scholarly journals Using High Throughput DNA Sequencing to Evaluate the Accuracy of Serial Dilution Based Tests of Microbial Activities in Oil Pipelines

2018 ◽  
Author(s):  
K. Khanipov ◽  
G. Golovko ◽  
M. Rojas ◽  
M. Pimenova ◽  
L. Albayrak ◽  
...  
Keyword(s):  
High Throughput ◽  
Biofilm Formation ◽  
High Throughput Sequencing ◽  
Serial Dilution ◽  
Metabolic Function ◽  
Microbial Activities ◽  
Microbial Composition ◽  
Oil Pipelines ◽  
High Throughput Dna Sequencing ◽  
Product Degradation

AbstractMicrobial activities have detrimental effects on industrial infrastructure. If not controlled, microbial presence can result in corrosion, biofilm formation, and product degradation. Serial dilution tests are routinely used for evaluating presence and abundance of microorganisms by diluting samples and culturing microbes in specific media designed to support microorganisms with particular properties, such as sulfate reduction.A high-throughput sequencing approach was used to evaluate changes in microbial composition during four standard serial dilution tests. Analysis of 159 isolates revealed significant differences in the microbial compositions of sequential serial dilution titers and identified several cases where: (a) bacteria known to have a detrimental metabolic function (such as acid production) were lost in the serial dilution medium designed to test for this function; (b) bacteria virtually absent in the original sample became dominant in the serial dilution medium. These observations raise concerns regarding the accuracy and overall usefulness of serial dilution tests.

scholarly journals High-throughput sequencing analysis of bacterial diversity in raw and pasteurized goat milk

2019 ◽  
Author(s):  
Feng Huang ◽  
Siqi Liu ◽  
Xiaokang Zhou ◽  
Pengfei Wang ◽  
Rengchun He ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Bacterial Species ◽  
Goat Milk ◽  
Raw Milk ◽  
Sequencing Analysis ◽  
Quality And Safety ◽  
Microbial Composition ◽  
High Throughput Dna Sequencing

AbstractThe aim of this study was to evaluate the microbial composition of both raw and pasteurized goat milk using high-throughput DNA sequencing. This analysis revealed that the dominant phylum found in the raw milk was Proteobacteria, and the dominant genus was Kluyvera; Proteobacteria and Kluyvera constituted up to 67.66% and 28.85% of the total bacteria population, respectively. The microorganisms in goat milk predominantly consist of Gram-negative bacteria. Notably, Akkermansia and Faecalibacterium were identified in goat milk for the first time. In addition, the results also indicate that some bacteria in pasteurized goat milk may exist in a viable but nonculturable (VBNC) state. This study provides a theoretical basis that may aid the community in better understanding bacterial diversity in goat milk. The results of this study will help us to improve the quality and safety of goat milk.ImportanceThe microbial diversity in goat milk and pasteurized goat milk at different refrigeration stages was described. Several bacterial species that have not previously been reported in goat milk were identified, including many VBNC bacteria. The findings provided the necessary microbial information for quality and safety of goat milk and dairy products.

scholarly journals A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood

2020 ◽  
Vol 6 (6) ◽  
pp. FSO476
Author(s):  
Ofir Israeli ◽  
Efi Makdasi ◽  
Inbar Cohen-Gihon ◽  
Anat Zvi ◽  
Shirley Lazar ◽  
...  
Keyword(s):  
High Throughput ◽  
Whole Blood ◽  
High Throughput Sequencing ◽  
High Background ◽  
Blood Samples ◽  
Bacterial Enrichment ◽  
Host Pathogen ◽  
High Throughput Dna Sequencing ◽  
Human Blood Samples ◽  
Pathogen Dna

High-throughput DNA sequencing (HTS) of pathogens in whole blood samples is hampered by the high host/pathogen nucleic acids ratio. We describe a novel and rapid bacterial enrichment procedure whose implementation is exemplified in simulated bacteremic human blood samples. The procedure involves depletion of the host DNA, rapid HTS and bioinformatic analyses. Following this procedure, Y. pestis, F. tularensis and B. anthracis spiked-in samples displayed an improved host/pathogen DNA ratio of 2.5–5.9 orders of magnitude, in samples with bacteria spiked-in at 103–105 CFU/ml. The procedure described in this study enables rapid and detailed metagenomic profiling of pathogens within 8–9 h, circumventing the challenges imposed by the high background present in the bacteremic blood and by the unknown nature of the sample.

scholarly journals High-Throughput, Sequence-Based Analysis of the Microbiota of Greek Kefir Grains from Two Geographic Regions

2020 ◽  
Vol 58 (2) ◽  
pp. 138-146
Author(s):  
Mary S. Kalamaki ◽  
Apostolos S. Angelidis
Keyword(s):  
Bacterial Community ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Geographic Origin ◽  
Probiotic Properties ◽  
Sequencing Analysis ◽  
Microbial Composition ◽  
Bacterial Phyla ◽  
Geographic Regions ◽  
Kefir Grains

Research background. Kefir is a natural probiotic drink traditionally produced by milk fermentation using kefir grains. Kefir grains are composed of a complex population of bacteria and yeasts embedded in a polysaccharide-protein matrix. The geographic origin of kefir grains may largely influence their microbial composition and the associated kefir drink properties. Although the detailed bacterial composition of kefir grains from several geographic regions has been reported, to date, analogous data about the microbiome of Greek kefir are lacking. Hence, the aim of this study is to investigate the structure and the diversity of the bacterial community of Greek kefir grains.Experimental approach. The bacterial community structure and diversity of two different kefir grains from distant geographic regions in Greece were examined via high-throughput sequencing analysis, a culture-independent metagenomic approach, targeting the 16S rRNA V4 variable region, in order to gain a deeper understanding of their bacterial population diversities.Results and conclusions. Firmicutes (a phylum that includes lactic acid bacteria) was strikingly dominant amongst the identified bacterial phyla, with over 99 % of the sequences from both kefir grains classified to this phylum. At the family level, Lactobacillaceae sequences accounted for more than 98 % of the operational taxonomic units (OTUs), followed by Ruminococcaceae, Lahnospiraceae, Bacteroidaceae and other bacterial families of lesser abundance. Α relatively small number of bacterial genera dominated, with Lactobacillus kefiranofaciens being the most abundant in both kefir grains (95.0 % of OTUs in kefir A and 96.3 % of OTUs in kefir B). However, a quite variable subdominant population was also present in both grains, including bacterial genera that have been previously associated with the gastrointestinal tract of humans and animals, some of which are believed to possess probiotic properties (Faecalibacterium spp., Bacteroides spp., Blautia spp.). Differences among the bacterial profiles of the two grains were very small indicating a high homogeneity despite the distant geographic origin.Novelty and scientific contribution. This is the first study to deeply explore and report on the bacterial diversity and species richness of Greek kefir.

scholarly journals Microbial Composition of Human Appendices from Patients following Appendectomy

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Caitriona M. Guinane ◽  
Amany Tadrous ◽  
Fiona Fouhy ◽  
C. Anthony Ryan ◽  
Eugene M. Dempsey ◽  
...  
Keyword(s):  
Human Health ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Evolutionary Development ◽  
Rrna Gene ◽  
Limited Data ◽  
Microbial Composition ◽  
Content Type ◽  
Study Results ◽  
Human Appendix

ABSTRACT The human appendix has historically been considered a vestige of evolutionary development with an unknown function. While limited data are available on the microbial composition of the appendix, it has been postulated that this organ could serve as a microbial reservoir for repopulating the gastrointestinal tract in times of necessity. We aimed to explore the microbial composition of the human appendix, using high-throughput sequencing of the 16S rRNA gene V4 region. Seven patients, 5 to 25 years of age, presenting with symptoms of acute appendicitis were included in this study. Results showed considerable diversity and interindividual variability among the microbial composition of the appendix samples. In general, however, Firmicutes was the dominant phylum, with the majority of additional sequences being assigned at various levels to Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria. Despite the large diversity in the microbiota found within the appendix, however, a few major families and genera were found to comprise the majority of the sequences present. Interestingly, also, certain taxa not generally associated with the human intestine, including the oral pathogens Gemella, Parvimonas, and Fusobacterium, were identified among the appendix samples. The prevalence of genera such as Fusobacterium could also be linked to the severity of inflammation of the organ. We conclude that the human appendix contains a robust and varied microbiota distinct from the microbiotas in other niches within the human microbiome. The microbial composition of the human appendix is subject to extreme variability and comprises a diversity of biota that may play an important, as-yet-unknown role in human health. IMPORTANCE There are currently limited data available on the microbial composition of the human appendix. It has been suggested, however, that it may serve as a “safe house” for commensal bacteria that can reinoculate the gut at need. The present study is the first comprehensive view of the microbial composition of the appendix as determined by high-throughput sequencing. We have determined that the human appendix contains a wealth of microbes, including members of 15 phyla. Important information regarding the associated bacterial diversity of the appendix which will help determine the role, if any, the appendix microbiota has in human health is presented.

scholarly journals Multi-Body-Site Microbiome and Culture Profiling of Military Trainees Suffering from Skin and Soft Tissue Infections at Fort Benning, Georgia

mSphere ◽  
2016 ◽  
Vol 1 (5) ◽  
Author(s):  
Jatinder Singh ◽  
Ryan C. Johnson ◽  
Carey D. Schlett ◽  
Emad M. Elassal ◽  
Katrina B. Crawford ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
Bacterial Community Composition ◽  
Soft Tissue Infections ◽  
Microbial Composition ◽  
Content Type ◽  
Microbial Dysbiosis ◽  
Lack Of Information ◽  
The Impact ◽  
Nasal Microbiota

ABSTRACT While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs. Skin and soft tissue infections (SSTIs) are common in the general population, with increased prevalence among military trainees. Previous research has revealed numerous nasal microbial signatures that correlate with SSTI development and Staphylococcus aureus colonization. Thus, we hypothesized that the ecology of the inguinal, oropharynx, and perianal regions may also be altered in response to SSTI and/or S. aureus colonization. We collected body site samples from 46 military trainees with purulent abscess (SSTI group) as well as from 66 asymptomatic controls (non-SSTI group). We also collected abscess cavity samples to assess the microbial composition of these infections. Samples were analyzed by culture, and the microbial communities were characterized by high-throughput sequencing. We found that the nasal, inguinal, and perianal regions were similar in microbial composition and significantly differed from the oropharynx. We also observed differences in Anaerococcus and Streptococcus abundance between the SSTI and non-SSTI groups for the nasal and oropharyngeal regions, respectively. Furthermore, we detected community membership differences between the SSTI and non-SSTI groups for the nasal and inguinal sites. Compared to that of the other regions, the microbial compositions of the nares of S. aureus carriers and noncarriers were dramatically different; we noted an inverse correlation between the presence of Corynebacterium and the presence of Staphylococcus in the nares. This correlation was also observed for the inguinal region. Culture analysis revealed elevated methicillin-resistant S. aureus (MRSA) colonization levels for the SSTI group in the nasal and inguinal body sites. Together, these data suggest significant microbial variability in patients with SSTI as well as between S. aureus carriers and noncarriers. IMPORTANCE While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs.

scholarly journals Large-scale, quantitative protein assays on a high-throughput DNA sequencing chip

2018 ◽  
Author(s):  
Curtis J Layton ◽  
Peter L McMahon ◽  
William J Greenleaf
Keyword(s):  
Amino Acid ◽  
Dna Sequencing ◽  
High Throughput ◽  
Protein Function ◽  
Large Scale ◽  
High Throughput Sequencing ◽  
Molecular Variants ◽  
Amino Acid Interaction ◽  
High Throughput Dna Sequencing ◽  
Protein Assays

SummaryHigh-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally-translated proteins and peptides, and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely-available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3×104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a “superFLAG” epitope variant. We also measured the enzymatic activity of 1.56×105 molecular variants of full-length of human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates linked to amino acid sequence perturbations revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively-cooperative interactions with histidine residues.

scholarly journals GrigoraSNPs: Optimized HTS DNA Forensic SNP Analysis

2017 ◽  
Author(s):  
Darrell O. Ricke ◽  
Anna Shcherbina ◽  
Adam Michaleas ◽  
Philip Fremont-Smith
Keyword(s):  
High Throughput ◽  
Tandem Repeats ◽  
High Throughput Sequencing ◽  
Dna Analysis ◽  
Sequence Data ◽  
Snp Analysis ◽  
Analysis Pipeline ◽  
Sequencing Technologies ◽  
High Throughput Dna Sequencing

AbstractHigh throughput DNA sequencing technologies enable improved characterization of forensic DNA samples enabling greater insights into DNA contributor(s). Current DNA forensics techniques rely upon allele sizing of short tandem repeats by capillary electrophoresis. High throughput sequencing enables forensic sample characterizations for large numbers of single nucleotide polymorphism loci. The slowest computational component of the DNA forensics analysis pipeline is the characterization of raw sequence data. This paper optimizes the SNP calling module of the DNA analysis pipeline with runtime results that scale linearly with the number of HTS sequences (patent pending)[1]. GrigoraSNPs can analyze 100 million reads in less than 5 minutes using 3 threads on a 4.0 GHz Intel i7-6700K laptop CPU.

scholarly journals Analysis of Sour Porridge Microbiota and Improvement of Cooking Quality via Pure Culture Fermentation Using Lacticaseibacillus paracasei Strain SZ02

2021 ◽  
Vol 12 ◽  
Author(s):  
Cheng Wang ◽  
Yunhe Xu ◽  
Bin Yu ◽  
Aibo Xiao ◽  
Yuhong Su ◽  
...  
Keyword(s):  
High Throughput ◽  
Pure Culture ◽  
Fermentation Process ◽  
High Throughput Sequencing ◽  
Cooking Quality ◽  
Microbial Composition ◽  
Natural Fermentation ◽  
Acid Producing Bacteria ◽  
Dominant Bacteria

The microbial composition of sour porridge at different fermentation times was analyzed through high-throughput sequencing, and a pure culture fermentation process was established to optimize production process and improve the edible quality of the porridge. In natural fermentation, Firmicutes and Proteobacteria were abundant throughout the process. Specifically, Aeromonas, Acinetobacter, and Klebsiella were dominant on fermentation days 1–5 (groups NF-1, NF-3, and NF-5), while Lactobacillus and Acetobacter gradually became the dominant bacteria on fermentation day 7 (group NF-7). Further, we isolated one strain of acid-producing bacteria from sour porridge, identified as Lacticaseibacillus paracasei by 16SrRNA sequencing and annotated as strain SZ02. Pure culture fermentation using this strain significantly increased the relative starch and amylose contents of the porridge, while decreasing the lipid, protein, and ash contents (P < 0.05). These findings suggest that sour porridge produced using strain SZ02 has superior edible qualities and this strategy may be exploited for its industrial production.

scholarly journals Assessment of microbiota diversity in dental unit waterline contamination

PeerJ ◽  
2022 ◽  
Vol 10 ◽  
pp. e12723
Author(s):  
Yun Dang ◽  
Qian Zhang ◽  
Jing Wang ◽  
Qian Wang ◽  
Meng Han ◽  
...  
Keyword(s):  
Community Structure ◽  
High Throughput ◽  
Water Samples ◽  
High Speed ◽  
High Throughput Sequencing ◽  
Mouth Rinse ◽  
Microbial Composition ◽  
Linear Discriminant ◽  
Rinse Water ◽  
Bacterial Concentrations

Background Dental unit waterlines (DUWLs) provide water for handpieces, air/water syringes, and mouth-rinse water outlets. DUWL contamination can negatively affect the operating environment and public health. Therefore, it is important to elucidate the bacterial concentrations and microbial composition in the DUWLs from different dental specialties. Methods We collected 350 5-mL dental water samples (from high-speed handpieces, air/water syringes, and mouth-rinse water outlets) from 60 dental chair units (DCUs) at a dental hospital to determine the bacterial concentrations by culture methods. Meanwhile, to investigate the diversity and community structure of microbe in the DUWLs, 17 high-quality DNA from 60 250-mL air/water syringe water samples, which were collected from the same 60 DCUs, were analyzed using 16S rDNA high-throughput sequencing. Results The median bacterial concentration was 166 (31.5, 672.5) CFU/mL and the range was 0–3,816,000 CFU/mL. Only 42.6% of the water samples had bacterial concentrations below 100 CFU/mL. The Kruskal–Wallis H-test revealed that the water samples from three dental specialties had significantly different bacterial concentrations (H = 27.441, P < 0.01). High-throughput sequencing results showed significant differences in bacterial community structure between periodontics and the other two dental specialties. In the samples from three dental specialties, 508 OTUs were detected, with 160, 182 and 176 OTUs unique to the periodontics, endodontics and prosthodontics specialties, respectively. Linear discriminant analysis (LDA) effect size (LEfSe) suggested that Hydrocarboniphaga, Zoogloea, Aquabacterium, and Hydrogenophaga were enriched in the periodontics specialty; Acinetobacter, Geothrix, and Desulfovibrio were enriched in the prosthodontics specialty; and Alistipes, Clostridium XIVa, and Serratia were enriched in the endodontics specialty. Seven potentially human-pathogenic genera (Pseudomonas, Acinetobacter, Sphingomonas, Ochrobactrum, Rhizobium, Brevundimonas, and Methylobacterium) with relative abundance exceeding 1% were also detected in the DUWLs. Conclusions The bacterial concentrations and microbial composition were influenced by different dental specialties, so a validated disinfection protocol should be used to control DUWL contamination in different dental specialties.

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