Enrichment of human nasopharyngeal bacteriome with bacteria from dust after short-term exposure to indoor environment: a pilot study.

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.

The Human Mycobiome: Colonization, Composition and the Role in Health and Disease.

The mycobiome is the fungal component of the human microbial ecosystem that represents only a small part of this environment but plays an essential role in maintaining homeostasis. Colonization by fungi begins immediately after birth. The initial mycobiome is influenced by the gestational age of a newborn, birth weight, delivery method and feeding method. During a human's life, the composition of the mycobiome is further influenced by a large number of endogenous and exogenous factors. The most important factors are diet, body weight, age, sex and antibiotic and antifungal therapy. The human mycobiome inhabits the oral cavity, gastrointestinal tract, respiratory tract, urogenital tract and skin. Its composition can influence the gut-brain axis through immune and non-immune mediated crosstalk systems. It also interacts with other commensals of the ecosystem through synergistic and antagonistic relationships. Moreover, colonization of the gut by opportunistic fungal pathogens in immunocompromised individuals can lead to clinically relevant disease states. Thus, the mycobiome represents an essential part of the microbiome associated with a variety of physiological and pathological processes. This review summarizes the current knowledge on the composition of the mycobiome in specific sites of the human body and its role in health and disease.

Colorectal Tumour Mucosa Microbiome Is Enriched in Oral Pathogens and Defines Three Subtypes That Correlate with Markers of Tumour Progression.

Long-term dysbiosis of the gut microbiome has a significant impact on colorectal cancer (CRC) progression and explains part of the observed heterogeneity of the disease. Even though the shifts in gut microbiome in the normal-adenoma-carcinoma sequence were described, the landscape of the microbiome within CRC and its associations with clinical variables remain under-explored. We performed 16S rRNA gene sequencing of paired tumour tissue, adjacent visually normal mucosa and stool swabs of 178 patients with stage 0-IV CRC to describe the tumour microbiome and its association with clinical variables. We identified new genera associated either with CRC tumour mucosa or CRC in general. The tumour mucosa was dominated by genera belonging to oral pathogens. Based on the tumour microbiome, we stratified CRC patients into three subtypes, significantly associated with prognostic factors such as tumour grade, sidedness and TNM staging, BRAF mutation and MSI status. We found that the CRC microbiome is strongly correlated with the grade, location and stage, but these associations are dependent on the microbial environment. Our study opens new research avenues in the microbiome CRC biomarker detection of disease progression while identifying its limitations, suggesting the need for combining several sampling sites (e.g., stool and tumour swabs).

Determination of Butyrate Synthesis Capacity in Gut Microbiota: Quantification of but Gene Abundance by qPCR in Fecal Samples.

Butyrate is formed in the gut during bacterial fermentation of dietary fiber and is attributed numerous beneficial effects on the host metabolism. We aimed to develop a method for the assessment of functional capacity of gut microbiota butyrate synthesis based on the qPCR quantification of bacterial gene coding butyryl-CoA:acetate CoA-transferase, the key enzyme of butyrate synthesis. In silico, we identified bacteria possessing but gene among human gut microbiota by searching but coding sequences in available databases. We designed and validated six sets of degenerate primers covering all selected bacteria, based on their phylogenetic nearness and sequence similarity, and developed a method for gene abundance normalization in human fecal DNA. We determined but gene abundance in fecal DNA of subjects with opposing dietary patterns and metabolic phenotypes-lean vegans (VG) and healthy obese omnivores (OB) with known fecal microbiota and metabolome composition. We found higher but gene copy number in VG compared with OB, in line with higher fecal butyrate content in VG group. We further found a positive correlation between the relative abundance of target bacterial genera identified by next-generation sequencing and groups of but gene-containing bacteria determined by specific primers. In conclusion, this approach represents a simple and feasible tool for estimation of microbial functional capacity.

