Evaluation of Human Milk Microbiota by 16S rRNA Gene Next-Generation Sequencing (NGS) and Cultivation/MALDI-TOF Mass Spectrometry Identification

ABSTRACTBackgroundThe endometrial cavity is an upper genital tract site largely heralded as sterile, however, advances in culture-independent, next generation sequencing technology have revealed that this site harbours a rich microbial community which includes multiple Lactobacillus species. These bacteria are considered to be the most common non-pathogenic genital tract commensals. Next-generation sequencing of the female lower genital tract has revealed significant variation amongst microbial community composition with respect to Lactobacillus sp. in samples collected from healthy and diseased women. The aim of this study was to evaluate the ability of the 16S rRNA gene to characterize genital tract lactobacilli to species-level taxonomy.MethodsSamples were interrogated for the presence of microbial DNA using two-step next generation sequencing technology to exploit the V5–V8 regions of the 16S rRNA gene and compared to standard speciation using qPCR.ResultsThe V5-V8 region of the 16S rRNA gene has sufficient sequence variation within frequently encountered genital tract lactobacilli to allow accurate determination of relative abundance within the community, and speciation for several key community members without completing additional experimentation.ConclusionsNext-generation sequencing of clinical genital tract isolates is an effective method for high throughput identification to species-level of key Lactobacillus sp.IMPORTANCEHuman microbiome experiments, including the low biomass organs such as the upper genital tract, require the development of consensus protocols to ensure accurate comparison between such studies and our data forms an important foundation for future protocols.This paper provides evidence to support the selection of the V5-V8 regions of the 16S rRNA gene improved Lactobacillus speciation using next generation sequencing technology. The choice of variable region for broad-range amplification in microbiome studies is important due to preferential primer binding associated with some genera based on nucleotide sequence patterns. By utilising the V5-V8 region, multiple species of Lactobacillus can be characterised with relative confidence.

Download Full-text

What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?

Pathogens ◽

10.3390/pathogens11010034 ◽

2021 ◽

Vol 11 (1) ◽

pp. 34

Author(s):

Paula Santibáñez ◽

Concepción García-García ◽

Aránzazu Portillo ◽

Sonia Santibáñez ◽

Lara García-Álvarez ◽

...

Keyword(s):

Next Generation Sequencing ◽

Infective Endocarditis ◽

16S Rrna ◽

16S Rrna Gene ◽

Heart Valve ◽

Rrna Gene ◽

Microbiological Diagnosis ◽

Next Generation ◽

Targeted Ngs ◽

Generation Sequencing

Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. Here, next-generation sequencing (NGS) of the V3–V4 region of the 16S rRNA gene was performed in 27 heart valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) from 27 patients diagnosed with IE (4 of them with negative blood cultures). Metataxonomics matched with conventional diagnostic techniques in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases; the same genus, to 23 cases; and the same species, to 13 cases. In 22 of them, the etiological agent was represented by percentages > 99% of the reads and in two cases, by ~70%. Staphylococcus aureus was detected in a previously microbiological undiagnosed patient. Thus, microbiological diagnosis with 16S rRNA gene targeted-NGS was possible in one more sample than using traditional techniques. The remaining two patients showed no coincidence between traditional and 16S rRNA gene-targeted NGS microbiological diagnoses. In addition, 16S rRNA gene-targeted NGS allowed us to suggest coinfections that were supported by clinical data in one patient, and minority records also verified mixed infections in three cases. In our series, metataxonomics was valid for the identification of the causative agents, although more studies are needed before implementation of 16S rRNA gene-targeted NGS for the diagnosis of IE.

Download Full-text

Quantitative PCR provides a simple and accessible method for quantitative microbiome profiling

10.1101/478685 ◽

2018 ◽

Cited By ~ 11

Author(s):

Ching Jian ◽

Panu Luukkonen ◽

Hannele Yki-Järvinen ◽

Anne Salonen ◽

Katri Korpela

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Quantitative Pcr ◽

Rrna Gene ◽

Microbial Community Structures ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Bacterial Groups ◽

Microbiome Profiling ◽

Generation Sequencing

AbstractThe use of relative next generation sequencing (NGS) abundance data can lead to misinterpretations of microbial community structures as the increase of one taxon leads to concurrent decrease of the other(s). To overcome compositionality, we provide a quantitative NGS solution, which is achieved by adjusting the relative 16S rRNA gene amplicon NGS data with quantitative PCR (qPCR-based) total bacterial counts. By comparing the enumeration of dominant bacterial groups on different taxonomic levels in human fecal samples using taxon-specific 16S rRNA gene-targeted qPCR we show that quantitative NGS is able to estimate absolute bacterial abundances accurately. We also observed a higher degree of correspondence in the estimated microbe-metabolite relationship when quantitative NGS was applied. Being conceptually and methodologically analogous to amplicon-based NGS, our qPCR-based method can be readily incorporated into the standard, high-throughput NGS sample processing pipeline for more accurate description of interactions within and between the microbes and host.

Download Full-text

Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions

Nucleic Acids Research ◽

10.1093/nar/gkq873 ◽

2010 ◽

Vol 38 (22) ◽

pp. e200-e200 ◽

Cited By ~ 518

Author(s):

Marcus J. Claesson ◽

Qiong Wang ◽

Orla O'Sullivan ◽

Rachel Greene-Diniz ◽

James R. Cole ◽

...

Keyword(s):

Next Generation Sequencing ◽

16S Rrna ◽

16S Rrna Gene ◽

Rrna Gene ◽

Microbiota Composition ◽

Next Generation ◽

Sequencing Technologies ◽

Generation Sequencing

Download Full-text

Unraveling the bacterial diversity of Cangar Hot Spring, Indonesia by Next Generation Sequencing of 16S rRNA gene

Biodiversitas Journal of Biological Diversity ◽

10.13057/biodiv/d220955 ◽

2021 ◽

Vol 22 (9) ◽

Author(s):

Almando Geraldi ◽

Chia Chay Tay ◽

Ni’matuzahroh Ni’matuzahroh ◽

Fatimah FATIMAH ◽

Wan Nurhayati Wan Hanafi

Keyword(s):

Next Generation Sequencing ◽

16S Rrna ◽

16S Rrna Gene ◽

Bacterial Diversity ◽

Hot Springs ◽

Hot Spring ◽

Rrna Gene ◽

Next Generation ◽

Acinetobacter Junii ◽

Generation Sequencing

Abstract. Geraldi A, Tay CC, Ni’matuzahroh, Fatimah, Hanafi WNW. 2021. Unraveling the bacterial diversity of Cangar Hot Spring, Indonesia by Next Generation Sequencing of 16S rRNA gene. Biodiversitas 22: 4060-4066. This study is the first attempt at using the Next Generation Sequencing (NGS) method with 16S rRNA to understand the bacterial community structure in an Indonesian hot spring. This study aims to unravel the bacterial diversity of the Cangar Hot Spring as one of the most explored natural hot springs in East Java, Indonesia. We found Proteobacteria and Bacteroidetes as the two most abundant phyla. We discovered the first occurrence of genera Cloacibacterium and Methylobacillus in the hot spring ecosystem, which was the most dominant genera at Cangar Hot Spring. We also found several potential bacteria for bioindustry and bioremediation, such as Acinetobacter junii and Pseudomonas alcaligenes. Besides that, we also observed opportunistic pathogens from genera Comamonas and Vogesella. This study result will provide valuable information for further bioprospecting of bacteria with commercial potential and the development of health and safety measures in the Cangar Hot Spring, among others. Hopefully, this report would encourage the use of NGS technology for studying other hot springs in Indonesia.

Download Full-text

A New Type of Chronic Wound Infection after Wisdom Tooth Extraction: A Diagnostic Approach with 16S-rRNA Gene Analysis, Next-Generation Sequencing, and Bioinformatics

Pathogens ◽

10.3390/pathogens9100798 ◽

2020 ◽

Vol 9 (10) ◽

pp. 798

Author(s):

Sebastian Böttger ◽

Silke Zechel-Gran ◽

Philipp Streckbein ◽

Michael Knitschke ◽

Torsten Hain ◽

...

Keyword(s):

Next Generation Sequencing ◽

16S Rrna ◽

16S Rrna Gene ◽

Third Molar ◽

Gene Analysis ◽

Rrna Gene ◽

Next Generation ◽

16S Rrna Gene Analysis ◽

New Type ◽

Generation Sequencing

Delayed-onset infections are rare postoperative complications of lower third molar extractions. This article presents a case of a chronic combined hard and soft tissue infection after the extraction of a third molar, where the causative organisms could only be elucidated by molecular methods. Experimental 16S-rRNA gene analysis with next-generation sequencing and bioinformatics was used to identify the bacterial spectrum of the infection. 16S-rRNA gene analysis delivered the microbiome of the abscessing inflammation while standard culture and laboratory examinations were all sterile. The microbiome showed a mixed bacterial infection with a dominance of Delftia and Alcanivorax (spp.) besides other bacteria of the normal oral flora. Using 16S-rRNA-gene analysis, next-generation sequencing, and bioinformatics, a new type of chronic wound infection after wisdom tooth extraction was found. The property of Delftia and Alcanivorax (spp.) as water-affine environmental bacteria raises suspicion of infection from contaminated water from a dental unit. Thus, osteotomies of teeth should only be done with sterile cooling water. The 16S-rRNA gene analysis should become a part of the routine diagnostics in medical microbiology.

Download Full-text