Assessing microbial growth monitoring methods for challenging strains and cultures

2021 ◽  
Author(s):  
Damon Brown ◽  
Raymond J. Turner
Keyword(s):  
16S Rrna ◽  
Optical Density ◽  
Pure Culture ◽  
Desulfovibrio Vulgaris ◽  
Rrna Gene ◽  
Growth Monitoring ◽  
Sample Type ◽  
Monitoring Method ◽  
Reliable Technique ◽  
Cell Counts

AbstractThis is a paper focusing on the comparison of growth curves using field relevant testing methods and moving away from colony counts. Challenges exist to explore antimicrobial growth of fastidious strains, poorly culturable bacterial and bacterial communities of environmental interest. Thus, various approaches have been explored to follow bacteria growth that can be an efficient surrogate for classical optical density or colony forming unit measurements.Here we tested optical density, ATP assays, DNA concentrations and 16S rRNA qPCR as means to monitor pure culture growth of six different species including Acetobacterium woodii, Bacillus subtilis, Desulfovibrio vulgaris, Geoalkalibacter subterraneus, Pseudomonas putida and Thauera aromatica. Optical density is and excellent, rapid monitoring method of pure culture planktonic cells but cannot be applied to environmental or complex samples. ATP assays provide rapid results but conversions to cell counts may be misleading for different species. DNA concentration is a very reliable technique which can be used for any sample type and provides genetic materials for downstream applications. qPCR of the 16S rRNA gene is a widely applicable technique for monitoring microbial cell concentrations but is susceptible to variation between replicates. DNA concentrations were found to correlate the best with the other three assays and provides the advantages of rapid extraction, consistency between replicates and potential for downstream analysis, DNA concentrations is determined to be the best universal monitoring method for complex environmental samples.

scholarly journals Phylogenetic Analysis and In Situ Identification of Bacteria Community Composition in an Acidic Sphagnum Peat Bog

2006 ◽  
Vol 72 (3) ◽  
pp. 2110-2117 ◽  
Author(s):  
Svetlana N. Dedysh ◽  
Timofei A. Pankratov ◽  
Svetlana E. Belova ◽  
Irina S. Kulichevskaya ◽  
Werner Liesack
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Community Composition ◽  
Significant Proportion ◽  
Peat Bog ◽  
Rrna Gene ◽  
Bacterial Phyla ◽  
Cell Counts ◽  
Novel Strategy

ABSTRACT The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (≥95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3 × 107 and 1.1 × 107 cells g−1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.

scholarly journals Composition and Origin of the Fermentation Microbiota of Mahewu, a Zimbabwean Fermented Cereal Beverage

2019 ◽  
Vol 85 (11) ◽  
Author(s):  
Felicitas Pswarayi ◽  
Michael G. Gänzle
Keyword(s):  
16S Rrna ◽  
Quantitative Pcr ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Starter Cultures ◽  
Rrna Gene ◽  
Culture Methods ◽  
Content Type ◽  
Cell Counts

ABSTRACTMahewu is a fermented cereal beverage produced in Zimbabwe. This study determined the composition and origin of mahewu microbiota. The microbiota of mahewu samples consisted of 3 to 7 dominant strains of lactobacilli and two strains of yeasts.Enterobacteriaceaewere not detected.Candida glabratawas present in high cell counts from samples collected in summer but not from samples collected in winter. Millet malt is the only raw ingredient used in the production of mahewu and is a likely source of fermentation microbiota; therefore, malt microbiota was also analyzed by culture-dependent and high-throughput 16S rRNA gene sequencing methodologies. Millet malt contained 8 to 19 strains ofEnterobacteriaceae, lactobacilli, bacilli, and very few yeasts. Strain-specific quantitative PCR assays were established on the basis of the genome sequences ofLactobacillus fermentumFUA3588 and FUA3589 andLactobacillus plantarumFUA3590 to obtain a direct assessment of the identity of strains from malt and mahewu.L. fermentumFUA3588 and FUA3589 were detected in millet malt, demonstrating that millet malt is a main source of mahewu microbiota. Strains which were detected in summer were not detected in samples produced at the same site in winter. Model mahewu fermentations conducted with a 5-strain inoculum consisting of lactobacilli,Klebsiella pneumoniae,andCronobacter sakazakiidemonstrated that lactobacilli outcompeteEnterobacteriaceae, which sharply decreased in the first 24 h. In conclusion, mahewu microbiota is mainly derived from millet malt microbiota, but minor components of malt microbiota rapidly outcompeteEnterobacteriaceaeandBacillusspecies during fermentation.IMPORTANCEThis study provides insight into the composition and origin of the microbiota of mahewu and the composition of millet malt microbiota. Fermentation microbiota are often hypothesized to be derived from the environment, but the evidence remains inconclusive. Our findings confirm that millet malt is the major source of mahewu microbiota. By complementing culture methods with high-throughput sequencing of 16S rRNA amplicons and strain-specific quantitative PCR, this study provides evidence about the source of mahewu microbiota, which can inform the development of starter cultures for mahewu production. The study also documents the fate ofEnterobacteriaceaeduring the fermentation of mahewu. There are concerns regarding the safety of traditionally prepared mahewu, and this requires in-depth knowledge of the fermentation process. Therefore, this study elucidated millet malt microbiota and identified cultures that are able to control the high numbers ofEnterobacteriaceaethat are initially present in mahewu fermentations.

