Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis

AbstractOne of the major methods to identify microbial community composition, to unravel microbial population dynamics, and to explore microbial diversity in environmental samples is DNA- or RNA-based 16S rRNA (gene) amplicon sequencing. Subsequent bioinformatics analyses are required to extract valuable information from the high-throughput sequencing approach. However, manifold bioinformatics tools complicate their choice and might cause differences in data interpretation, making the selection of the pipeline a crucial step.Here, we compared the performance of most widely used 16S rRNA gene amplicon sequencing analysis tools (i.e. Mothur, QIIME1, QIIME2, and MEGAN) using mock datasets and environmental samples from contrasting terrestrial and freshwater sites. Our results showed that QIIME2 outcompeted all other investigated tools in sequence recovery (>10 times less false positives), taxonomic assignments (>22% better F-score) and diversity estimates (>5% better assessment), while there was still room for improvement e.g. imperfect sequence recovery (recall up to 87%) or detection of additional false sequences (precision up to 72%). Furthermore, we found that microbial diversity estimates and highest abundant taxa varied among analysis pipelines (i.e. only one in five genera was shared among all analysis tools) when analyzing environmental samples, which might skew biological conclusions.Our findings were subsequently implemented in a high-performance computing conformant workflow following the FAIR (Findable, Accessible, Interoperable, and Re-usable) principle, allowing reproducible 16S rRNA gene amplicon sequence analysis starting from raw sequence files. Our presented workflow can be utilized for future studies, thereby facilitating the analysis of high-throughput DNA- or RNA-based 16S rRNA (gene) sequencing data substantially.ImportanceMicroorganisms play an essential role in biogeochemical cycling events across the globe. Phylogenetic marker gene analysis is a widely used method to explore microbial community dynamics in space and time, to predict the ecological relevance of microbial populations, or to identify microbial key players in biogeochemical cycles. Several computational analysis methods were developed to aid 16S rRNA gene analysis but choosing the best method is not trivial. In this study, we compared popular analysis methods (i.e. Mothur, QIIME1 and 2, and MEGAN) using samples with known microbial composition (i.e. mock community samples) and environmental samples from contrasting habitats (i.e. groundwater, soil, sediment, and river water). Our findings provide guidance for choosing the currently optimal 16S rRNA gene sequencing analysis method and we implemented our recommended pipeline into a reproducible workflow, which follows highest bioinformatics standards and is open source and free to use.

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Much ado about nothing? Off-target amplification can lead to false-positive bacterial brain microbiome detection in healthy and Parkinson’s disease individuals

Microbiome ◽

10.1186/s40168-021-01012-1 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Janis R. Bedarf ◽

Naiara Beraza ◽

Hassan Khazneh ◽

Ezgi Özkurt ◽

David Baker ◽

...

Keyword(s):

Parkinson’S Disease ◽

16S Rrna ◽

16S Rrna Gene ◽

False Positive ◽

Gene Sequencing ◽

Bacterial Biomass ◽

16S Rrna Gene Sequencing ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Rrna Gene Sequencing

Abstract Background Recent studies suggested the existence of (poly-)microbial infections in human brains. These have been described either as putative pathogens linked to the neuro-inflammatory changes seen in Parkinson’s disease (PD) and Alzheimer’s disease (AD) or as a “brain microbiome” in the context of healthy patients’ brain samples. Methods Using 16S rRNA gene sequencing, we tested the hypothesis that there is a bacterial brain microbiome. We evaluated brain samples from healthy human subjects and individuals suffering from PD (olfactory bulb and pre-frontal cortex), as well as murine brains. In line with state-of-the-art recommendations, we included several negative and positive controls in our analysis and estimated total bacterial biomass by 16S rRNA gene qPCR. Results Amplicon sequencing did detect bacterial signals in both human and murine samples, but estimated bacterial biomass was extremely low in all samples. Stringent reanalyses implied bacterial signals being explained by a combination of exogenous DNA contamination (54.8%) and false positive amplification of host DNA (34.2%, off-target amplicons). Several seemingly brain-enriched microbes in our dataset turned out to be false-positive signals upon closer examination. We identified off-target amplification as a major confounding factor in low-bacterial/high-host-DNA scenarios. These amplified human or mouse DNA sequences were clustered and falsely assigned to bacterial taxa in the majority of tested amplicon sequencing pipelines. Off-target amplicons seemed to be related to the tissue’s sterility and could also be found in independent brain 16S rRNA gene sequences. Conclusions Taxonomic signals obtained from (extremely) low biomass samples by 16S rRNA gene sequencing must be scrutinized closely to exclude the possibility of off-target amplifications, amplicons that can only appear enriched in biological samples, but are sometimes assigned to bacterial taxa. Sequences must be explicitly matched against any possible background genomes present in large quantities (i.e., the host genome). Using close scrutiny in our approach, we find no evidence supporting the hypothetical presence of either a brain microbiome or a bacterial infection in PD brains.