Actinotignum schaalii: Relation to Concomitants and Connection to Patients' Conditions in Polymicrobial Biofilms of Urinary Tract Catheters and Urines.

Actinotignum schaalii is an emerging, opportunistic pathogen and its connection to non-infectious diseases and conditions, such as prostate or bladder cancer, or chronic inflammation has been proposed. Here, we analyzed 297 urine, ureteral and urinary catheter samples from 128 patients by Polymerase Chain Reaction followed by Denaturing Gradient Gel Electrophoresis and Sequencing (PCR-DGGE-S), and culture, and 29 of these samples also by 16S rRNA Illumina sequencing, to establish A. schaalii's prevalence in urinary tract-related samples, its relation to other bacteria, and its potential association with patients' conditions and samples' characteristics. A. schaalii-positive samples were significantly more diverse than A. schaalii negative and between-group diversity was higher than intra-group. Propionimicrobium lymphophilum, Fusobacterium nucleatum, Veillonella sp., Morganella sp., and Aerococcus sp. were significantly more often present in A. schaalii-positive samples; thus, we suggest these species are A. schaalii's concomitants, while Enterobacter and Staphylococcaceae were more often identified in A. schaalii-negative samples; therefore, we propose A. schaalii and these species are mutually exclusive. Additionally, a significantly higher A. schaalii prevalence in patients with ureter stricture associated hydronephrosis (p = 0.020) was noted. We suggest that A. schaalii could be an early polybacterial biofilm colonizer, together with concomitant species, known for pro-inflammatory features.

Vegan Diet Is Associated With Favorable Effects on the Metabolic Performance of Intestinal Microbiota: A Cross-Sectional Multi-Omics Study.

Background and Aim: Plant-based diets are associated with potential health benefits, but the contribution of gut microbiota remains to be clarified. We aimed to identify differences in key features of microbiome composition and function with relevance to metabolic health in individuals adhering to a vegan vs. omnivore diet. Methods: This cross-sectional study involved lean, healthy vegans (n = 62) and omnivore (n = 33) subjects. We assessed their glucose and lipid metabolism and employed an integrated multi-omics approach (16S rRNA sequencing, metabolomics profiling) to compare dietary intake, metabolic health, gut microbiome, and fecal, serum, and urine metabolomes. Results: The vegans had more favorable glucose and lipid homeostasis profiles than the omnivores. Long-term reported adherence to a vegan diet affected only 14.8% of all detected bacterial genera in fecal microbiome. However, significant differences in vegan and omnivore metabolomes were observed. In feces, 43.3% of all identified metabolites were significantly different between the vegans and omnivores, such as amino acid fermentation products p-cresol, scatole, indole, methional (lower in the vegans), and polysaccharide fermentation product short- and medium-chain fatty acids (SCFAs, MCFAs), and their derivatives (higher in the vegans). Vegan serum metabolome differed markedly from the omnivores (55.8% of all metabolites), especially in amino acid composition, such as low BCAAs, high SCFAs (formic-, acetic-, propionic-, butyric acids), and dimethylsulfone, the latter two being potential host microbiome co-metabolites. Using a machine-learning approach, we tested the discriminative power of each dataset. Best results were obtained for serum metabolome (accuracy rate 91.6%). Conclusion: While only small differences in the gut microbiota were found between the groups, their metabolic activity differed substantially. In particular, we observed a significantly different abundance of fermentation products associated with protein and carbohydrate intakes in the vegans. Vegans had significantly lower abundances of potentially harmful (such as p-cresol, lithocholic acid, BCAAs, aromatic compounds, etc.) and higher occurrence of potentially beneficial metabolites (SCFAs and their derivatives).

Carbapenemase-Producing Gram-Negative Bacteria from American Crows in the United States.

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.

Different Bacteroides Species Colonise Human and Chicken Intestinal Tract.