scholarly journals Effects of sampling strategy and DNA extraction on human skin microbiome investigations

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rie Dybboe Bjerre ◽  
Luisa Warchavchik Hugerth ◽  
Fredrik Boulund ◽  
Maike Seifert ◽  
Jeanne Duus Johansen ◽  
...  
Keyword(s):  
16S Rrna ◽  
Dna Extraction ◽  
Human Skin ◽  
Alpha Diversity ◽  
Sampling Strategy ◽  
Sampling Technique ◽  
Rrna Gene ◽  
Skin Microbiome ◽  
Library Preparation ◽  
Reliable Technique

AbstractThe human skin is colonized by a wide array of microorganisms playing a role in skin disorders. Studying the skin microbiome provides unique obstacles such as low microbial biomass. The objective of this study was to establish methodology for skin microbiome analyses, focusing on sampling technique and DNA extraction. Skin swabs and scrapes were collected from 9 healthy adult subjects, and DNA extracted using 12 commercial kits. All 165 samples were sequenced using the 16S rRNA gene. Comparing the populations captured by eSwabs and scrapes, 99.3% of sequences overlapped. Using eSwabs yielded higher consistency. The success rate of library preparation applying different DNA extraction kits ranged from 39% to 100%. Some kits had higher Shannon alpha-diversity. Metagenomic shotgun analyses were performed on a subset of samples (N = 12). These data indicate that a reduction of human DNA from 90% to 57% is feasible without lowering the success of 16S rRNA library preparation and without introducing taxonomic bias. Using swabs is a reliable technique to investigate the skin microbiome. DNA extraction methodology is crucial for success of sequencing and adds a substantial amount of variation in microbiome analyses. Reduction of host DNA is recommended for interventional studies applying metagenomics.

scholarly journals Enumeration and Characterization of Iron(III)-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)

2003 ◽  
Vol 69 (12) ◽  
pp. 7467-7479 ◽  
Author(s):  
Lainie Petrie ◽  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
David L. Balkwill ◽  
Joel E. Kostka
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Contaminated Sediments ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Enrichment Cultures ◽  
Cell Counts ◽  
Subsurface Sediments ◽  
Reducing Bacteria

ABSTRACT Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml−1 for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.

scholarly journals Evaluation of Oxford Nanopore MinIONTM Sequencing for 16S rRNA Microbiome Characterization

2017 ◽  
Author(s):  
Xiao Ma ◽  
Elyse Stachler ◽  
Kyle Bibby
Keyword(s):  
16S Rrna ◽  
Pure Culture ◽  
Community Sample ◽  
Operational Taxonomic Unit ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Ribosomal Database Project ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Oxford Nanopore

AbstractIn this manuscript we evaluate the potential for microbiome characterization by sequencing of near-full length 16S rRNA gene region fragments using the Oxford Nanopore MinION (hereafter ‘Nanopore’) sequencing platform. We analyzed pure-culture E. coli and P. fluorescens, as well as a low-diversity mixed community sample from hydraulic fracturing produced water. Both closed and open reference operational taxonomic unit (OTU) picking failed, necessitating the direct use of sequences without OTU picking. The Ribosomal Database Project classifier against the Green Genes database was found to be the optimal annotation approach, with average pure-culture annotation accuracies of 93.8% and 82.0% at the phyla and genus levels, respectively. Comparative analysis of an environmental sample using Nanopore and Illumina MiSeq sequencing identified high taxonomic similarity when using a weighted metric (Bray-Curtis), and significantly reduced similarity when using an unweighted metric (Jaccard). These results highlight the great potential of Nanopore sequencing to analyze broad microbial community trends, and the challenge of applying Nanopore sequencing to discern rare taxa in mixed microbial communities. Finally, we observed that between-run carryover following washes on the same flowcell accounted for >10% of sequence reads, necessitating future development to either prevent carryover or filter sequences of interest (e.g. barcoding).

scholarly journals The microbiome of the pelagic tunicate Dolioletta gegenbauri: a potential link between the grazing and microbial food web

2022 ◽  
Author(s):  
Tiago Pereira ◽  
Tina Walters ◽  
Hisham El-Shaffey ◽  
Holly Bik ◽  
Marc Frischer
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Rrna Gene ◽  
Natural Seawater ◽  
Sample Type ◽  
Fecal Pellets ◽  
Feeding Experiments ◽  
Gene Copies ◽  
Order Of Magnitude ◽  
Pelagic Tunicate