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Microbiome of Odontogenic Abscesses

Microorganisms ◽

10.3390/microorganisms9061307 ◽

2021 ◽

Vol 9 (6) ◽

pp. 1307

Author(s):

Sebastian Böttger ◽

Silke Zechel-Gran ◽

Daniel Schmermund ◽

Philipp Streckbein ◽

Jan-Falco Wilbrand ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Oral Microbiome ◽

Minor Role ◽

Rrna Gene ◽

Sequencing Analysis ◽

Bacterial Strains ◽

16S Rrna Gene Analysis ◽

Odontogenic Infections ◽

Odontogenic Abscess

Severe odontogenic abscesses are regularly caused by bacteria of the physiological oral microbiome. However, the culture of these bacteria is often prone to errors and sometimes does not result in any bacterial growth. Furthermore, various authors found completely different bacterial spectra in odontogenic abscesses. Experimental 16S rRNA gene next-generation sequencing analysis was used to identify the microbiome of the saliva and the pus in patients with a severe odontogenic infection. The microbiome of the saliva and the pus was determined for 50 patients with a severe odontogenic abscess. Perimandibular and submandibular abscesses were the most commonly observed diseases at 15 (30%) patients each. Polymicrobial infections were observed in 48 (96%) cases, while the picture of a mono-infection only occurred twice (4%). On average, 31.44 (±12.09) bacterial genera were detected in the pus and 41.32 (±9.00) in the saliva. In most cases, a predominantly anaerobic bacterial spectrum was found in the pus, while saliva showed a similar oral microbiome to healthy individuals. In the majority of cases, odontogenic infections are polymicrobial. Our results indicate that these are mainly caused by anaerobic bacterial strains and that aerobic and facultative anaerobe bacteria seem to play a more minor role than previously described by other authors. The 16S rRNA gene analysis detects significantly more bacteria than conventional methods and molecular methods should therefore become a part of routine diagnostics in medical microbiology.

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Handling of spurious sequences affects the outcome of high-throughput 16S rRNA gene amplicon profiling

ISME Communications ◽

10.1038/s43705-021-00033-z ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Sandra Reitmeier ◽

Thomas C. A. Hitch ◽

Nicole Treichel ◽

Nikolaos Fikas ◽

Bela Hausmann ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Diversity Analysis ◽

Careful Attention ◽

Operational Taxonomic Units ◽

Basic Concepts ◽

Gnotobiotic Mice ◽

Mock Communities

Abstract16S rRNA gene amplicon sequencing is a popular approach for studying microbiomes. However, some basic concepts have still not been investigated comprehensively. We studied the occurrence of spurious sequences using defined microbial communities based on data either from the literature or generated in three sequencing facilities and analyzed via both operational taxonomic units (OTUs) and amplicon sequence variants (ASVs) approaches. OTU clustering and singleton removal, a commonly used approach, delivered approximately 50% (mock communities) to 80% (gnotobiotic mice) spurious taxa. The fraction of spurious taxa was generally lower based on ASV analysis, but varied depending on the gene region targeted and the barcoding system used. A relative abundance of 0.25% was found as an effective threshold below which the analysis of spurious taxa can be prevented to a large extent in both OTU- and ASV-based analysis approaches. Using this cutoff improved the reproducibility of analysis, i.e., variation in richness estimates was reduced by 38% compared with singleton filtering using six human fecal samples across seven sequencing runs. Beta-diversity analysis of human fecal communities was markedly affected by both the filtering strategy and the type of phylogenetic distances used for comparison, highlighting the importance of carefully analyzing data before drawing conclusions on microbiome changes. In summary, handling of artifact sequences during bioinformatic processing of 16S rRNA gene amplicon data requires careful attention to avoid the generation of misleading findings. We propose the concept of effective richness to facilitate the comparison of alpha-diversity across studies.

