scholarly journals Isolation of SAR11 marine bacteria from cryopreserved seawater

2020 ◽  
Author(s):  
Elizabeth A. Monaghan ◽  
Kelle C. Freel ◽  
Michael S. Rappé
Keyword(s):  
Growth Medium ◽  
High Throughput ◽  
Marine Bacteria ◽  
Mixed Cultures ◽  
Rrna Gene ◽  
Culture Collections ◽  
Wide Range ◽  
Long Time ◽  
Dilution Culture ◽  
Cultivation Experiment

AbstractIn this study, we sought a means to increase current culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. In July 2017, raw seawater was collected outside of Kāne‘ohe Bay, Hawai‘i, in the tropical Pacific Ocean. A portion of this sample was diluted in seawater-based growth medium to create 576 × 2 mL dilution cultures containing 5 cells each and incubated for a high-throughput cultivation experiment, while another portion was cryopreserved in 10% glycerol. After ten months, a cryopreserved aliquot of seawater was thawed, diluted in seawater-based growth medium, and distributed to create a second high-throughput cultivation experiment of 480 × 2 mL dilution cultures containing 5 cells each and 94 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed-cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (115 of 125) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though isolates of subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater sample used to create both culture experiments were isolated by both approaches.ImportanceHigh-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing in the presence of cryoprotectants such as glycerol has been shown to be an effective method of storing viable cells over long time periods (i.e. years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months, at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings expand the potential of high-throughput cultivation experiments to include opportunities where immediate isolation experiments are impractical, allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and enable cultivation experiments across a wide range of other conditions that would benefit from having source inoculum available over extended periods of time.

scholarly journals Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
pp. e00954-20
Author(s):  
Elizabeth A. Monaghan ◽  
Kelle C. Freel ◽  
Michael S. Rappé
Keyword(s):  
High Throughput ◽  
Marine Bacteria ◽  
Mixed Cultures ◽  
Rrna Gene ◽  
Marine Microorganisms ◽  
Culture Collections ◽  
Natural Systems ◽  
Wide Range ◽  
Long Time ◽  
Cultivation Experiment

ABSTRACTWhile marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches.IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.

scholarly journals Microbiome meets classical microbiology: quantifying sample CFU using 16S rRNA gene sequencing data

2020 ◽  
Author(s):  
Giuliano Netto Flores Cruz ◽  
Ana Paula Christoff ◽  
Luiz Felipe Valter de Oliveira
Keyword(s):  
High Throughput ◽  
Cross Validation ◽  
Microbial Load ◽  
Cumulative Probability ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Probability Models ◽  
Microbial Composition ◽  
Wide Range ◽  
Ngs Data

Abstract Background Next-generation sequencing (NGS) has been extensively employed to perform microbiome characterization worldwide. As a culture-independent methodology, it has allowed high-level profiling of sample microbial composition. However, most studies are limited to sample information regarding relative bacterial abundances (sample proportions), ignoring scenarios in which sample microbe biomass can vary widely. Here, we develop an equivolumetric protocol for amplicon library preparation capable of generating NGS data responsive to input DNA, recovering proportionality between observed read counts and absolute bacterial abundances within each sample. Within a determined range, we show that the estimation of sample colony-forming units (CFU), the most common unit of bacterial abundance in classical microbiology, is challenged mostly by resolution and taxon-to-taxon variation. We propose Bayesian cumulative probability models to address such issues.Results Observed read counts were consistently proportional to input DNA, total microbial load, and bacterium-specific sample abundances, although a saturation tendency was observed as abundances increased. Using Bayesian cumulative probability models, predictive errors in sample CFU estimation varied constantly below one order of magnitude - as measured by the mean absolute log10-ratio (MALR). For total microbial load, observed MALR was no greater than 0.2 during both cross-validation and validation on a test dataset. For observed bacteria, estimation of taxon-specific CFU showed MALR values of at most 0.5. We also performed leave-one-group-out cross-validation to assess predictive performance for previously unseen bacteria. While most bacteria showed MALR no greater than 1, such a threshold was exceeded only by Bacillus cereus.Conclusions Being able to estimate sample CFU in a high-throughput fashion has a wide range of applications, from the study of built environments to public health surveillance. This study shows that equivolumetric protocols along with cumulative probability models allow sample CFU estimation from microbiome datasets. Further, our approach has the potential to generalize to previously unmodeled bacteria, an important feature in high-throughput settings. Lastly, it remains clear that NGS data are not inherently restricted to sample proportions only, and microbiome science can finally meet the working scales of classical microbiology.

