Úvod a cíl: Apikální periodontitida (AP) je zánětlivé onemocnění zubních periradikulárních tkání vyvolané bakteriální infekcí. Ke stanovení bakterií osídlujících kořenové kanálky jsou využívány různé metodické přístupy, analýza bakteriomu celého kořenového systému zubu postiženého AP je však zatím stále výzvou. Cílem naší přehledové práce bylo vytvořit literární rešerši zaměřenou na postupy pro odběr vzorku a metodiku pro analýzu bakteriomu a poté navrhnout vhodný metodický přístup k účelu studia etiopatogeneze tohoto onemocnění. Metodika: Po vyhledávání v databázi PubMed jsme do rešerše vybrali pouze publikační výstupy typu původní práce, ve kterých byla analyzována bakteriální DNA lidských zubů. Výsledky: Metodicky se studie mezi sebou velmi liší, a to jak způsobem odběru vzorku, izolací DNA, tak i samotnou analýzou bakteriální DNA. Častým způsobem odběru vzorku je využití sterilních endodontických papírových čepů. Tento způsob odběru vzorku je sice vhodný v rámci klinické praxe, avšak pro komplexní analýzu prostředí kořenového systému je považován za nedostatečný, a to kvůli samotné morfologii zubu a přítomnosti ramifikací. Jiným způsobem odběru vzorku je resekce kořenového hrotu pomocí sterilních fréz a následné rozemletí apexu nebo provedení stěru sterilními endodontickými papírovými čepy. Ke stanovení bakteriomu je tak využívána pouze apikální část zubu, tudíž bakterie, které kolonizují koronární část zubu a podílejí se na etiopatogenezi onemocnění, nemohou být analyzovány. V recentních studiích je využívána metoda, při které je celý extrahovaný zub postižený AP nadrcen na jemný homogenní prach pomocí kryogenního mletí. Z prachu nadrceného zubu je možné stanovit komplexní bakteriom kořenového systému i dřeňové dutiny, a proto se tento způsob z pohledu přípravy vzorku pro experimentální studii jeví jako optimální. Nejčastěji je při izolaci DNA využívána efektivní kolonková metoda různými purifikačními soupravami a k následné analýze DNA slouží většinou metodiky založené na principu polymerázové řetězové reakce. Sekvenování variabilních oblastí genu pro 16S rRNA je v dnešní době již zlatým standardem pro kategorizaci bakterií a charakterizaci bakteriálních komunit. Závěr: Ke studiu bakteriomu AP se jeví jako nejvhodnější použít vzorky extrahovaných zubů a bezprostředně je zamrazit bez dalších preanalytických kroků. Nadrcený zub je při dodržení sterilních podmínek při kryogenním mletí vhodnou matricí pro izolaci mikrobiální DNA komerčně dostupnými kity. V současné době je pro stanovení bakteriomu a získání informace o relativní abundanci bakteriálních rodů, a to z analytického i ekonomického hlediska, sekvenování nové generace nejlepší volbou.

Introduction, aim: Apical periodontitis (AP) is an inflammatory disease of the dental periradicular tissues caused by a bacterial infection. Various methodological approaches are used to determine the bacteria inhabiting the root canals, however, the analysis of the entire root system of a tooth affected by AP still remains a challenge. The aim of our study was to perform a literature search focused on sample collection procedures and methodologies for bacteriome analysis, and then propose a suitable methodological approach for the purpose of studying the etiopathogenesis of this disease. Methods: After searching the PubMed database, we selected only publications of the original work type in which the bacterial DNA of human teeth was analyzed, for the search. Results: Methodologically, the studies differ greatly, in terms of sample collection, DNA isolation, and bacterial DNA analysis itself. A common method of sample collection is the use of sterile endodontic paper points. Although this method of sampling is suitable in clinical practice, it is considered insufficient for a comprehensive analysis of the environment of the root canal system, due to the morphology of the tooth itself and the presence of ramifications. Another method of sampling is resection of the root tip using sterile burs and subsequent grinding of the apex or smearing with sterile endodontic paper pins. Only the apical part of the tooth is used to determine the bacteriome, therefore bacteria that colonize the coronal part of the tooth and participate in the etiopathogenesis of the disease cannot be analyzed. In recent studies, a method is used in which the entire extracted tooth affected by AP is ground into a fine homogeneous powder using cryogenic grinding. It is possible to determine the complex bacteriome of the root canal system and the pulp chamber from the dust of a crushed tooth, and therefore this method seems optimal for the sample preparation from an experimental study point of view. Most often, an effective column method with various purification kits is used for DNA isolation, and for subsequent DNA analysis, methodologies based on the principle of the polymerase chain reaction are mostly used. Sequencing the variable regions of the gene for 16S rRNA is nowadays already the gold standard for categorizing bacteria and characterizing bacterial communities. Conclusion: To study the AP bacteriome, it seems most appropriate to use extracted tooth samples and immediate freezing of the sample without further pre-analytical steps. The crushed tooth is a suitable matrix for the isolation of microbial DNA with commercially available kits, provided that sterile conditions are maintained during cryogenic grinding. Currently, next-generation sequencing is the best choice for determining the bacteriome and obtaining information about the relative abundance of bacterial genera, both analytically and economically.

