Thermal degradation of agar: Mechanism and toxicity of products.
Ouyang Qian-Qian,Hu Zhang,Li Si-Dong,Quan Wei-Yan,Wen Li-Li,Yang Zi-Ming,Li Pu-Wang
The mechanism of the thermal degradation and the toxicity of the thermal degradation products of agar were studied using TG/DTA, Fourier-transform infrared spectroscopy and pyrolysis gas chromatography/mass spectrometry. It was found that the thermal degradation of agar is a single-step reaction, the thermal degradation temperature (T, T, T) increases with increasing gel strength (P) and the influence of P on the thermal degradation rate is modest. The thermal degradation of agar is an exothermic reaction, and the activation energy of the reaction increases with increasing P. In the thermal degradation, agar is first decomposed into 3,6-anhydropyran galactopyranose and galactopyranose, then 3,6-anhydropyran galactopyranose, and finally furyl hydroxymethyl ketone, through loop opening, dehydration and hydrogen transfer. Galactopyranose follows three degradation pathways, and its final degradation products are 3,4-atrosan, d-allose, furfural and 5-(hydroxymethyl)-2-furancarboxaldehyde. Of the degradation products, furyl hydroxymethyl ketone, furfural, and 5-(hydroxymethyl)-2-furancarboxaldehyde show some toxicity to humans.
Optimization of Culture Medium for the Growth of Candida sp. and Blastobotrys sp. as Starter Culture in Fermentation of Cocoa Beans (Theobroma cacao) Using Response Surface Methodology (RSM).
Mahazar N H,Zakuan Z,Norhayati H,MeorHussin A S,Rukayadi Y
Pakistan journal of biological sciences : PJBS
BACKGROUND AND OBJECTIVE:Inoculation of starter culture in cocoa bean fermentation produces consistent, predictable and high quality of fermented cocoa beans. It is important to produce healthy inoculum in cocoa bean fermentation for better fermented products. Inoculum could minimize the length of the lag phase in fermentation. The purpose of this study was to optimize the component of culture medium for the maximum cultivation of Candida sp. and Blastobotrys sp. MATERIALS AND METHODS:Molasses and yeast extract were chosen as medium composition and Response Surface Methodology (RSM) was then employed to optimize the molasses and yeast extract. RESULTS:Maximum growth of Candida sp. (7.63 log CFU mL-1) and Blastobotrys sp. (8.30 log CFU mL-1) were obtained from the fermentation. Optimum culture media for the growth of Candida sp., consist of 10% (w/v) molasses and 2% (w/v) yeast extract, while for Blastobotrys sp., were 1.94% (w/v) molasses and 2% (w/v) yeast extract. CONCLUSION:This study shows that culture medium consists of molasses and yeast extract were able to produce maximum growth of Candida sp. and Blastobotrys sp., as a starter culture for cocoa bean fermentation.
Characterization, culture medium optimization and antioxidant activity of an endophytic vitexin-producing fungus Dichotomopilus funicola Y3 from pigeon pea [Cajanus cajan (L.) Millsp.].
Gu C B,Ma H,Ning W J,Niu L L,Han H Y,Yuan X H,Fu Y J
Journal of applied microbiology
AIMS:The aim of this study was to characterize a fungal endophyte Y3 from pigeon pea (Cajanus cajan [L.] Millsp), as a novel producer of vitexin, and its culture medium optimization and antioxidant activity. METHODS AND RESULTS:The endophyte from the leaves of pigeon pea was identified as Dichotomopilus funicola by the morphological and molecular characteristics. The most important medium variables affecting vitexin production in liquid culture of D. funicola Y3 were screened by Plackett-Burman design, and three culture medium constituents (i.e. l-phenylalanine, salicylic acid and CuSO ·5H O) were identified to play significant roles in vitexin production. The most significant factors were further optimized using by central composite design with response surface methodology. The DPPH radical-scavenging assay indicated that fungal vitexin exhibited notable antioxidant activity with an EC value of 164 μg l . CONCLUSIONS:First, a novel endophyte vitexin-producing Dichotomopilus funicola Y3 was isolated from pigeon pea (Cajanus cajan[L.] Millsp.). The maximum vitexin yield was obtained as 78·86 mg l under the optimum culture medium constituents: 0·06 g l l-phenylalanine, 0·21 g l salicylic acid, and 0·19 g l CuSO ·5H O in medium, which is 4·59-fold higher than that in the unoptimized medium. Also, fungal vitexin clearly demonstrated its antioxidant potential. SIGNIFICANCE AND IMPACT OF THE STUDY:These findings provide an alternative source for large-scale production of vitexin by endophytic fungal fermentation and have a promising prospect in food and pharmaceutical industry.