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Detection of Legionella species, the influence of precipitation on the amount of Legionella DNA, and bacterial microbiome in aerosols from outdoor sites near asphalt roads in Toyama Prefecture, Japan

BMC Microbiology ◽

10.1186/s12866-021-02275-2 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Jun-ichi Kanatani ◽

Masanori Watahiki ◽

Keiko Kimata ◽

Tomoko Kato ◽

Kaoru Uchida ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Indoor Environment ◽

Geometric Mean ◽

Amplicon Sequencing ◽

Positive Sample ◽

Outdoor Environment ◽

Rrna Gene ◽

Total Precipitation ◽

Monthly Total Precipitation

Abstract Background Legionellosis is caused by the inhalation of aerosolized water contaminated with Legionella bacteria. In this study, we investigated the prevalence of Legionella species in aerosols collected from outdoor sites near asphalt roads, bathrooms in public bath facilities, and other indoor sites, such as buildings and private homes, using amoebic co-culture, quantitative PCR, and 16S rRNA gene amplicon sequencing. Results Legionella species were not detected by amoebic co-culture. However, Legionella DNA was detected in 114/151 (75.5%) air samples collected near roads (geometric mean ± standard deviation: 1.80 ± 0.52 log10 copies/m3), which was comparable to the numbers collected from bathrooms [15/21 (71.4%), 1.82 ± 0.50] but higher than those collected from other indoor sites [11/30 (36.7%), 0.88 ± 0.56] (P < 0.05). The amount of Legionella DNA was correlated with the monthly total precipitation (r = 0.56, P < 0.01). It was also directly and inversely correlated with the daily total precipitation for seven days (r = 0.21, P = 0.01) and one day (r = − 0.29, P < 0.01) before the sampling day, respectively. 16S rRNA gene amplicon sequencing revealed that Legionella species were detected in 9/30 samples collected near roads (mean proportion of reads, 0.11%). At the species level, L. pneumophila was detected in 2/30 samples collected near roads (the proportion of reads, 0.09 and 0.11% of the total reads number in each positive sample). The three most abundant bacterial genera in the samples collected near roads were Sphingomonas, Streptococcus, and Methylobacterium (mean proportion of reads; 21.1%, 14.6%, and 1.6%, respectively). In addition, the bacterial diversity in outdoor environment was comparable to that in indoor environment which contains aerosol-generating features and higher than that in indoor environment without the features. Conclusions DNA from Legionella species was widely present in aerosols collected from outdoor sites near asphalt roads, especially during the rainy season. Our findings suggest that there may be a risk of exposure to Legionella species not only in bathrooms but also in the areas surrounding asphalt roads. Therefore, the possibility of contracting legionellosis in daily life should be considered.

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First case of an invasive Bacteroides dorei infection detected in a patient with a mycotic aortic aneurysm—raising a rebellion of major indigenous bacteria in humans: a case report and review

BMC Infectious Diseases ◽

10.1186/s12879-021-06345-8 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Takayuki Matsuoka ◽

Takuya Shimizu ◽

Tadanori Minagawa ◽

Wakiko Hiranuma ◽

Miki Takeda ◽

...

Keyword(s):

Aortic Aneurysm ◽

16S Rrna ◽

16S Rrna Gene ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Resected Specimen ◽