• Klíčová slova
• kryomlýnek,
• MeSH
• DNA izolace a purifikace   MeSH
• kavita zubní dřeně mikrobiologie   patologie   MeSH
• lidé MeSH
• periapikální periodontitida * mikrobiologie   patologie   terapie   MeSH
• sekvenční analýza DNA MeSH
• terapie kořenového kanálku MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH
• přehledy MeSH

OBJECTIVES: Gut bacteriome profiling studies in type 1 diabetes (T1D) to date are mostly limited to populations of Europe, with two studies from China and one study each from Mexico and the USA. We therefore sought to characterize the stool bacteriome in children after onset of T1D along with age- and place-matched control subjects from four geographically distant African and Asian countries. METHODS: Samples were collected from 73 children and adolescents shortly after T1D onset (Azerbaijan 19, Jordan 20, Nigeria 14, Sudan 20) and 104 matched control subjects of similar age and locale. Genotyping of major T1D susceptibility genes was performed using saliva or blood samples. The bacteriome was profiled by next-generation sequencing of 16S rDNA. Negative binomial regression was used to model associations, with adjustment for the matched structure of the study. RESULTS: A significant positive association with T1D was noted for the genus Escherichia (class Gammaproteobacteria, phylum Proteobacteria), whereas Eubacterium and Roseburia, two genera of class Clostridia, phylum Firmicutes, were inversely associated with T1D. We also confirmed a previously observed inverse association with Clostridium clusters IV or XIVa. No associations were noted for richness, evenness, or enterotypes. CONCLUSIONS: Based on our results, some type of distortion of the gut bacteriome appears to be a global feature of T1D, and our findings for four distant populations add new candidates to the existing list of bacteria. It remains to be established whether the observed associations are markers or causative factors.

• MeSH
• Bacteria genetika   MeSH
• bakteriální RNA genetika   MeSH
• diabetes mellitus 1. typu epidemiologie   genetika   mikrobiologie   MeSH
• dítě MeSH
• dospělí MeSH
• feces mikrobiologie   MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• předškolní dítě MeSH
• RNA ribozomální 16S genetika   MeSH
• střevní mikroflóra genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Afrika MeSH
• Asie MeSH

AIMS/HYPOTHESIS: We previously detected indications that beta cell function is protected by gluten-free diet (GFD) introduced shortly after the onset of childhood type 1 diabetes. The present aim was to assess whether GFD was associated with changes in the gut bacteriome composition and in its functional capacity, and whether such changes mediated the observed effects of GFD on beta cell function. METHODS: Forty-five children (aged 10.2 ± 3.3 years) were recruited into a self-selected intervention trial primarily focused on determining the role of GFD on beta cell preservation ( ClinicalTrials.gov NCT02867436). Stool samples were collected prior to the dietary intervention and then at 3-month intervals. A total of 128 samples from the GFD group and 112 from the control group were analysed for bacteriome 16S rDNA community profiles, the bacteriome functional capacity was predicted using PICRUSt2 and actual gut metabolome profiles measured using NMR. Intestinal permeability was assessed using serum zonulin concentrations at 1, 6 and 12 months and lactulose/mannitol tests at the end of intervention. Dietary questionnaires were used to ensure that the dietary intervention did not result in differences in energy or nutrient intake. RESULTS: The bacteriome community composition changed during the intervention with GFD: of abundant genera, a 3.3-fold decrease was noted for Bifidobacterium genus (adjusted p=1.4 × 10-4 in a DESeq2 model, p=0.026 in generalised estimating equations model), whereas a 2.4-fold increase was observed in Roseburia (adjusted p=0.02 in DESeq2 model, p=0.002 in generalised estimating equations model). The within-sample (alpha) diversity did not change, and there was no statistically significant clustering of GFD samples in the ordination graphs of beta diversity. Neither of the genera changes upon GFD intervention showed any association with the pace of beta cell loss (p>0.50), but of the remaining taxa, several genera of Bacteroidaceae family yielded suggestive signals. The faecal metabolome profile ordination correlated with that of bacteriomes but did not associate with GFD or categories of beta cell preservation. There was no indication of changes in gut permeability. CONCLUSIONS/INTERPRETATION: The bacteriome reacted to GFD, but the changes were unrelated to the pace of beta cell capacity loss. The previously observed moderately protective effect of GFD is therefore mediated through other pathways.