Effect of cell culture medium additives on color and acidic charge variants of a monoclonal antibody.
Vijayasankaran Natarajan,Varma Sharat,Yang Yi,Meier Steven,Kiss Robert
This manuscript summarizes the effect of certain cell culture medium additives on antibody drug substance coloration and acidic charge variants. It has been shown previously that B-vitamins and iron in the cell culture medium could significantly impact color intensity. In this manuscript, we detail the effect of several other cell culture components that have been shown to impact coloration. It is shown that if cystine is used instead of cysteine in the cell culture medium, coloration was reduced. Hydrocortisone has been shown to reduce coloration and boost specific productivity. The effect of a peptone/hydrolysate on coloration was investigated in cell culture experiments, which showed its use can lead to reduced coloration. Mechanisms by which these compounds influence coloration will be briefly discussed. Since it has been previously shown that antibody oxidation could potentially lead to coloration, the current effort was focused on screening for specific antioxidant additives to the culture medium to reduce coloration. An in-vitro incubation model was used to screen antioxidant compounds, several of which were found to significantly reduce antibody color, while some led to significantly increased color. Hypotaurine and carboxymethylcysteine, which had the most significant color reducing effect in the incubation study, were further tested in small-scale bioreactor cell culture experiments. These studies demonstrated that these compounds lead to reduced coloration in cell culture without affecting cell growth and titer. Hypotaurine, hydrocortisone, peptone, and cystine were also shown to reduce the acidic charge variant levels, which was previously shown to correlate with color. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1298-1307, 2018.
A novel culture medium designed for the simultaneous enhancement of biomass and lipid production by Chlorella vulgaris UTEX 26.
Ramírez-López Citlally,Chairez Isaac,Fernández-Linares Luis
A novel culture medium to enhance the biomass and lipid production simultaneously by Chlorella vulgaris UTEX 26 was designed in three stages of optimization. Initially, a culture medium was inferred applying the response surface method to adjust six factors [NaNO3, NH4HCO3, MgSO4·7H2O, KH2PO4, K2HPO4 and (NH4)2HPO4], which were selected on the basement of BBM (Bold's Basal Medium) and HAMGM (Highly Assimilable Minimal Growth Medium) culture media. Afterwards, the nitrogen source compound was optimized to reduce both, ammonium and nitrate concentrations. As result of the optimization process, the proposed culture medium improved 40% the biomass (0.73gL(-1)) compared with the BBM medium and 85% the lipid concentration (281mgL(-1)), with respect to HAMGM medium. Some culture media components concentrations were reduced up to 50%. Gas chromatography analysis revealed that C16:0, C18:0, C18:1, C18:2 and C18:3 were the major fatty acids produced by C. vulgaris UTEX 26.
Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing.
Pfannebecker Jens,Schiffer-Hetz Claudia,Fröhlich Jürgen,Becker Barbara
Journal of microbiological methods
In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (a)-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1-0.2% ammonium salts ((NH)HPO, (NH)SO or NHNO), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 10CFU/g within a time period of 2-3days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124h in YEG50, 95.5h in MYG50 and 55h in OM bouillon. Compared to YEG50 the maximum CO transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3days for Z. rouxii and T. delbrueckii can be realized by using OM in combination with the automated test system even if low initial counts (10CFU/g) are present in the products. In conclusion, the presented data suggest that the OM culture medium is appropriate for the enrichment of osmotolerant yeasts from high-sugar food products.
Implications in studies of environmental risk assessments: Does culture medium influence the results of toxicity tests of marine bacteria?
Díaz-García Alejandra,Borrero-Santiago Ana R,Riba Inmaculada
Two marine bacterial populations (Roseobacter sp. and Pseudomonas litoralis) were exposed to different concentrations of zinc (300, 625, 1250, 2000, 2500 and 5000 mg L) and cadmium (75, 250, 340, 500 and 1000 mg L) using two culture media (full nutrient Marine Broth 2216 "MB" and 1:10 (vol/vol) dilution with seawater of Marine Broth 2216 "MB"), in order to assess population responses depending on the culture medium and also potential adverse effects associated with these two metals. Different responses were found depending on the culture medium (Bacterial abundance (cells·mL), growth rates (μ, hours), and production of Extracellular Polysaccharides Substances (EPS) (μg glucose·cells). Results showed negative effects in both strains after the exposure to Zn treatments. Both strains showed highest metal sensitivity at low concentrations using both culture media. However, different results were found when exposing the bacterial populations to Cd treatments depending on the culture medium. Highest toxicity was observed using MB at low levels of Cd concentrations, whereas MB showed toxicity to bacteria at higher concentrations of Cd. Results not only showed adverse effects on Roseobacter sp. and Pseudomonas litoralis associated with the concentration of Zn and Cd, but also confirm that depending on the culture medium results can differ. This work suggests MB as an adequate culture medium to study metal toxicity bioassays in order to predict realistic effects on marine bacterial populations.
