Sequencing 16S rRNA gene amplicons is the gold standard to uncover the composition of prokaryotic communities. The presence of multiple copies of this gene makes the community abundance data distorted and gene copy normalization (GCN) necessary for correction. Even though GCN of 16S data provided a picture closer to the metagenome before, it should also be compared with communities of known composition due to the fact that library preparation is prone to methodological biases. Here, we process 16S rRNA gene amplicon data from eleven simple mock communities with DADA2 and estimate the impact of GCN. In all cases, the mock community composition derived from the 16S sequencing differs from those expected, and GCN fails to improve the classification for most of the analysed communities. Our approach provides empirical evidence that GCN does not improve the 16S target sequencing analyses in real scenarios. We therefore question the use of GCN for metataxonomic surveys until a more comprehensive catalogue of copy numbers becomes available.

• Klíčová slova
• 16S rRNA, Gene, Metataxonomic surveys,
• MeSH
• genová dávka MeSH
• genová knihovna MeSH
• metagenom genetika   MeSH
• metagenomika normy   MeSH
• mikrobiota genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

The application of zero-valent iron particles (ZVI) for the treatment of heavily polluted environment and its biological effects have been studied for at least two decades. Still, information on the impact on bacterial metabolic pathways is lacking. This study describes the effect of microscale and nanoscale ZVI (mZVI and nZVI) on the abundance of different metabolic pathways in freshwater bacterial communities. The metabolic pathways were inferred from metabolism modelling based on 16S rRNA gene sequence data using paprica pipeline. The nZVI changed the abundance of numerous metabolic pathways compared to a less influencing mZVI. We identified the 50 most affected pathways, where 31 were related to degradation, 17 to biosynthesis, and 2 to detoxification. The linkage between pathways was two times higher in nZVI samples compared to mZVI, and was specifically higher considering the arsenate detoxification II pathway. Limnohabitans and Roseiflexus were linked to the same pathways in both nZVI and mZVI. The prediction of metabolic pathways increases our knowledge of the impacts of nZVI and mZVI on freshwater bacterioplankton.

• MeSH
• chemické látky znečišťující vodu * MeSH
• Chloroflexi * MeSH
• geny rRNA MeSH
• RNA ribozomální 16S genetika   MeSH
• sladká voda MeSH
• železo MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chemické látky znečišťující vodu * MeSH
• RNA ribozomální 16S MeSH
• železo MeSH

16S rRNA amplicon sequencing or, more recently, metatranscriptomic analysis are currently the only preferred methods for microbial profiling of samples containing a predominant ratio of human to bacterial DNA. However, due to the off-target amplification of human DNA, current protocols are inadequate for bioptic samples. Here we present an efficient, reliable, and affordable method for the bacteriome analysis of clinical samples human DNA content predominates. We determined the microbiota profile in a total of 40 human biopsies of the esophagus, stomach, and duodenum using 16S rRNA amplicon sequencing with the widely used 515F-806R (V4) primers targeting the V4 region, 68F-338R primers and a modified set of 68F-338R (V1-V2M) primers targeting the V1-V2 region. With the V4 primers, on average 70% of amplicon sequence variants (ASV) mapped to the human genome. On the other hand, this off-target amplification was absent when using the V1-V2M primers. Moreover, the V1-V2M primers provided significantly higher taxonomic richness and reproducibility of analysis compared to the V4 primers. We conclude that the V1-V2M 16S rRNA sequencing method is reliable, cost-effective, and applicable for low-bacterial abundant human samples in medical research.

• MeSH
• biopsie MeSH
• gastrointestinální trakt MeSH
• geny rRNA MeSH
• lidé MeSH
• mikrobiota * genetika   MeSH
• reprodukovatelnost výsledků MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA ribozomální 16S MeSH