• MeSH
• bezlepková dieta * MeSH
• dítě MeSH
• lidé MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The lung in cystic fibrosis (CF) is home to numerous pathogens that shorten the lives of patients. The aim of the present study was to assess changes in the lung bacteriome following antibiotic therapy targeting Pseudomonas aeruginosa in children with CF. The study included nine children (9-18 years) with CF who were treated for their chronic or intermittent positivity for Pseudomonas aeruginosa. The bacteriomes were determined in 16 pairs of sputa collected at the beginning and at the end of a course of intravenous antibiotic therapy via deep sequencing of the variable region 4 of the 16S rRNA gene, and the total bacterial load and selected specific pathogens were assessed using quantitative real-time PCR. The effect of antipseudomonal antibiotics was observable as a profound decrease in the total 16S rDNA load (p = 0.001) as well as in a broad range of individual taxa including Staphylococcus aureus (p = 0.03) and several members of the Streptococcus mitis group (S. oralis, S. mitis, and S. infantis) (p = 0.003). Improvements in forced expiratory volume (FEV1) were associated with an increase in Granulicatella sp. (p = 0.004), whereas a negative association was noted between the total bacterial load and white blood cell count (p = 0.007). In conclusion, the data show how microbial communities differ in reaction to antipseudomonal treatment, suggesting that certain rare species may be associated with clinical parameters. Our work also demonstrates the utility of absolute quantification of bacterial load in addition to the 16S rDNA profiling.

• MeSH
• antibakteriální látky aplikace a dávkování   MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• cystická fibróza farmakoterapie   mikrobiologie   MeSH
• dítě MeSH
• DNA bakterií genetika   MeSH
• lidé MeSH
• mikrobiota účinky léků   MeSH
• mladiství MeSH
• plíce mikrobiologie   MeSH
• pseudomonádové infekce farmakoterapie   mikrobiologie   MeSH
• Pseudomonas aeruginosa účinky léků   genetika   izolace a purifikace   fyziologie   MeSH
• RNA ribozomální 16S genetika   MeSH
• sputum mikrobiologie   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Indoor dust particles are an everyday source of human exposure to microorganisms and their inhalation may directly affect the microbiota of the respiratory tract. We aimed to characterize the changes in human nasopharyngeal bacteriome after short-term exposure to indoor (workplace) environments. METHODS: In this pilot study, nasopharyngeal swabs were taken from 22 participants in the morning and after 8 h of their presence at the workplace. At the same time points, indoor dust samples were collected from the participants' households (16 from flats and 6 from houses) and workplaces (8 from a maternity hospital - NEO, 6 from a pediatric hospital - ENT, and 8 from a research center - RCX). 16S rRNA sequencing analysis was performed on these human and environmental matrices. RESULTS: Staphylococcus and Corynebacterium were the most abundant genera in both indoor dust and nasopharyngeal samples. The analysis indicated lower bacterial diversity in indoor dust samples from flats compared to houses, NEO, ENT, and RCX (p < 0.05). Participants working in the NEO had the highest nasopharyngeal bacterial diversity of all groups (p < 0.05). After 8 h of exposure to the workplace environment, enrichment of the nasopharynx with several new bacterial genera present in the indoor dust was observed in 76% of study participants; however, no significant changes were observed at the level of the nasopharyngeal bacterial diversity (p > 0.05, Shannon index). These "enriching" bacterial genera overlapped between the hospital workplaces - NEO and ENT but differed from those in the research center - RCX. CONCLUSIONS: The results suggest that although the composition of nasopharyngeal bacteriome is relatively stable during the day. Short-term exposure to the indoor environment can result in the enrichment of the nasopharynx with bacterial DNA from indoor dust; the bacterial composition, however, varies by the indoor workplace environment.

