the 16S rRNA gene to characterize host-associated microbial communities (i.e., the microbiome) and to detect specific bacteria. Building upon this work, we evaluated the utility of high throughput 16S rRNA gene sequencing for potential bacterial pathogen detection in barn swallows (Hirundo rustica) and used these data to explore relationships between potential pathogens and microbiota. To accomplish this, we first compared the detection of Salmonella spp. in swallows using 16S rRNA data with standard culture techniques. Second, we examined the prevalence of Salmonella using 16S rRNA data and examined the relationship between Salmonella-presence or -absence and individual host factors. Lastly, we evaluated host-associated bacterial diversity and community composition in Salmonella-present vs. -absent birds. Out of 108 samples, we detected Salmonella in six (5.6%) samples based on culture, 25 (23.1%) samples with unrarefied 16S rRNA gene sequencing data, and three (2.8%) samples with both techniques. We found that sex, migratory status, and weight were correlated with Salmonella presence in swallows. In addition, bacterial community composition and diversity differed between birds based on Salmonella status. This study highlights the value of 16S rRNA gene sequencing data for monitoring pathogens in wild birds and investigating the ecology of host microbe-pathogen relationships, data which are important for prediction and mitigation of disease spillover into domestic animals and humans.

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A 16S rRNA gene sequencing and analysis protocol for the Illumina MiniSeq platform

10.1101/227108 ◽

2017 ◽

Author(s):

Monica Pichler ◽

Ömer K. Coskun ◽

Ana Sofia Ortega ◽

Nicola Conci ◽

Gert Wörheide ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Environmental Samples ◽

High Throughput Sequencing ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Mock Community ◽

Rrna Gene Sequencing

ABSTRACTHigh-throughput sequencing of the 16S rRNA gene is widely used in microbial ecology, with Illumina platforms being widely used in recent studies. The MiniSeq, Illumina’s latest benchtop sequencer, enables more cost-efficient DNA sequencing relative to larger sequencing platforms (e.g. MiSeq). Here we used a modified custom primer sequencing approach to test the fidelity of the MiniSeq for high-throughput sequencing of the V4 hypervariable region of 16S rRNA genes from complex communities in environmental samples. To this end, we designed an additional sequencing primer that enabled application of a dual-index barcoding method on the MiniSeq. A mock community was sequenced alongside the environmental samples as a quality control benchmark. After careful filtering procedures, we were able to recapture a realistic richness of the mock community, and identify meaningful differences in alpha and beta diversity in the environmental samples. These results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq, yet is a cost-effective option for any laboratory interested in performing high-throughput 16S rRNA gene sequencing.IMPORTANCEWe modified a custom sequencing approach and used a mock community to test the fidelity of high-throughput sequencing on the Illumina MiniSeq platform. Our results show that the MiniSeq can produce similar quantities of high quality V4 reads compared to the MiSeq. In addition, our protocol increases feasibility for small laboratories to perform their own high-throughput sequencing of the 16S rRNA marker gene.

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Dynamics of Soil Microbial Communities During Diazepam and Oxazepam Biodegradation in Soil Flooded by Water From a WWTP

Frontiers in Microbiology ◽

10.3389/fmicb.2021.742000 ◽

2021 ◽

Vol 12 ◽

Author(s):

Marc Crampon ◽

Coralie Soulier ◽

Pauline Sidoli ◽

Jennifer Hellal ◽

Catherine Joulian ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Soil Microbial Communities ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Treated Wastewater ◽

Rrna Gene ◽

Sequencing Data ◽

Rrna Gene Sequencing

The demand for energy and chemicals is constantly growing, leading to an increase of the amounts of contaminants discharged to the environment. Among these, pharmaceutical molecules are frequently found in treated wastewater that is discharged into superficial waters. Indeed, wastewater treatment plants (WWTPs) are designed to remove organic pollution from urban effluents but are not specific, especially toward contaminants of emerging concern (CECs), which finally reach the natural environment. In this context, it is important to study the fate of micropollutants, especially in a soil aquifer treatment (SAT) context for water from WWTPs, and for the most persistent molecules such as benzodiazepines. In the present study, soils sampled in a reed bed frequently flooded by water from a WWTP were spiked with diazepam and oxazepam in microcosms, and their concentrations were monitored for 97 days. It appeared that the two molecules were completely degraded after 15 days of incubation. Samples were collected during the experiment in order to follow the dynamics of the microbial communities, based on 16S rRNA gene sequencing for Archaea and Bacteria, and ITS2 gene for Fungi. The evolution of diversity and of specific operating taxonomic units (OTUs) highlighted an impact of the addition of benzodiazepines, a rapid resilience of the fungal community and an evolution of the bacterial community. It appeared that OTUs from the Brevibacillus genus were more abundant at the beginning of the biodegradation process, for diazepam and oxazepam conditions. Additionally, Tax4Fun tool was applied to 16S rRNA gene sequencing data to infer on the evolution of specific metabolic functions during biodegradation. It finally appeared that the microbial community in soils frequently exposed to water from WWTP, potentially containing CECs such as diazepam and oxazepam, may be adapted to the degradation of persistent contaminants.