Bacteroidaceae are common gut microbiota members in all warm-blooded animals. However, if Bacteroidaceae are to be used as probiotics, the species selected for different hosts should reflect the natural distribution. In this study, we therefore evaluated host adaptation of bacterial species belonging to the family Bacteroidaceae. B. dorei, B. uniformis, B. xylanisolvens, B. ovatus, B. clarus, B. thetaiotaomicron and B. vulgatus represented human-adapted species while B. gallinaceum, B. caecigallinarum, B. mediterraneensis, B. caecicola, M. massiliensis, B. plebeius and B. coprocola were commonly detected in chicken but not human gut microbiota. There were 29 genes which were present in all human-adapted Bacteroides but absent from the genomes of all chicken isolates, and these included genes required for the pentose cycle and glutamate or histidine metabolism. These genes were expressed during an in vitro competitive assay, in which human-adapted Bacteroides species overgrew the chicken-adapted isolates. Not a single gene specific for the chicken-adapted species was found. Instead, chicken-adapted species exhibited signs of frequent horizontal gene transfer, of KUP, linA and sugE genes in particular. The differences in host adaptation should be considered when the new generation of probiotics for humans or chickens is designed.

Hymenobacter terrestris sp. nov. and Hymenobacter lapidiphilus sp. nov., isolated from regoliths in Antarctica.

A group of four psychrotrophic bacterial strains was isolated on James Ross Island (Antarctica) in 2013. All isolates, originating from different soil samples, were collected from the ice-free northern part of the island. They were rod-shaped, Gram-stain-negative, and produced moderately slimy red-pink pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, MALDI-TOF MS, rep-PCR analyses, chemotaxonomic methods and extensive biotyping was used to clarify the taxonomic position of these isolates. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates belonged to the genus Hymenobacter. The closest relative was Hymenobacter humicola CCM 8763T, exhibiting 98.3 and 98.9% 16S rRNA pairwise similarity with the reference isolates P5342T and P5252T, respectively. Average nucleotide identity, digital DNA-DNA hybridization and core gene distances calculated from the whole-genome sequencing data confirmed that P5252T and P5342T represent two distinct Hymenobacter species. The menaquinone systems of both strains contained MK-7 as the major respiratory quinone. The predominant polar lipids for both strains were phosphatidylethanolamine and one unidentified glycolipid. The major components in the cellular fatty acid composition were summed feature 3 (C16:1 ω7c/C16:1ω6c), C16:1ω5c, summed feature 4 (anteiso-C17:1 B/iso-C17:1 I), anteiso-C15:0 and iso-C15 : 0 for all isolates. Based on the obtained results, two novel species are proposed, for which the names Hymenobacter terrestris sp. nov. (type strain P5252T=CCM 8765T=LMG 31495T) and Hymenobacter lapidiphilus sp. nov. (type strain P5342T=CCM 8764T=LMG 30613T) are suggested.

Stool sampling and DNA isolation kits affect DNA quality and bacterial composition following 16S rRNA gene sequencing using MiSeq Illumina platform.

Many studies correlate changes in human gut microbiome with the onset of various diseases, mostly by 16S rRNA gene sequencing. Setting up the optimal sampling and DNA isolation procedures is crucial for robustness and reproducibility of the results. We performed a systematic comparison of several sampling and DNA isolation kits, quantified their effect on bacterial gDNA quality and the bacterial composition estimates at all taxonomic levels. Sixteen volunteers tested three sampling kits. All samples were consequently processed by two DNA isolation kits. We found that the choice of both stool sampling and DNA isolation kits have an effect on bacterial composition with respect to Gram-positivity, however the isolation kit had a stronger effect than the sampling kit. The proportion of bacteria affected by isolation and sampling kits was larger at higher taxa levels compared to lower taxa levels. The PowerLyzer PowerSoil DNA Isolation Kit outperformed the QIAamp DNA Stool Mini Kit mainly due to better lysis of Gram-positive bacteria while keeping the values of all the other assessed parameters within a reasonable range. The presented effects need to be taken into account when comparing results across multiple studies or computing ratios between Gram-positive and Gram-negative bacteria.