BACKGROUND: Distribution and evolutionary history of resistance genes in environmental actinobacteria provide information on intensity of antibiosis and evolution of specific secondary metabolic pathways at a given site. To this day, actinobacteria producing biologically active compounds were isolated mostly from soil but only a limited range of soil environments were commonly sampled. Consequently, soil remains an unexplored environment in search for novel producers and related evolutionary questions. RESULTS: Ninety actinobacteria strains isolated at contrasting soil sites were characterized phylogenetically by 16S rRNA gene, for presence of erm and ABC transporter resistance genes and antibiotic production. An analogous analysis was performed in silico with 246 and 31 strains from Integrated Microbial Genomes (JGI_IMG) database selected by the presence of ABC transporter genes and erm genes, respectively. In the isolates, distances of erm gene sequences were significantly correlated to phylogenetic distances based on 16S rRNA genes, while ABC transporter gene distances were not. The phylogenetic distance of isolates was significantly correlated to soil pH and organic matter content of isolation sites. In the analysis of JGI_IMG datasets the correlation between phylogeny of resistance genes and the strain phylogeny based on 16S rRNA genes or five housekeeping genes was observed for both the erm genes and ABC transporter genes in both actinobacteria and streptomycetes. However, in the analysis of sequences from genomes where both resistance genes occurred together the correlation was observed for both ABC transporter and erm genes in actinobacteria but in streptomycetes only in the erm gene. CONCLUSIONS: The type of erm resistance gene sequences was influenced by linkage to 16S rRNA gene sequences and site characteristics. The phylogeny of ABC transporter gene was correlated to 16S rRNA genes mainly above the genus level. The results support the concept of new specific secondary metabolite scaffolds occurring more likely in taxonomically distant producers but suggest that the antibiotic selection of gene pools is also influenced by site conditions.

• MeSH
• ABC transportéry genetika   MeSH
• Actinobacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• antibakteriální látky biosyntéza   MeSH
• bakteriální léková rezistence * MeSH
• fylogeneze * MeSH
• geny rRNA MeSH
• methyltransferasy genetika   MeSH
• molekulární sekvence - údaje MeSH
• půdní mikrobiologie MeSH
• ribozomální DNA chemie   genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• shluková analýza MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportéry MeSH
• antibakteriální látky MeSH
• methyltransferasy MeSH
• ribozomální DNA MeSH
• RNA ribozomální 16S MeSH
• rRNA (adenosine-O-2'-)methyltransferase MeSH Prohlížeč

16S ribosomal RNA currently represents the most important target of study in bacterial ecology. Its use for the description of bacterial diversity is, however, limited by the presence of variable copy numbers in bacterial genomes and sequence variation within closely related taxa or within a genome. Here we use the information from sequenced bacterial genomes to explore the variability of 16S rRNA sequences and copy numbers at various taxonomic levels and apply it to estimate bacterial genome and DNA abundances. In total, 7,081 16S rRNA sequences were in silico extracted from 1,690 available bacterial genomes (1-15 per genome). While there are several phyla containing low 16S rRNA copy numbers, in certain taxa, e.g., the Firmicutes and Gammaproteobacteria, the variation is large. Genome sizes are more conserved at all tested taxonomic levels than 16S rRNA copy numbers. Only a minority of bacterial genomes harbors identical 16S rRNA gene copies, and sequence diversity increases with increasing copy numbers. While certain taxa harbor dissimilar 16S rRNA genes, others contain sequences common to multiple species. Sequence identity clusters (often termed operational taxonomic units) thus provide an imperfect representation of bacterial taxa of a certain phylogenetic rank. We have demonstrated that the information on 16S rRNA copy numbers and genome sizes of genome-sequenced bacteria may be used as an estimate for the closest related taxon in an environmental dataset to calculate alternative estimates of the relative abundance of individual bacterial taxa in environmental samples. Using an example from forest soil, this procedure would increase the abundance estimates of Acidobacteria and decrease these of Firmicutes. Using the currently available information, alternative estimates of bacterial community composition may be obtained in this way if the variation of 16S rRNA copy numbers among bacteria is considered.

• MeSH
• délka genomu genetika   MeSH
• DNA bakterií genetika   MeSH
• ekosystém * MeSH
• fylogeneze MeSH
• genetická variace * MeSH
• genom bakteriální genetika   MeSH
• genová dávka genetika   MeSH
• počítačová simulace MeSH
• půdní mikrobiologie MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• stromy mikrobiologie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

Nowadays, dental diseases are one of the most common illnesses in the world. Some of them can lead to translocation of oral bacteria to the bloodstream causing intermittent bacteraemia. Therefore, a potential association between oral infection and cardiovascular diseases has been discussed in recent years as a result of adhesion of oral microbes to the heart valves. The aim of this study was to detect oral bacteria on pathologically changed heart valves not caused by infective endocarditis. In the study, patients with pathologically changed heart valves were involved. Samples of heart valves removed during heart valve replacement surgery were cut into two parts. One aliquot was cultivated aerobically and anaerobically. Bacterial DNA was extracted using Ultra-Deep Microbiome Prep (Molzym GmbH, Bremen, Germany) followed by a 16S rRNA gene PCR amplification using Mastermix 16S Complete kit (Molzym GmbH, Bremen, Germany). Positive PCR products were sequenced and the sequences were analyzed using BLAST database ( http://www.ncbi.nlm.nih/BLAST ). During the study period, 41 samples were processed. Bacterial DNA of the following bacteria was detected in 21 samples: Cutibacterium acnes (formerly Propionibacterium acnes) (n = 11; 52.38% of patients with positive bacterial DNA detection), Staphylococcus sp. (n = 9; 42.86%), Streptococcus sp. (n = 1; 4.76%), Streptococcus sanguinis (n = 4; 19.05%), Streptococcus oralis (n = 1; 4.76%), Carnobacterium sp. (n = 1; 4.76%), Bacillus sp. (n = 2; 9.52%), and Bergeyella sp. (n = 1; 4.76%). In nine samples, multiple bacteria were found. Our results showed significant appearance of bacteria on pathologically changed heart valves in patients with no symptoms of infective endocarditis.

• MeSH
• amplifikace genu * MeSH
• Bacteria klasifikace   genetika   MeSH
• bakteriální endokarditida mikrobiologie   mortalita   patologie   terapie   MeSH
• chirurgická náhrada chlopně metody   MeSH
• DNA bakterií * MeSH
• lidé středního věku MeSH
• lidé MeSH
• polymerázová řetězová reakce MeSH
• RNA ribozomální 16S * MeSH
• sekvenční analýza DNA MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• srdeční chlopně mikrobiologie   patologie   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií * MeSH
• RNA ribozomální 16S * MeSH

Transplantation experiments and genome comparisons were used to determine if lineages of planktonic Polynucleobacter almost indistinguishable by their 16S ribosomal RNA (rRNA) sequences differ distinctively in their ecophysiological and genomic traits. The results of three transplantation experiments differing in complexity of biotic interactions revealed complete ecological isolation between some of the lineages. This pattern fits well to the previously detected environmental distribution of lineages along chemical gradients, as well as to differences in gene content putatively providing adaptation to chemically distinct habitats. Patterns of distribution of iron transporter genes across 209 Polynucleobacter strains obtained from freshwater systems and representing a broad pH spectrum further emphasize differences in habitat-specific adaptations. Genome comparisons of six strains sharing ⩾99% 16S rRNA similarities suggested that each strain represents a distinct species. Comparison of sequence diversity among genomes with sequence diversity among 240 cultivated Polynucleobacter strains indicated a large cryptic species complex not resolvable by 16S rRNA sequences. The revealed ecological isolation and cryptic diversity in Polynucleobacter bacteria is crucial in the interpretation of diversity studies on freshwater bacterioplankton based on ribosomal sequences.

• MeSH
• Burkholderiaceae genetika   izolace a purifikace   MeSH
• DNA bakterií genetika   MeSH
• ekologie MeSH
• ekosystém MeSH
• fylogeneze MeSH
• genomika * MeSH
• plankton genetika   izolace a purifikace   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA MeSH
• sladká voda mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

Electronic waste is an evolving source of harmful pollutants in our surrounding environments and considered to be perilous as it contains toxic metals such as chromium, cadmium, lead, mercury, zinc, and nickel in huge quantities. Heavy metals are harmful contaminants and accumulated in the environment due to various anthropogenic activities. The present study was conducted to isolate and characterize different heavy metal tolerant bacterial species, based on molecular techniques from soil contaminated by electronic waste. The contaminated soil samples were analyzed for various physicochemical properties such as pH, electrical conductivity, soil moisture, water holding capacity, organic carbon, organic matter, available phosphorus, total nitrogen, and potassium using standard procedures. The soil samples were found to contain a higher amount of different heavy metals such as copper, chromium, lead, iron, cadmium, and nickel. Serial dilution and spread plate techniques have been used for bacterial isolation. The identification and molecular characterization of isolated bacterial species were done by biochemical tests and 16S rRNA gene sequencing technique. The 16S rRNA sequencing analysis confirmed the presence of different bacterial species as, Micrococcus aloeverae, Kocuria turfanensis, Bacillus licheniformis, Bacillus jeotgali, Bacillus velezensis, and Bacillus haikouensis. The findings indicated that the e-waste dumping sites are the storehouse of elite bacterial species. The present research study offers a platform for systematic analysis of e-waste sites by microbial profiling that may help in the innovation of novel microorganisms of scientific importance and better biotechnological potential.

• Klíčová slova
• 16S rRNA gene sequencing, Electronic waste, Heavy metals, Microbial diversity, Phylogenetic tree analysis,
• MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• DNA bakterií MeSH
• elektronický odpad analýza   MeSH
• genom bakteriální MeSH
• látky znečišťující půdu analýza   MeSH
• monitorování životního prostředí MeSH
• půda chemie   MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• těžké kovy toxicita   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA bakterií MeSH
• látky znečišťující půdu MeSH
• půda MeSH
• RNA ribozomální 16S MeSH
• těžké kovy MeSH

The objectives of this study were (i) to isolate and characterize of cultivable denitrifying bacteria using classic microbiological and molecular methods, (ii) to compare of 16S rRNA and nosZ genes as molecular markers, (iii) to determine bacterial community structure and diversity in soil samples using single-strand conformation polymorphism (SSCP) analysis. In this study, 49 bacterial isolates were cultivated and phylogenetic analyses grouped them into two phyla: Proteobacteria (37 species) and Firmicutes (12 species). Our study showed that the nosZ functional gen could be used to identify denitrifying bacteria abundance in environment but could not be used to identify pure bacterial cultures. In addition, the bacterial community structure showed significant differences among the various soil types. Phylogenetic analysis of community structure indicated that 51 clones could be divided into 2 phylotypes. Uncultured bacteria (80.4%) and Gammaproteobacteria (19.6%) were the dominant components of the soil bacterial community. For 16S rRNA, PCR products of 49 bacteria were obtained with 27F-1492R primer pairs. For nosZ, PCR products were obtained with primers 1F-1R (259 bp), 2F-2R (267 bp), and F-1622R (453 bp) of 39 bacteria that the single nosZ band provided on the agarose gel. The bacterial 16S rRNA gene clone library was dominated by Gammaproteobacteria and Bacilli. The nosZ clone sequences did not represent the bacteria from which they were obtained but were found to be closer to the environmental clones. Our study showed that the nosZ functional gene could be used to identify denitrification abundance in environment but could not be used to identify pure bacterial cultures. It was also found that the nosZ sequences showed uncultured denitrifier species.

• MeSH
• Bacillus klasifikace   izolace a purifikace   MeSH
• Bacteria klasifikace   izolace a purifikace   MeSH
• bakteriální geny MeSH
• denitrifikace * MeSH
• DNA bakterií genetika   MeSH
• fylogeneze MeSH
• Gammaproteobacteria klasifikace   izolace a purifikace   MeSH
• mikrobiota * MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• půdní mikrobiologie * MeSH
• RNA ribozomální 16S genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA bakterií MeSH
• RNA ribozomální 16S MeSH

As an important source of human food, milk can be a carrier of human pathogenic bacteria, including tuberculous and nontuberculous mycobacteria (NTM), in its raw and unpasteurized state. In this research, 175 raw milk samples and 175 traditional cheese samples were collected from traditional dairy stores in 22 regions of Tehran in a 9- month period from August 2019 to May 2020. Samples were prepared and transferred to a specialized laboratory, where they were inoculated in Lowenstein-Jensen (LJ) medium containing glycerol or sodium pyruvate, as well as Herrold's egg-yolk with and without Mycobactin J. to determine the sample's identity of samples. The recommended 16S rRNA (1436 bp) and hsp65 (644 bp) gene fragments from the positive isolates identified in Ziehl-Neelsen (Z-N) staining were amplified and sequenced using PCR and compared with the sequences of the gene fragments of reference strains available in the global GenBank database. No mycobacterial species were isolated from traditional cheese samples in microbial culture. In case of raw milk samples, a total of four bacteria were collected, all of which were found in the genetic differential testing to be NTM, including n = 1 Mycobacterium heraklionense, n = 2 Mycolicibacterium fortuitum, and n = 1 Mycobacterium thermoresistibile. The analysis of the results obtained by isolate sequencing using the 16S rRNA gene showed higher discriminatory power and percentage similarities in the identification of the isolates than the hsp65 gene.

• Klíčová slova
• 16S rRNA gene, Nontuberculous mycobacteria isolates, Traditional dairy milk and cheese, hsp65 gene,
• MeSH
• atypické mykobakteriální infekce * mikrobiologie   MeSH
• lidé MeSH
• mléko mikrobiologie   MeSH
• netuberkulózní mykobakterie genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sýr * mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Írán MeSH
• Názvy látek
• RNA ribozomální 16S MeSH