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Discriminating activated sludge flocs from biofilm microbial communities in a novel pilot-scale reciprocation MBR using high-throughput 16S rRNA gene sequencing

Journal of Environmental Management ◽

10.1016/j.jenvman.2018.03.081 ◽

2018 ◽

Vol 217 ◽

pp. 268-277 ◽

Cited By ~ 8

Author(s):

Ryan De Sotto ◽

Jaeho Ho ◽

Woonyoung Lee ◽

Sungwoo Bae

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

High Throughput ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Pilot Scale ◽

Rrna Gene ◽

Sludge Flocs ◽

Rrna Gene Sequencing

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High-throughput 16S rRNA gene sequencing reveals alterations of mouse intestinal microbiota after radiotherapy

Anaerobe ◽

10.1016/j.anaerobe.2015.01.004 ◽

2015 ◽

Vol 33 ◽

pp. 1-7 ◽

Cited By ~ 40

Author(s):

Young Suk Kim ◽

Jinu Kim ◽

Soo-Je Park

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Intestinal Microbiota ◽

High Throughput ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Rrna Gene Sequencing

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Stratified Bacterial and Archaeal Community in Mangrove and Intertidal Wetland Mudflats Revealed by High Throughput 16S rRNA Gene Sequencing

Frontiers in Microbiology ◽

10.3389/fmicb.2017.02148 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 29

Author(s):

Zhichao Zhou ◽

Han Meng ◽

Yang Liu ◽

Ji-Dong Gu ◽

Meng Li

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

High Throughput ◽

Gene Sequencing ◽

Archaeal Community ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Intertidal Wetland ◽

Rrna Gene Sequencing

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16S rRNA gene sequencing data of the human skin microbiome before and after swimming in the ocean

Data in Brief ◽

10.1016/j.dib.2021.107207 ◽

2021 ◽

pp. 107207

Author(s):

Marisa C. Nielsen ◽

Sunny C. Jiang

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Human Skin ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Skin Microbiome ◽

Sequencing Data ◽

Before And After ◽

Rrna Gene Sequencing

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Deciphering the low abundance microbiota of presumed aseptic hip and knee implants

PLoS ONE ◽

10.1371/journal.pone.0257471 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0257471

Author(s):

Charles Carr ◽

Hannah Wilcox ◽

Jeremy P. Burton ◽

Sharanya Menon ◽

Kait F. Al ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Sequencing Data ◽

Computational Tools ◽

Cutibacterium Acnes ◽

Joint Implants ◽

Rrna Gene Sequencing

16S rRNA gene sequencing of DNA extracted from clinically uninfected hip and knee implant samples has revealed polymicrobial populations. However, previous studies assessed 16S rRNA gene sequencing as a technique for the diagnosis of periprosthetic joint infections, leaving the microbiota of presumed aseptic hip and knee implants largely unstudied. These communities of microorganisms might play important roles in aspects of host health, such as aseptic loosening. Therefore, this study sought to characterize the bacterial composition of presumed aseptic joint implant microbiota using next generation 16S rRNA gene sequencing, and it evaluated this method for future investigations. 248 samples were collected from implants of 41 patients undergoing total hip or knee arthroplasty revision for presumed aseptic failure. DNA was extracted using two methodologies—one optimized for high throughput and the other for human samples—and amplicons of the V4 region of the 16S rRNA gene were sequenced. Sequencing data were analyzed and compared with ancillary specific PCR and microbiological culture. Computational tools (SourceTracker and decontam) were used to detect and compensate for environmental and processing contaminants. Microbial diversity of patient samples was higher than that of open-air controls and differentially abundant taxa were detected between these conditions, possibly reflecting a true microbiota that is present in clinically uninfected joint implants. However, positive control-associated artifacts and DNA extraction methodology significantly affected sequencing results. As well, sequencing failed to identify Cutibacterium acnes in most culture- and PCR-positive samples. These challenges limited characterization of bacteria in presumed aseptic implants, but genera were identified for further investigation. In all, we provide further support for the hypothesis that there is likely a microbiota present in clinically uninfected joint implants, and we show that methods other than 16S rRNA gene sequencing may be ideal for its characterization. This work has illuminated the importance of further study of microbiota of clinically uninfected joint implants with novel molecular and computational tools to further eliminate contaminants and artifacts that arise in low bacterial abundance samples.

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