Role of the Microbiome in the Formation and Development of Colorectal Cancer.

BACKGROUND: The clinical, histopathological, and molecular characteristics of colorectal cancer vary considerably. Factors associated with the heterogeneity of this disease and with understanding the effects of heterogeneity on disease progression and response to therapy are critical for the better stratification of patients and the development of new therapeutic methods. Although studies have focused mainly on tumor molecular profiling, current molecular predictive and prognostic factors are relevant to specific groups of colorectal cancer patients and are mostly used to predict the applicability of targeted biological agents rather than to predict their benefits. Molecular profiling fails to capture aspects important for tumor growth and aggressiveness, including the tumor microenvironment. The gut microbiome, consisting of specific communities of all commensal, symbiotic, and pathogenic microorganisms, has been shown to have a significant impact on the development of many diseases, including Crohns disease, type II diabetes, and obesity. Recent studies have indicated that long-term dysbiosis of the intestinal microflora can influence the development and progression of colorectal cancer, as well as tumor aggressiveness and response to treatment. CONCLUSION: This review article summarizes current knowledge of the gut microbiome in colorectal cancer, including the various mechanisms by which the gut microbiome affects the intestinal wall, thereby contributing to the development and progression of colorectal cancer. This work was supported by Ministry of Health of the Czech Republic (project AZV 16-31966A), project of Ministry of Education, Youth and Sports of the Czech Republic - NPU I - LO1413 a Ministry of Health of the Czech Republic - RVO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 15. 4. 2019 Accepted: 17. 6. 2019.

Molecular Techniques Complement Culture-Based Assessment of Bacteria Composition in Mixed Biofilms of Urinary Tract Catheter-Related Samples.

Urinary or ureteral catheter insertion remains one of the most common urological procedures, yet is considered a predisposing factor for urinary tract infection. Diverse bacterial consortia adhere to foreign body surfaces and create various difficult to treat biofilm structures. We analyzed 347 urinary catheter- and stent-related samples, treated with sonication, using both routine culture and broad-range 16S rDNA PCR followed by Denaturing Gradient Gel Electrophoresis and Sanger sequencing (PCR-DGGE-S). In 29 selected samples, 16S rRNA amplicon Illumina sequencing was performed. The results of all methods were compared. In 338 positive samples, from which 86.1% were polybacterial, 1,295 representatives of 153 unique OTUs were detected. Gram-positive microbes were found in 46.5 and 59.1% of catheter- and stent-related samples, respectively. PCR-DGGE-S was shown as a feasible method with higher overall specificity (95 vs. 85%, p < 0.01) though lower sensitivity (50 vs. 69%, p < 0.01) in comparison to standard culture. Molecular methods considerably widened a spectrum of microbes detected in biofilms, including the very prevalent emerging opportunistic pathogen Actinotignum schaalii. Using massive parallel sequencing as a reference method in selected specimens, culture combined with PCR-DGGE was shown to be an efficient and reliable tool for determining the composition of urinary catheter-related biofilms. This might be applicable particularly to immunocompromised patients, in whom catheter-colonizing bacteria may lead to severe infectious complications. For the first time, broad-range molecular detection sensitivity and specificity were evaluated in this setting. This study extends the knowledge of biofilm consortia composition by analyzing large urinary catheter and stent sample sets using both molecular and culture techniques, including the widest dataset of catheter-related samples characterized by 16S rRNA amplicon Illumina sequencing.

The Effect of Butyrate-Supplemented Parenteral Nutrition on Intestinal Defence Mechanisms and the Parenteral Nutrition-Induced Shift in the Gut Microbiota in the Rat Model.

Butyrate produced by the intestinal microbiota is essential for proper functioning of the intestinal immune system. Total dependence on parenteral nutrition (PN) is associated with numerous adverse effects, including severe microbial dysbiosis and loss of important butyrate producers. We hypothesised that a lack of butyrate produced by the gut microbiota may be compensated by its supplementation in PN mixtures. We tested whether i.v. butyrate administration would (a) positively modulate intestinal defence mechanisms and (b) counteract PN-induced dysbiosis. Male Wistar rats were randomised to chow, PN, and PN supplemented with 9 mM butyrate (PN+But) for 12 days. Antimicrobial peptides, mucins, tight junction proteins, and cytokine expression were assessed by RT-qPCR. T-cell subpopulations in mesenteric lymph nodes (MLN) were analysed by flow cytometry. Microbiota composition was assessed in caecum content. Butyrate supplementation resulted in increased expression of tight junction proteins (ZO-1, claudin-7, E-cadherin), antimicrobial peptides (Defa 8, Rd5, RegIIIγ), and lysozyme in the ileal mucosa. Butyrate partially alleviated PN-induced intestinal barrier impairment and normalised IL-4, IL-10, and IgA mRNA expression. PN administration was associated with an increase in Tregs in MLN, which was normalised by butyrate. Butyrate increased the total number of CD4+ and decreased a relative amount of CD8+ memory T cells in MLN. Lack of enteral nutrition and PN administration led to a shift in caecal microbiota composition. Butyrate did not reverse the altered expression of most taxa but did influence the abundance of some potentially beneficial/pathogenic genera, which might contribute to its overall beneficial effect.

Bipartite Graphs for Visualization Analysis of Microbiome Data.

Visualization analysis plays an important role in metagenomics research. Proper and clear visualization can help researchers get their first insights into data and by selecting different features, also revealing and highlighting hidden relationships and drawing conclusions. To prevent the resulting presentations from becoming chaotic, visualization techniques have to properly tackle the high dimensionality of microbiome data. Although a number of different methods based on dimensionality reduction, correlations, Venn diagrams, and network representations have already been published, there is still room for further improvement, especially in the techniques that allow visual comparison of several environments or developmental stages in one environment. In this article, we represent microbiome data by bipartite graphs, where one partition stands for taxa and the other stands for samples. We demonstrated that community detection is independent of taxonomical level. Moreover, focusing on higher taxonomical levels and the appropriate merging of samples greatly helps improving graph organization and makes our presentations clearer than other graph and network visualizations. Capturing labels in the vertices also brings the possibility of clearly comparing two or more microbial communities by showing their common and unique parts.

Seasonal changes in microbial community composition in river water studied using 454-pyrosequencing.

The aims of this study were to determine the microbial community in five rivers in the proximity of a city in the Czech Republic using 454-pyrosequencing, as well as to assess seasonal variability over the course of 1 year and to identify the factors influencing the structure of bacterial communities. Samples from five rivers around the city of Brno were obtained during four seasons and analysed using 454 pyrosequencing of the 16S rRNA gene. The core composition of bacterial communities consisted of Actinobacteria, Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, TM7 and others. Our approach enabled us to more closely study the correlation between the abundance of different families and environmental factors. Overall, Actinobacteria negatively correlated with phosphorus, sulphate, dissolved particle and chloride levels. In contrast, Proteobacteria positively correlated with sulphate, dissolved particle, chloride, dissolved oxygen and nitrite levels. Future work should focus on the dynamics of bacterial communities present in river water and their relation to the overall stability of the water ecosystem.

Faecal bacterial composition in dairy cows shedding Mycobacterium avium subsp. paratuberculosis in faeces in comparison with nonshedding cows.

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

Complete Sequences of IncU Plasmids Harboring Quinolone Resistance Genes qnrS2 and aac(6')-Ib-cr in Aeromonas spp. from Ornamental Fish.

The nucleotide sequences of three IncU plasmids from Aeromonas spp. isolated from ornamental fish are described. They had a typical IncU backbone for plasmid replication and maintenance functions, but conjugative transfer modules were disrupted. The gene qnrS2 was inserted into mpR as a mobile insertion cassette. Novel Tn3 family transposons carrying putative toxin-antitoxin and plasmid stability genes were identified. The study demonstrates high plasticity of IncU plasmids from aquatic environments.

Changes in Microbial Composition of Wastewater During Treatment in a Full-Scale Plant.

The monitoring of wastewater treatment plants is important for their proper functioning as well as for re-use of water and also to avoid possible circulation of human or animal pathogens in our environment. The samples in this study originated from a full-scale wastewater treatment plant where the structure of the bacterial community was monitored using 454-pyrosequencing. The composition differed in different parts of the plant. In the effluent, bacteria belonging to phyla Proteobacteria, Actinobacteria, TM7 and Bacteroidetes were most frequently detected. The presence of Mycobacterium sp., Mycobacterium avium, Norovirus, Hepatitis A and E viruses was examined using quantitative real-time PCR. Mycobacterium sp. was detected in the effluent in quantities of up to 10(4) cells/ml. Mycobacterium avium subsp. paratuberculosis and subsp. hominissuis were detected in amounts of up to 10(3) cells/ml, and Norovirus group 1 and 2 were also detected. Our findings show the importance of monitoring and controlling the occurrence of specific pathogens in effluent, mainly because of the negative impact on human health when the water is reused.

Characterization of Antibiotic Resistance Gene Abundance and Microbiota Composition in Feces of Organic and Conventional Pigs from Four EU Countries.

One of the recent trends in animal production is the revival of interest in organic farming. The increased consumer interest in organic animal farming is mainly due to concerns about animal welfare and the use of antibiotics in conventional farming. On the other hand, providing animals with a more natural lifestyle implies their increased exposure to environmental sources of different microorganisms including pathogens. To address these concerns, we determined the abundance of antibiotic resistance and diversity within fecal microbiota in pigs kept under conventional and organic farming systems in Sweden, Denmark, France and Italy. The abundance of sul1, sul2, strA, tet(A), tet(B) and cat antibiotic resistance genes was determined in 468 samples by real-time PCR and the fecal microbiota diversity was characterized in 48 selected samples by pyrosequencing of V3/V4 regions of 16S rRNA. Contrary to our expectations, there were no extensive differences between the abundance of tested antibiotic resistance genes in microbiota originating from organic or conventionally housed pigs within individual countries. There were also no differences in the microbiota composition of organic and conventional pigs. The only significant difference was the difference in the abundance of antibiotic resistance genes in the samples from different countries. Fecal microbiota in the samples originating from southern European countries (Italy, France) exhibited significantly higher antibiotic resistance gene abundance than those from northern parts of Europe (Denmark, Sweden). Therefore, the geographical location of the herd influenced the antibiotic resistance in the fecal microbiota more than farm's status as organic or conventional.

Succession and replacement of bacterial populations in the caecum of egg laying hens over their whole life.

In this study we characterised the development of caecal microbiota in egg laying hens over their commercial production lifespan, from the day of hatching until 60 weeks of age. Using pyrosequencing of V3/V4 variable regions of 16S rRNA genes for microbiota characterisation, we were able to define 4 different stages of caecal microbiota development. The first stage lasted for the first week of life and was characterised by a high prevalence of Enterobacteriaceae (phylum Proteobacteria). The second stage lasted from week 2 to week 4 and was characterised by nearly an absolute dominance of Lachnospiraceae and Ruminococcaceae (both phylum Firmicutes). The third stage lasted from month 2 to month 6 and was characterised by the succession of Firmicutes at the expense of Bacteroidetes. The fourth stage was typical for adult hens in full egg production aged 7 months or more and was characterised by a constant ratio of Bacteroidetes and Firmicutes formed by equal numbers of the representatives of both phyla.