• MeSH
• Bacteria genetika   MeSH
• dítě MeSH
• lidé MeSH
• nazofarynx MeSH
• pilotní projekty MeSH
• prach * analýza   MeSH
• RNA ribozomální 16S genetika   analýza   MeSH
• těhotenství MeSH
• znečištění vzduchu ve vnitřním prostředí * analýza   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Blastocystis is a human gut symbiont of yet undefined clinical significance. In a set of faecal samples collected from asymptomatic children of six distant populations, we first assessed the community profiles of protist 18S rDNA and then characterized Blastocystis subtypes and tested Blastocystis association with the faecal bacteriome community. METHODS: Stool samples were collected from 244 children and young persons (mean age 11.3 years, interquartile range 8.1-13.7) of six countries (Azerbaijan 51 subjects, Czechia 52, Jordan 40, Nigeria 27, Sudan 59 and Tanzania 15). The subjects showed no symptoms of infection. Amplicon profiling of the 18S rDNA was used for verification that Blastocystis was the most frequent protist, whereas specific real-time PCR showed its prevalence and quantity, and massive parallel amplicon sequencing defined the Blastocystis subtypes. The relation between Blastocystis and the stool bacteriome community was characterized using 16S rDNA profiling. RESULTS: Blastocystis was detected by specific PCR in 36% (88/244) stool samples and was the most often observed faecal protist. Children from Czechia and Jordan had significantly lower prevalence than children from the remaining countries. The most frequent subtype was ST3 (49%, 40/81 sequenced samples), followed by ST1 (36%) and ST2 (25%). Co-infection with two different subtypes was noted in 12% samples. The faecal bacteriome had higher richness in Blastocystis-positive samples, and Blastocystis was associated with significantly different community composition regardless of the country (p < 0.001 in constrained redundancy analysis). Several taxa differed with Blastocystis positivity or quantity: two genera of Ruminococcaceae were more abundant, while Bifidobacterium, Veillonella, Lactobacillus and several other genera were undrerrepresented. CONCLUSIONS: Asymptomatic children frequently carry Blastocystis, and co-infection with multiple distinct subtypes is not exceptional. Prevalence and quantity of the organism clearly differ among populations. Blastocystis is linked to both faecal bacteriome diversity and its composition.

• MeSH
• asymptomatické infekce epidemiologie   MeSH
• Blastocystis klasifikace   genetika   izolace a purifikace   MeSH
• blastocystóza epidemiologie   parazitologie   MeSH
• dítě MeSH
• feces parazitologie   MeSH
• genetická variace MeSH
• lidé MeSH
• mladiství MeSH
• prevalence MeSH
• protozoální DNA genetika   MeSH
• ribozomální DNA genetika   MeSH
• střevní mikroflóra genetika   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Ázerbájdžán MeSH
• Československo MeSH
• Jordánsko MeSH
• Nigérie MeSH
• Súdán MeSH
• Tanzanie MeSH

BACKGROUND: We set out to explore associations between the stool bacteriome profiles and early-onset islet autoimmunity, taking into account the interactions with the virus component of the microbiome. METHODS: Serial stool samples were longitudinally collected from 18 infants and toddlers with early-onset islet autoimmunity (median age 17.4 months) followed by type 1 diabetes, and 18 tightly matched controls from the Finnish Diabetes Prediction and Prevention (DIPP) cohort. Three stool samples were analyzed, taken 3, 6, and 9 months before the first detection of serum autoantibodies in the case child. The risk of islet autoimmunity was evaluated in relation to the composition of the bacteriome 16S rDNA profiles assessed by mass sequencing, and to the composition of DNA and RNA viromes. RESULTS: Four operational taxonomic units were significantly less abundant in children who later on developed islet autoimmunity as compared to controls-most markedly the species of Bacteroides vulgatus and Bifidobacterium bifidum. The alpha or beta diversity, or the taxonomic levels of bacterial phyla, classes or genera, showed no differences between cases and controls. A correlation analysis suggested a possible relation between CrAssphage signals and quantities of Bacteroides dorei. No apparent associations were seen between development of islet autoimmunity and sequences of yet unknown origin. CONCLUSIONS: The results confirm previous findings that an imbalance within the prevalent Bacteroides genus is associated with islet autoimmunity. The detected quantitative relation of the novel "orphan" bacteriophage CrAssphage with a prevalent species of the Bacteroides genus may exemplify possible modifiers of the bacteriome.