Doliolids often form massive blooms during upwelling conditions in sub-tropical shelves. However, their trophic role, including their nutritious fecal pellets, in pelagic marine food webs remains poorly investigated. In this study, we performed three independent feeding experiments of cultured Dolioletta gegenbauri and used qPCR analysis and 16S rRNA metabarcoding to characterize the microbial community associated with full gut (FG) and empty (EG) doliolids, fresh (FP2Hrs) and senescing (FP24Hrs) fecal pellets, and the surrounding natural seawater (SW). Bacterial abundance (i.e., 16S rRNA gene copies) in EG samples was an order of magnitude lower than in SW and three orders lower than in FP24Hrs. Diversity analyses, based on the 16S rRNA metabarcoding data, supported a richer microbial community in SW, FP2Hrs, FP24Hrs, and FG samples. Furthermore, microbial community structure was determined by sample type, with FG samples appearing more similar to either FP2Hrs or FP24Hrs. These patterns resulted from the higher number of shared ASVs and consequently the contribution of similar major bacterial taxa (e.g., Rhodobacteraceae, Pirellulaceae). These observations support the hypothesis that there are significant ecological and trophic interactions between D. gegenbauri and the ocean microbiome. Predicted gene function recovered many genes related to key processes in the marine environment and supported greater similarity between FP2Hrs, FP24Hrs, and FG samples. These observations suggest that pelagic marine bacteria are utilized by D. gegenbauri to digest captured prey particles, and the subsequent release of fecal pellets supports the rapid proliferation of distinct microbial communities which likely influence key biogeochemical processes in the ocean.

scholarly journals Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

2020 ◽  
Author(s):  
Michael E.C. Abundo ◽  
John M. Ngunjiri ◽  
Kara J.M. Taylor ◽  
Hana Ji ◽  
Amir Ghorbani ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Extraction Process ◽  
Production Performance ◽  
Rrna Gene ◽  
Sample Type ◽  
Alpha And Beta Diversity ◽  
Gene Copies ◽  
Diversity Metrics

AbstractCharacterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. Furthermore, between-kit differences in alpha and beta diversity metrics were statistically indistinguishable. Therefore, both kits perform similarly with regard to 16S rRNA gene-based poultry microbiome analysis.

scholarly journals Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0241732
Author(s):  
Michael E. C. Abundo ◽  
John M. Ngunjiri ◽  
Kara J. M. Taylor ◽  
Hana Ji ◽  
Amir Ghorbani ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Dna Extraction ◽  
Production Performance ◽  
Rrna Gene ◽  
Sequence Variant ◽  
Sample Type ◽  
Alpha And Beta Diversity ◽  
Gene Copies ◽  
Diversity Metrics

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

scholarly journals Identification and Isolation of Anaerobic, Syntrophic Phthalate Isomer-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephthalate Manufacturing

2004 ◽  
Vol 70 (3) ◽  
pp. 1617-1626 ◽  
Author(s):  
Yan-Ling Qiu ◽  
Yuji Sekiguchi ◽  
Hiroyuki Imachi ◽  
Yoichi Kamagata ◽  
I-Cheng Tseng ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pure Culture ◽  
Initial Step ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Enrichment Cultures ◽  
Cluster Group ◽  
Degrading Bacterium ◽  
High Concentrations

ABSTRACT The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37�C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group ‘Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-Proteobacteria. Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group ‘Desulfotomaculum lineage I', but it was only distantly related to other known species.

scholarly journals Bacteria and sputum inflammatory cell counts; a COPD cohort analysis

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Augusta S. Beech ◽  
Simon Lea ◽  
Umme Kolsum ◽  
Zhang Wang ◽  
Bruce E. Miller ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Airway Inflammation ◽  
Bacterial Load ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Cell Counts ◽  
Copd Patients ◽  
Rrna Gene Sequencing

Abstract Background There is evidence that bacterial colonisation in chronic obstructive pulmonary disease (COPD) is associated with increased neutrophilic airway inflammation. This study tested the hypothesis that different bacterial phyla and species cause different inflammatory profiles in COPD patients. Methods Sputum was analysed by quantitative polymerase chain reaction (qPCR) to quantify bacterial load and 16S rRNA gene sequencing to identify taxonomic composition. Sputum differential cell counts (DCC) and blood DCC were obtained at baseline and 6 months. Patients were categorised into five groups based on bacterial load defined by genome copies/ml of ≥ 1 × 104, no colonisation and colonisation by Haemophilus influenzae (H. influenzae), Moraxella catarrhalis (M. catarrhalis), Streptococcus pneumoniae (S. pneumoniae), or > 1 potentially pathogenic microorganism (PPM). Results We observed an increase in sputum neutrophil (%), blood neutrophil (%) and neutrophil–lymphocyte ratio (NLR) in patients colonised with H. influenzae (82.6, 67.1, and 3.29 respectively) compared to those without PPM colonisation at baseline (69.5, 63.51 and 2.56 respectively) (p < 0.05 for all analyses), with similar findings at 6 months. The bacterial load of H. influenzae and Haemophilus determined by qPCR and 16s rRNA gene sequencing respectively, and sputum neutrophil % were positively correlated between baseline and 6 months visits (p < 0.0001, 0.0150 and 0.0002 with r = 0.53, 0.33 and 0.44 respectively). Conclusions These results demonstrate a subgroup of COPD patients with persistent H. influenzae colonisation that is associated with increased airway and systemic neutrophilic airway inflammation, and less eosinophilic airway inflammation.

Sign in / Sign up

Export Citation Format

Share Document