The Problem of Identifying and Modeling the Relationship between Monetary Factor and Inflation in the Russian Economy

2008 ◽  
pp. 61-76
Author(s):  
A. Porshakov ◽  
A. Ponomarenko
Keyword(s):  
Money Supply ◽  
Money Demand ◽  
Russian Economy ◽  
Long Run ◽  
Complex Approach ◽  
Wide Range ◽  
Long Time ◽  
The Relationship ◽  
Supply Side Factors

The role of monetary factor in generating inflationary processes in Russia has stimulated various debates in social and scientific circles for a relatively long time. The authors show that identification of the specificity of relationship between money and inflation requires a complex approach based on statistical modeling and involving a wide range of indicators relevant for the price changes in the economy. As a result a model of inflation for Russia implying the decomposition of inflation dynamics into demand-side and supply-side factors is suggested. The main conclusion drawn is that during the recent years the volume of inflationary pressures in the Russian economy has been determined by the deviation of money supply from money demand, rather than by money supply alone. At the same time, monetary factor has a long-run spread over time impact on inflation.

Comperehencive review of Tankana

2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Dr. Jyotsna Sankpal ◽  
Dr. Jyotsna Takalikar
Keyword(s):  
Heart Disease ◽  
Medical Science ◽  
Menstrual Disorders ◽  
Therapeutic Applications ◽  
Indian Medical ◽  
Wide Range ◽  
Long Time

Rasa Shastra and Bhaishajya Kalpana is branch of the ancient Indian medical science based on herbs and herbo-mineral preparation. Tankana has been described under Uparasa Tankana, which is one among the Kshara Trayas has been used since very long time in Ayurveda. It has a wide range of therapeutic applications, including diseases like Varna (ulcers), Shvasa (asthma), Kasa (cough), Hrudya (beneficial to heart disease), Streepushpajanana (menstrual disorders) etc. It is used in the form of compound formulations like Parpati, Kupipakwa, Khalvee Rasayana, Churna, Vati, Lepa etc. In this paper Tankana Shodhana procedure, different synonyms, dose, Anupana, indications and different formulations containing Tankana Bhasma has been discussed.

scholarly journals Impact of Diagnosed and Undiagnosed Respiratory Pseudomonas on VAP and VAE During Long-Term Acute Care

2020 ◽  
Vol 41 (S1) ◽  
pp. s258-s259
Author(s):  
James Harrigan ◽  
Ebbing Lautenbach ◽  
Emily Reesey ◽  
Magda Wernovsky ◽  
Pam Tolomeo ◽  
...  
Keyword(s):  
Bacterial Community ◽  
Acute Care ◽  
Statistical Significance ◽  
Rrna Gene ◽  
Care Hospital ◽  
16S Sequencing ◽  
Increased Risk ◽  
Wide Range ◽  
The Impact

Background: Clinically diagnosed ventilator-associated pneumonia (VAP) is common in the long-term acute-care hospital (LTACH) setting and may contribute to adverse ventilator-associated events (VAEs). Pseudomonas aeruginosa is a common causative organism of VAP. We evaluated the impact of respiratory P. aeruginosa colonization and bacterial community dominance, both diagnosed and undiagnosed, on subsequent P. aeruginosa VAP and VAE events during long-term acute care. Methods: We enrolled 83 patients on LTACH admission for ventilator weaning, performed longitudinal sampling of endotracheal aspirates followed by 16S rRNA gene sequencing (Illumina HiSeq), and bacterial community profiling (QIIME2). Statistical analysis was performed with R and Stan; mixed-effects models were fit to relate the abundance of respiratory Psa on admission to clinically diagnosed VAP and VAE events. Results: Of the 83 patients included, 12 were diagnosed with P. aeruginosa pneumonia during the 14 days prior to LTACH admission (known P. aeruginosa), and 22 additional patients received anti–P. aeruginosa antibiotics within 48 hours of admission (suspected P. aeruginosa); 49 patients had no known or suspected P. aeruginosa (unknown P. aeruginosa). Among the known P. aeruginosa group, all 12 patients had P. aeruginosa detectable by 16S sequencing, with elevated admission P. aeruginosa proportional abundance (median, 0.97; IQR, 0.33–1). Among the suspected P. aeruginosa group, all 22 patients had P. aeruginosa detectable by 16S sequencing, with a wide range of admission P. aeruginosa proportional abundance (median, 0.0088; IQR, 0.00012–0.31). Of the 49 patients in the unknown group, 47 also had detectable respiratory Psa, and many had high P. aeruginosa proportional abundance at admission (median, 0.014; IQR, 0.00025–0.52). Incident P. aeruginosa VAP was observed within 30 days in 4 of the known P. aeruginosa patients (33.3%), 5 of the suspected P. aeruginosa patients (22.7%), and 8 of the unknown P. aeruginosa patients (16.3%). VAE was observed within 30 days in 1 of the known P. aeruginosa patients (8.3%), 2 of the suspected P. aeruginosa patients (9.1%), and 1 of the unknown P. aeruginosa patients (2%). Admission P. aeruginosa abundance was positively associated with VAP and VAE risk in all groups, but the association only achieved statistical significance in the unknown group (type S error <0.002 for 30-day VAP and <0.011 for 30-day VAE). Conclusions: We identified a high prevalence of unrecognized respiratory P. aeruginosa colonization among patients admitted to LTACH for weaning from mechanical ventilation. The admission P. aeruginosa proportional abundance was strongly associated with increased risk of incident P. aeruginosa VAP among these patients.Funding: NoneDisclosures: None

scholarly journals Collection of a Bacterial Community Reconstructed from Marine Metagenomes Derived from Jinhae Bay, South Korea

Data ◽  
2021 ◽  
Vol 6 (5) ◽  
pp. 44
Author(s):  
Jae-Hyun Lim ◽  
Il-Nam Kim
Keyword(s):  
Microbial Community ◽  
South Korea ◽  
Microbial Communities ◽  
Marine Bacteria ◽  
Sampling Site ◽  
Community Change ◽  
Rrna Gene ◽  
Water Column Stratification ◽  
Metagenomics Data ◽  
Jinhae Bay

Marine bacteria are known to play significant roles in marine biogeochemical cycles regarding the decomposition of organic matter. Despite the increasing attention paid to the study of marine bacteria, research has been too limited to fully elucidate the complex interaction between marine bacterial communities and environmental variables. Jinhae Bay, the study area in this work, is the most anthropogenically eutrophied coastal bay in South Korea, and while its physical and biogeochemical characteristics are well described, less is known about the associated changes in microbial communities. In the present study, we reconstructed a metagenomics data based on the 16S rRNA gene to investigate temporal and vertical changes in microbial communities at three depths (surface, middle, and bottom) during a seven-month period from June to December 2016 at one sampling site (J1) in Jinhae Bay. Of all the bacterial data, Proteobacteria, Bacteroidetes, and Cyanobacteria were predominant from June to November, whereas Firmicutes were predominant in December, especially at the middle and bottom depths. These results show that the composition of the microbial community is strongly associated with temporal changes. Furthermore, the community compositions were markedly different between the surface, middle, and bottom depths in summer, when water column stratification and bottom water hypoxia (low dissolved oxygen level) were strongly developed. Metagenomics data contribute to improving our understanding of important relationships between environmental characteristics and microbial community change in eutrophication-induced and deoxygenated coastal areas.

scholarly journals Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
K. Böhme ◽  
P. Cremonesi ◽  
M. Severgnini ◽  
Tomás G. Villa ◽  
I. C. Fernández-No ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Cost Effective ◽  
Rrna Gene ◽  
Bacterial Identification ◽  
Dot Blot ◽  
Culture Collections ◽  
Ligation Detection Reaction ◽  
Flow Through ◽  
Food Safety And Quality

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific forAeromonasspp.,Pseudomonasspp.,Shewanellaspp., andMorganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

scholarly journals Exploring protocol bias in airway microbiome studies: one versus two PCR steps and 16S rRNA gene region V3 V4 versus V4

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Christine Drengenes ◽  
Tomas M. L. Eagan ◽  
Ingvild Haaland ◽  
Harald G. Wiker ◽  
Rune Nielsen
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Bacterial Load ◽  
Marker Gene ◽  
Gene Region ◽  
Lower Airway ◽  
Rrna Gene ◽  
Wide Range ◽  
Airway Microbiome ◽  
The Impact

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

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