• MeSH
• autoimunita * MeSH
• autoimunitní nemoci krev   epidemiologie   etiologie   imunologie   MeSH
• Bacteroides klasifikace   imunologie   izolace a purifikace   virologie   MeSH
• bakteriální RNA chemie   metabolismus   MeSH
• bakteriofágy klasifikace   imunologie   izolace a purifikace   MeSH
• diabetes mellitus 1. typu krev   epidemiologie   etiologie   imunologie   MeSH
• dítě MeSH
• dysbióza imunologie   mikrobiologie   patofyziologie   virologie   MeSH
• feces mikrobiologie   virologie   MeSH
• fylogeneze MeSH
• kohortové studie MeSH
• Langerhansovy ostrůvky imunologie   MeSH
• lidé MeSH
• longitudinální studie MeSH
• molekulární typizace MeSH
• nemocnice univerzitní MeSH
• prospektivní studie MeSH
• riziko MeSH
• RNA ribozomální 16S chemie   metabolismus   MeSH
• RNA virová chemie   metabolismus   MeSH
• střevní mikroflóra imunologie   MeSH
• studie případů a kontrol MeSH
• výpočetní biologie MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• multicentrická studie MeSH
• Geografické názvy
• Finsko epidemiologie   MeSH

• Publikační typ
• tisková chyba MeSH

BACKGROUND: The vertebrate gastrointestinal tract is colonised by microbiota that have a major effect on the host's health, physiology and phenotype. Once introduced into captivity, however, the gut microbial composition of free-living individuals can change dramatically. At present, little is known about gut microbial changes associated with adaptation to a synanthropic lifestyle in commensal species, compared with their non-commensal counterparts. Here, we compare the taxonomic composition and diversity of bacterial and fungal communities across three gut sections in synanthropic house mouse (Mus musculus) and a closely related non-synanthropic mound-building mouse (Mus spicilegus). RESULTS: Using Illumina sequencing of bacterial 16S rRNA amplicons, we found higher bacterial diversity in M. spicilegus and detected 11 bacterial operational taxonomic units with significantly different proportions. Notably, abundance of Oscillospira, which is typically higher in lean or outdoor pasturing animals, was more abundant in non-commensal M. spicilegus. ITS2-based barcoding revealed low diversity and high uniformity of gut fungi in both species, with the genus Kazachstania clearly dominant. CONCLUSIONS: Though differences in gut bacteria observed in the two species can be associated with their close association with humans, changes due to a move from commensalism to captivity would appear to have caused larger shifts in microbiota.

• MeSH
• Bacteria klasifikace   genetika   izolace a purifikace   MeSH
• ekologie MeSH
• feces mikrobiologie   MeSH
• fylogeneze MeSH
• houby klasifikace   genetika   izolace a purifikace   MeSH
• mikrobiota MeSH
• mykobiom MeSH
• myši MeSH
• ribozomální DNA genetika   MeSH
• RNA ribozomální 16S genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

The honey bee, Apis mellifera, is a globally important species that suffers from a variety of pathogens and parasites. These parasites and pathogens may have sublethal effects on their bee hosts via an array of mechanisms, including through a change in symbiotic bacterial taxa. Our aim was to assess the influence of four globally widespread parasites and pathogens on the honey bee bacteriome. We examined the effects of the ectoparasitic mite Varroa destructor, the fungal pathogens Nosema apis and Nosema ceranae, and the trypanosome Lotmaria passim. Varroa was detected by acaricidal treatment, Nosema and L. passim by PCR, and the bacteriome using MiSeq 16S rRNA gene sequencing. Overall, the 1,858,850 obtained sequences formed 86 operational taxonomic units (OTUs) at 3 % dissimilarity. Location, time of year, and degree of infestation by Varroa had significant effects on the composition of the bacteriome of honey bee workers. Based on statistical correlations, we found varroosis more important factor than N. ceranae, N. apis, and L. passim infestation influencing the honey bee bacteriome and contributing to the changes in the composition of the bacterial community in adult bees. At the population level, Varroa appeared to modify 20 OTUs. In the colonies with high Varroa infestation levels (varroosis), the relative abundance of the bacteria Bartonella apis and Lactobacillus apis decreased. In contrast, an increase in relative abundance was observed for several taxa including Lactobacillus helsingborgensis, Lactobacillus mellis, Commensalibacter intestini, and Snodgrassella alvi. The results showed that the "normal" bacterial community is altered by eukaryotic parasites as well as displaying temporal changes and changes associated with the geographical origin of the beehive.

• MeSH
• Bartonella klasifikace   genetika   izolace a purifikace   MeSH
• infestace roztoči patologie   MeSH
• Kinetoplastida patogenita   MeSH
• Lactobacillus klasifikace   genetika   izolace a purifikace   MeSH
• mikrobiota genetika   MeSH
• Nosema patogenita   MeSH
• RNA ribozomální 16S genetika   MeSH
• symbióza MeSH
• Varroidae patogenita   MeSH
• včely mikrobiologie   parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH