Effect of cryopreservation and lyophilization on viability and growth of strict anaerobic human gut microbes.
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ABSTRACT:
SUBMITTER: Bircher L
PROVIDER: S-EPMC6011992 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundNovel strategies for anaerobic bacterial isolations from human faecal samples and various initiatives to generate culture collections of gut-derived bacteria have instigated considerable interest for the development of novel microbiota-based treatments. Early in the process of building a culture collection, optimal faecal sample preservation is essential to safeguard the viability of the broadest taxonomic diversity range possible. In contrast to the much more established faecal storage conditions for meta-omics applications, the impact of stool sample preservation conditions on bacterial growth recovery and isolation remains largely unexplored. In this study, aliquoted faecal samples from eleven healthy human volunteers selected based on a range of physicochemical and microbiological gradients were cryopreserved at - 80 °C either without the addition of any medium (dry condition) or in different Cary-Blair medium conditions with or without a cryoprotectant, i.e. 20% (v/v) glycerol or 5% (v/v) DMSO. Faecal aliquots were subjected to bulk 16S rRNA gene sequencing as well as dilution plating on modified Gifu Anaerobic Medium after preservation for culturable fraction profiling and generation of bacterial culture collections.ResultsAnalyses of compositional variation showed that cryopreservation medium conditions affected quantitative recovery but not the overall community composition of cultured fractions. Post-preservation sample dilution and richness of the uncultured source samples were the major drivers of the cultured fraction richness at genus level. However, preservation conditions differentially affected recovery of specific genera. Presence-absence analysis indicated that twenty-two of the 45 most abundant common genera (>0.01% abundance, dilution 10-4) were recovered in cultured fractions from all preservation conditions, while nine genera were only detected in fractions from a single preservation condition. Overall, the highest number of common genera (i.e. 35/45) in cultured fractions were recovered from sample aliquots preserved without medium and in the presence of Cary-Blair medium containing 5% (v/v) DMSO. Also, in the culture collection generated from the cultured fractions, these two preservation conditions yielded the highest species richness (72 and 66, respectively).ConclusionOur results demonstrate that preservation methods partly determine richness and taxonomic diversity of gut anaerobes recovered from faecal samples. Complementing the current standard practice of cryopreserving stool samples in dry conditions with other preservation conditions, such as Cary-Blair medium with DMSO, could increase the species diversity of gut-associated culture collections. Video abstract.
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Project description:The succession from aerobic and facultative anaerobic bacteria to obligate anaerobes in the infant gut along with the differences between the compositions of the mucosally adherent vs. luminal microbiota suggests that the gut microbes consume oxygen, which diffuses into the lumen from the intestinal tissue, maintaining the lumen in a deeply anaerobic state. Remarkably, measurements of luminal oxygen levels show nearly identical pO2 (partial pressure of oxygen) profiles in conventional and germ-free mice, pointing to the existence of oxygen consumption mechanisms other than microbial respiration. In vitro experiments confirmed that the luminal contents of germ-free mice are able to chemically consume oxygen (e.g., via lipid oxidation reactions), although at rates significantly lower than those observed in the case of conventionally housed mice. For conventional mice, we also show that the taxonomic composition of the gut microbiota adherent to the gut mucosa and in the lumen throughout the length of the gut correlates with oxygen levels. At the same time, an increase in the biomass of the gut microbiota provides an explanation for the reduction of luminal oxygen in the distal vs. proximal gut. These results demonstrate how oxygen from the mammalian host is used by the gut microbiota, while both the microbes and the oxidative chemical reactions regulate luminal oxygen levels, shaping the composition of the microbial community throughout different regions of the gut.
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Project description:Humans have evolved intimate symbiotic relationships with a consortium of gut microbes (microbiome) and individual variations in the microbiome influence host health, may be implicated in disease etiology, and affect drug metabolism, toxicity, and efficacy. However, the molecular basis of these microbe-host interactions and the roles of individual bacterial species are obscure. We now demonstrate a"transgenomic" approach to link gut microbiome and metabolic phenotype (metabotype) variation. We have used a combination of spectroscopic, microbiomic, and multivariate statistical tools to analyze fecal and urinary samples from seven Chinese individuals (sampled twice) and to model the microbial-host metabolic connectivities. At the species level, we found structural differences in the Chinese family gut microbiomes and those reported for American volunteers, which is consistent with population microbial cometabolic differences reported in epidemiological studies. We also introduce the concept of functional metagenomics, defined as "the characterization of key functional members of the microbiome that most influence host metabolism and hence health." For example, Faecalibacterium prausnitzii population variation is associated with modulation of eight urinary metabolites of diverse structure, indicating that this species is a highly functionally active member of the microbiome, influencing numerous host pathways. Other species were identified showing different and varied metabolic interactions. Our approach for understanding the dynamic basis of host-microbiome symbiosis provides a foundation for the development of functional metagenomics as a probe of systemic effects of drugs and diet that are of relevance to personal and public health care solutions.
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Project description:To evaluate the toxicity of smokeless tobacco products (STPs) on oral bacteria, seven smokeless tobacco aqueous extracts (STAEs) from major brands of STPs and three tobacco-specific N-nitrosamines (TSNAs) were used in a growth and viability test against 38 oral bacterial species or subspecies. All seven STAEs showed concentration-dependent effects on the growth and viability of tested oral bacteria under anaerobic culture conditions, although there were strain-to-strain variations. In the presence of 1 mg/ml STAEs, the growth of 4 strains decreased over 0.32-2.14 log10 fold, while 14 strains demonstrated enhanced growth of 0.3-1.76 log10 fold, and the growth of 21 strains was not significantly affected. In the presence of 10 mg/ml STAEs, the growth of 17 strains was inhibited 0.3-2.11 log10 fold, 18 strains showed enhanced growth of 0.3-0.97 log10 fold, and 4 strains were not significantly affected. In the presence of 50 mg/ml STAEs, the growth of 32 strains was inhibited 0.3-2.96 log10 fold, 8 strains showed enhanced growth of 0.3-1.0 log10 fold, and 2 strains were not significantly affected. All seven STAEs could promote the growth of 4 bacterial strains, including Eubacterium nodatum, Peptostreptococcus micros, Streptococcus anginosus, and Streptococcus constellatus. Exposure to STAEs modulated the viability of some bacterial strains, with 21.1-66.5% decrease for 4 strains at 1 mg/ml, 20.3-85.7% decrease for 10 strains at 10 mg/ml, 20.0-93.3% decrease for 27 strains at 50 mg/ml, and no significant effect for 11 strains at up to 50 mg/ml. STAEs from snuffs inhibited more tested bacterial strains than those from snus indicating that the snuffs may be more toxic to the oral bacteria than snus. For TSNAs, cell growth and viability of 34 tested strains were not significantly affected at up to 100 ?g/ml; while the growth of P. micros was enhanced 0.31-0.54 log10 fold; the growth of Veillonella parvula was repressed 0.33-0.36 log10 fold; and the cell viabilities of 2 strains decreased 56.6-69.9%. The results demonstrate that STAEs affected the growth of some types of oral bacteria, which may affect the healthy ecological balance of oral bacteria in humans. On the other hand, TSNAs did not significantly affect the growth of the oral bacteria.
| S-EPMC5693344 | biostudies-literature
Project description:Emerging studies have revealed a strong link between the gut microbiome and several human diseases. Since human gut microbiome mirrors variations in lifestyle and environment, whether associations between disease conditions and gut microbiome are consistent across populations-particularly in communities practicing traditional subsistence strategies whose microbiomes differ markedly from industrialists-remains unknown. Cardiovascular diseases are the leading cause of mortality in India affecting 55 million people, and high blood pressure is one of the primary risk factors for cardiovascular diseases. We examined associations between gut microbiome and blood pressure along with 14 other variables associated with lifestyle, dietary habits, disease conditions, and clinical blood markers in the three Assamese populations. Our analysis reveals a robust link between the gut microbiome diversity and composition and systolic blood pressure. Moreover, several genera previously associated with hypertension in non-Indian populations were also associated with systolic blood pressure in this cohort and these genera were predictors of elevated blood pressure in these populations. These findings confer opportunities to design personalized, preventative, and targeted interventions harnessing the gut microbiome to tackle the burden of cardiovascular diseases in India.
| S-EPMC8831042 | biostudies-literature
Project description:Human milk contains large amounts of complex oligosaccharides that putatively modulate the intestinal microbiota of breast-fed infants by acting as decoy binding sites for pathogens and as prebiotics for enrichment of beneficial bacteria. Several bifidobacterial species have been shown to grow well on human milk oligosaccharides. However, few data exist on other bacterial species. This work examined 16 bacterial strains belonging to 10 different genera for growth on human milk oligosaccharides. For this propose, a chemically defined medium, ZMB1, was used, which allows vigorous growth of a number of gut-related microorganisms in a fashion similar to complex media. Interestingly, Bifidobacterium longum subsp. infantis, Bacteroides fragilis , and Bacteroides vulgatus strains were able to metabolize milk oligosaccharides with high efficiency, whereas Enterococcus , Streptococcus , Veillonella , Eubacterium , Clostridium , and Escherichia coli strains grew less well or not at all. Mass spectrometry-based glycoprofiling of the oligosaccharide consumption behavior revealed a specific preference for fucosylated oligosaccharides by Bi. longum subsp. infantis and Ba. vulgatus. This work expands the current knowledge of human milk oligosaccharide consumption by gut microbes, revealing bacteroides as avid consumers of this substrate. These results provide insight on how human milk oligosaccharides shape the infant intestinal microbiota.
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Project description:ObjectiveTo develop a protocol for cryopreservation and recovery of human endometrial epithelial cells (eECs) retaining molecular and functional characteristics of endometrial epithelium in vivo.DesignIn vitro study using human endometrial cells.SettingUniversity research laboratory.Patient(s)Endometrial biopsies were obtained from premenopausal women undergoing benign gynecologic procedures.Intervention(s)Primary eECs were cryopreserved in 1% fetal bovine serum/10% dimethylsulfoxide in Defined Keratinocyte Serum-Free Medium (KSFM). Recovered cells were observed for endometrial stromal fibroblast (eSF) contamination and subsequently evaluated for morphology, gene expression, and functional characteristics of freshly cultured eECs and in vivo endometrial epithelium.Main outcome measure(s)Analysis of eEC morphology and the absence of eSF contamination; evaluation of epithelial-specific gene and protein expression; assessment of epithelial polarity.Result(s)Endometrial epithelial cells recovered after cryopreservation (n = 5) displayed epithelial morphology and expressed E-cadherin (CDH1), occludin (OCLN), claudin1 (CLDN1), and keratin18 (KRT18). Compared with eSF, recovered eECs displayed increased (P<.05) expression of epithelial-specific genes AREG, CDH1, DEFB4A, MMP7, and WNT7A, while exhibiting low-to-undetectable (P<.05) stromal-specific genes COL6A3, HOXA11, MMP2, PDGFRB, and WNT5A. Recovered eECs secreted levels of cytokines and growth factors similarly to freshly cultured eECs. Recovered eECs could form a polarized monolayer with high transepithelial electrical resistance (TER) and impermeability to small molecules, and expressed apical/basolateral localization of CDH1 and apical localization of OCLN.Conclusion(s)We have developed a protocol for cryopreservation of eECs in which recovered cells after thawing demonstrate morphologic, transcriptomic, and functional characteristics of human endometrial epithelium in vivo.
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Project description:Sound and timely microbial gut colonization completes newborn's healthy metabolic programming and manifests in infant appropriate growth and weight development. Feces, collected at 3, 30, and 90 days after birth from 60 breastfed Slovenian newborns, was submitted to microbial DNA extraction and qPCR quantification of selected gut associated taxa. Multivariate regression analysis was applied to evaluate microbial dynamics with respect to infant demographic, environmental, clinical characteristics and first year growth data. Early microbial variability was marked by the proportion of Bacilli, but diminished and converged in later samples, as bifidobacteria started to prevail. The first month proportions of enterococci were associated with maternity hospital locality and supplementation of breastfeeding with formulae, while Enterococcus faecalis proportion reflected the mode of delivery. Group Bacteroides-Prevotella proportion was associated with infant weight and ponderal index at first month. Infant mixed feeding pattern and health issues within the first month revealed the most profound and extended microbial perturbations. Our findings raise concerns over the ability of the early feeding supplementation to emulate and support the gut microbiota in a way similar to the exclusively breastfed infants. Additionally, practicing supplementation beyond the first month also manifested in higher first year weight and weight gain Z-score.
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Project description:The extreme variation in gene content among phylogenetically related microorganisms suggests that gene acquisition, expansion, and loss are important evolutionary forces for adaptation to new environments. Accordingly, phylogenetically disparate organisms that share a habitat may converge in gene content as they adapt to confront shared challenges. This response should be especially pronounced for functional genes that are important for survival in a particular habitat. We illustrate this principle by showing that the repertoires of two different types of carbohydrate-active enzymes, glycoside hydrolases and glycosyltransferases, have converged in bacteria and archaea that live in the human gut and that this convergence is largely due to horizontal gene transfer rather than gene family expansion. We also identify gut microbes that may have more similar dietary niches in the human gut than would be expected based on phylogeny. The techniques used to obtain these results should be broadly applicable to understanding the functional genes and evolutionary processes important for adaptation in many environments and useful for interpreting the large number of reference microbial genome sequences being generated for the International Human Microbiome Project.
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Project description:Recent studies have linked human gut microbes to obesity and inflammatory bowel disease, but consistent signals have been difficult to identify. Here we test for indicator taxa and general features of the microbiota that are generally consistent across studies of obesity and of IBD, focusing on studies involving high-throughput sequencing of the 16S rRNA gene (which we could process using a common computational pipeline). We find that IBD has a consistent signature across studies and allows high classification accuracy of IBD from non-IBD subjects, but that although subjects can be classified as lean or obese within each individual study with statistically significant accuracy, consistent with the ability of the microbiota to experimentally transfer this phenotype, signatures of obesity are not consistent between studies even when the data are analyzed with consistent methods. The results suggest that correlations between microbes and clinical conditions with different effect sizes (e.g. the large effect size of IBD versus the small effect size of obesity) may require different cohort selection and analysis strategies.
| S-EPMC5050012 | biostudies-literature