Rrna Gene ◽

Sequencing Analysis ◽

First Case ◽

Rrna Gene Sequencing

Abstract Background Bacteroides dorei is an anaerobic gram-negative bacterium first described in 2006. Because of the high similarity in mass spectra between B. dorei and Bacteroides vulgatus, discriminating between these species is arduous in clinical practice. In recent decades, 16S rRNA gene sequencing has been a complementary method for distinguishing taxonomically close bacteria, including B. dorei and B. vulgatus, at the genus and species levels. Consequently, B. dorei has been shown to contribute to some diseases, including type 1 autoimmune diabetes mellitus and atherosclerotic diseases. However, there are no reports on invasive infectious diseases caused by B. dorei. This report describes the first case of direct invasion and colonisation of human tissue by B. dorei, thus providing a warning regarding the previously proposed application of B. dorei as a live biotherapeutic for atherosclerotic diseases. Case presentation A 78-year-old Japanese man complained of intermittent chest/back pain and was diagnosed with a mycotic thoracic aortic aneurysm by enhanced computed tomography on admission. Despite strict blood pressure control and empirical antibiotic therapy, the patient’s condition worsened. To prevent aneurysmal rupture and eliminate infectious foci, the patient underwent surgical treatment. The resected specimen was subjected to tissue culture and 16S rRNA gene sequencing analysis to identify pathogenic bacteria. A few days after the surgery, culture and sequencing results revealed that the pathogen was B. dorei/B. vulgatus and B. dorei, respectively. The patient was successfully treated with appropriate antibacterial therapy and after improvement, was transferred to another hospital for rehabilitation on postoperative day 34. There was no recurrence of infection or aneurysm after the patient transfer. Conclusions This report describes the first case of invasive infectious disease caused by B. dorei, casting a shadow over its utilisation as a probiotic for atherosclerotic diseases.

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Diversity of microbial communities in hot springs of Sri Lanka as revealed by 16S rRNA gene high-throughput sequencing analysis

Gene ◽

10.1016/j.gene.2021.146103 ◽

2021 ◽

pp. 146103

Author(s):

Dilini Sadeepa ◽

Kosala Sirisena ◽

Pathmalal M. Manage

Keyword(s):

Sri Lanka ◽

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

High Throughput ◽

High Throughput Sequencing ◽

Hot Springs ◽

Rrna Gene ◽

Sequencing Analysis

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Metagenome-validated Parallel Amplicon Sequencing and Text Mining-based Annotations for Simultaneous Profiling of Bacteria and Fungi: Vaginal Microbiome and Mycobiota in Healthy Women

10.21203/rs.3.rs-321778/v1 ◽

2021 ◽

Author(s):

Seppo Virtanen ◽

Schahzad Saqib ◽

Tinja Kanerva ◽

Pekka Nieminen ◽

Ilkka Kalliala ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Cost Effective ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Extensive Literature ◽

Metagenomic Sequencing ◽

Text Extraction ◽

Healthy Women ◽

Bacteria And Fungi

Abstract Background: Amplicon sequencing of kingdom-specific tags such as 16S rRNA gene for bacteria and internal transcribed spacer (ITS) region for fungi are widely used for investigating microbial populations. So far most human studies have focused on bacteria while studies on host-associated fungi in health and disease have only recently started to accumulate. To enable cost-effective parallel analysis of bacterial and fungal communities in human and environmental samples, we developed a method where 16S rRNA gene and ITS-1 amplicons were pooled together for a single Illumina MiSeq or HiSeq run and analysed after primer-based segregation. Taxonomic assignments were performed with Blast in combination with an iterative text-extraction based filtration approach, which uses extensive literature records from public databases to select the most probable hits that were further validated by shotgun metagenomic sequencing. Results: Using 50 vaginal samples, we show that the combined run provides comparable results on bacterial composition and diversity to conventional 16S rRNA gene amplicon sequencing. The text-extraction-based taxonomic assignment guided tool provided ecosystem specific annotations that were confirmed by Metagenomic Phylogenetic Analysis (MetaPhlAn). The metagenome analysis revealed distinct functional differences between the bacterial community types while fungi were undetected, despite being identified in all samples based on ITS amplicons. Co-abundance analysis of bacteria and fungi did not show strong between-kingdom correlations within the vaginal ecosystem of healthy women.Conclusion: Combined amplicon sequencing for bacteria and fungi provides a simple and cost-effective method for simultaneous analysis of microbiota and mycobiota within the same samples. Text extraction-based annotation tool facilitates the characterization and interpretation of defined microbial communities from rapidly accumulating sequencing and metadata readily available through public databases.

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Apis andreniformis associated Actinomycetes show antimicrobial activity against black rot pathogen (Xanthomonas campestris pv. campestris)

PeerJ ◽

10.7717/peerj.12097 ◽

2021 ◽

Vol 9 ◽

pp. e12097

Author(s):

Yaowanoot Promnuan ◽

Saran Promsai ◽

Wasu Pathom-aree ◽

Sujinan Meelai

Keyword(s):

Antimicrobial Activity ◽

16S Rrna ◽

16S Rrna Gene ◽

Honey Bee ◽

Pseudomonas Syringae ◽

Xanthomonas Campestris ◽

Pathogenic Bacteria ◽

Rrna Gene ◽

Apis Andreniformis

This study aimed to investigate cultivable actinomycetes associated with rare honey bee species in Thailand and their antagonistic activity against plant pathogenic bacteria. Actinomycetes were selectively isolated from the black dwarf honey bee (Apis andreniformis). A total of 64 actinomycete isolates were obtained with Streptomyces as the predominant genus (84.4%) followed by Micromonospora (7.8%), Nonomuraea (4.7%) and Actinomadura (3.1%). All isolates were screened for antimicrobial activity against Xanthomonas campestris pv. campestris, Pectobacterium carotovorum and Pseudomonas syringae pv. sesame. Three isolates inhibited the growth of X. campestris pv. campestris during in vitro screening. The crude extracts of two isolates (ASC3-2 and ASC5-7P) had a minimum inhibitory concentration (MIC) of 128 mg L−1against X. campestris pv. campestris. For isolate ACZ2-27, its crude extract showed stronger inhibitory effect with a lower MIC value of 64 mg L−1 against X. campestris pv. campestris. These three active isolates were identified as members of the genus Streptomyces based on their 16S rRNA gene sequences. Phylogenetic analysis based on the maximum likelihood algorithm showed that isolate ACZ2-27, ASC3-2 and ASC5-7P were closely related to Streptomyces misionensis NBRC 13063T (99.71%), Streptomyces cacaoi subsp. cacaoi NBRC 12748T (100%) and Streptomyces puniceus NBRC 12811T (100%), respectively. In addition, representative isolates from non-Streptomyces groups were identified by 16S rRNA gene sequence analysis. High similarities were found with members of the genera Actinomadura, Micromonospora and Nonomuraea. Our study provides evidence of actinomycetes associated with the black dwarf honey bee including members of rare genera. Antimicrobial potential of these insect associated Streptomyces was also demonstrated especially the antibacterial activity against phytopathogenic bacteria.

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Flavobacterium cutihirudinis sp. nov., isolated from the skin of the medical leech Hirudo verbana

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.048736-0 ◽

2013 ◽

Vol 63 (Pt_8) ◽

pp. 2841-2847 ◽

Cited By ~ 18

Author(s):

S. P. Glaeser ◽

H. Galatis ◽

K. Martin ◽

P. Kämpfer

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Type Species ◽

Biochemical Characterization ◽

Rrna Gene ◽

Sequencing Analysis ◽

Respiratory Quinone ◽

Content Type ◽

Link Type ◽

Hirudo Verbana

A Gram-staining-negative, non-endospore-forming, yellow-pigmented strain (E89T) was isolated from the skin of the medical leech Hirudo verbana obtained from a leech farm located in Biebertal, Germany. 16S rRNA gene sequencing analysis showed that the isolate was grouped in the genus Flavobacterium . Strain E89T was most closely related to Flavobacterium chilense LM-09-FpT (98.2 %), Flavobacterium chungangense CJ7T (98.1 %), and Flavobacterium oncorhynchi 631-08T (98.1 %). 16S rRNA gene sequence similarities to all other species of the genus Flavobacterium were ≤97.4 %. A menaquinone of the type MK-6 was found to be the predominant respiratory quinone and the polar lipid profile consisted of the major compounds phosphatidylethanolamine, phosphatidylserine, two unidentified aminolipids, one unknown phospholipid and two unknown lipids. The fatty acid profile was composed of iso-C15 : 0, C15 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) found in major amounts and several hydroxylated fatty acids in smaller amounts, among them iso-C15 : 0 3-OH and iso-C17 : 0 3-OH. All these data support the allocation of the isolate in the genus Flavobacterium . Physiological/biochemical characterization and DNA–DNA hybridizations with the type strains of the most closely related species allowed a clear phenotypic and genotypic differentiation of the strain. Based on these data, strain E89T represents a novel species of the genus Flavobacterium , for which the name Flavobacterium cutihirudinis sp. nov. is proposed. The type strain is E89T ( = DSM 25795T = LMG 26922T = CIP 110374T).

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