An experimental strategy validated to design cost-effective culture media based on response surface methodology.
Navarrete-Bolaños J L,Téllez-Martínez M G,Miranda-López R,Jiménez-Islas H
Preparative biochemistry & biotechnology
For any fermentation process, the production cost depends on several factors, such as the genetics of the microorganism, the process condition, and the culture medium composition. In this work, a guideline for the design of cost-efficient culture media using a sequential approach based on response surface methodology is described. The procedure was applied to analyze and optimize a culture medium of registered trademark and a base culture medium obtained as a result of the screening analysis from different culture media used to grow the same strain according to the literature. During the experiments, the procedure quantitatively identified an appropriate array of micronutrients to obtain a significant yield and find a minimum number of culture medium ingredients without limiting the process efficiency. The resultant culture medium showed an efficiency that compares favorably with the registered trademark medium at a 95% lower cost as well as reduced the number of ingredients in the base culture medium by 60% without limiting the process efficiency. These results demonstrated that, aside from satisfying the qualitative requirements, an optimum quantity of each constituent is needed to obtain a cost-effective culture medium. Study process variables for optimized culture medium and scaling-up production for the optimal values are desirable.
Crosstalk Between Culturomics and Microbial Profiling of Egyptian Mongoose () Gut Microbiome.
Pereira André C,Bandeira Victor,Fonseca Carlos,Cunha Mónica V
Recently, we unveiled taxonomical and functional differences in Egyptian mongoose () gut microbiota across sex and age classes by microbial profiling. In this study, we generate, through culturomics, extended baseline information on the culturable bacterial and fungal microbiome of the species using the same specimens as models. Firstly, this strategy enabled us to explore cultivable microbial community differences across sexes and to ascertain the influence exerted by biological and environmental contexts of each host in its microbiota signature. Secondly, it permitted us to compare the culturomics and microbial profiling approaches and their ability to provide information on mongoose gut microbiota. In agreement with microbial profiling, culturomics showed that the core gut cultivable microbiota of the mongoose is dominated by and, as previously found, is able to distinguish sex- and age class-specific genera. Additional information could be obtained by culturomics, with six new genera unveiled. Richness indices and the Shannon index were concordant between culture-dependent and culture-independent approaches, highlighting significantly higher values when using microbial profiling. However, the Simpson index underlined higher values for the culturomics-generated data. These contrasting results were due to a differential influence of dominant and rare taxa on those indices. Beta diversity analyses of culturable microbiota showed similarities between adults and juveniles, but not in the data series originated from microbial profiling. Additionally, whereas the microbial profiling indicated that there were several bioenvironmental features related to the bacterial gut microbiota of the Egyptian mongoose, a clear association between microbiota and bioenvironmental features could not be established through culturomics. The discrepancies found between the data generated by the two methodologies and the underlying inferences, both in terms of β-diversity and role of bioenvironmental features, confirm that culture-independent, sequence-based methods have a higher ability to assess, at a fine scale, the influence of abiotic and biotic factors on the microbial community composition of mongoose' gut. However, when used in a complementary perspective, this knowledge can be expanded by culturomics.
"" Culturing of Plant Microbiota: A Novel Simulated Environmental Method Based on Plant Leaf Blades as Nutritional Pads.
Nemr Rahma A,Khalil Mohab,Sarhan Mohamed S,Abbas Mohamed,Elsawey Hend,Youssef Hanan H,Hamza Mervat A,Morsi Ahmed T,El-Tahan Mahmoud,Fayez Mohamed,Patz Sascha,Witzel Katja,Ruppel Silke,El-Sahhar Kassem F,Hegazi Nabil A
Frontiers in microbiology
High-throughput cultivation methods have recently been developed to accelerate the recovery of microorganisms reluctant to cultivation. They simulate environmental conditions for the isolation of environmental microbiota through the exchange of growth substrates during cultivation. Here, we introduce leaf-based culture media adopting the concept of the plant being the master architect of the composition of its microbial community. Pre-physical treatments of sunflower plant leaves, namely punching, freezing, and/or autoclavation, allowed the diffusion of electrolytes and other nutrients to configure the leaf surface as a natural pad, i.e., creating an "" environment suitable for the growth of rarely isolated microbiota. We used surface inoculation and membrane-filtration methods to assess the culturability of endophytic bacteria from the sunflower phyllosphere and rhizosphere. Both methods supported excellent colony-forming unit (CFU) development when compared to standard R2A medium, with a special affinity to support better growth of epiphytic and endophytic populations of the phyllosphere compared with the rhizosphere. A 16S rRNA gene analysis of >122 representative isolates indicated the cultivation of a diverse set of microorganisms by application of the new methods. It indicated the predominance of 13 genera of >30 potential species, belonging to Firmicutes, Proteobacteria, and Actinobacteria, and especially genera not commonly reported for sunflower, e.g., , , , , , , , and . The strategy successfully extended diversity and richness in the endophyllosphere compared to the endorhizosphere, while CFUs grown on the standard R2A medium mainly pertain to Firmicutes, especially spp. MALDI-TOF MS analysis clustered the isolates according to their niche and potential functions, where the majority of isolates of the endorhizosphere were clustered away from those of the endophyllosphere. Isolates identified as Gammaproteobacteria and Alphaproteobacteria were distinguishably sub-clustered, which was in contrast to the heterogeneous isolates of Firmicutes ( spp.). In conclusion, leaf cultivation is an effective strategy to support the future application of culturomics of plant microbiota. This is an effort to access novel isolates that are more adapted and competitive in their natural environments, especially those subjected to abiotic stresses like those prevailing in arid/semi-arid zones, and, consequently, to support the application of agro-biotechnologies, among other technologies, to improving agriculture in such zones.
Alcohol pretreatment of stools effect on culturomics.
Afouda Pamela,Hocquart Marie,Pham Thi-Phuong-Thao,Kuete Edmond,Ngom Issa Isaac,Dione Niokhor,Valles Camille,Bellali Sara,Lagier Jean-Christophe,Dubourg Grégory,Raoult Didier
Recent studies have used ethanol stool disinfection as a mean of promoting valuable species' cultivation in bacteriotherapy trials for Clostridium difficile infections (CDI) treatment with a particular focus on sporulating bacteria. Moreover, the culturomic approach has considerably enriched the repertoire of cultivable organisms in the human gut in recent years. This study aimed to apply this culturomic approach on fecal donor samples treated with ethanol disinfection to evidence potential beneficial microbes that could be used in bacteriotherapy trials for the treatment of CDI. Thereby, a total of 254 bacterial species were identified, 9 of which were novel. Of these, 242 have never been included in clinical trials for the treatment of CDIs, representing potential new candidates for bacteriotherapy trials. While non-sporulating species were nevertheless more affected by the ethanol pretreatment than sporulating species, the ethanol disinfection technique did not specifically select bacteria able to sporulate, as suggested by previous studies. Furthermore, some bacteria previously considered as potential candidates for bacteriotherapy have been lost after ethanol treatment. This study, while enriching the bacterial repertoire of the human intestine, would nevertheless require determining the exact contribution of each of species composing the bacterial consortia intended to be administered for CDI treatment.
Microbial culturomics: paradigm shift in the human gut microbiome study.
Lagier J-C,Armougom F,Million M,Hugon P,Pagnier I,Robert C,Bittar F,Fournous G,Gimenez G,Maraninchi M,Trape J-F,Koonin E V,La Scola B,Raoult D
Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases
Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.
Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media - A review.
Sarhan Mohamed S,Hamza Mervat A,Youssef Hanan H,Patz Sascha,Becker Matthias,ElSawey Hend,Nemr Rahma,Daanaa Hassan-Sibroe A,Mourad Elhussein F,Morsi Ahmed T,Abdelfadeel Mohamed R,Abbas Mohamed T,Fayez Mohamed,Ruppel Silke,Hegazi Nabil A
Journal of advanced research
Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding microbial gene functioning , since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel procedures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels.
Identification of Clostridioides difficile-Inhibiting Gut Commensals Using Culturomics, Phenotyping, and Combinatorial Community Assembly.
Ghimire Sudeep,Roy Chayan,Wongkuna Supapit,Antony Linto,Maji Abhijit,Keena Mitchel Chan,Foley Andrew,Scaria Joy
A major function of the gut microbiota is to provide colonization resistance, wherein pathogens are inhibited or suppressed below infectious levels. However, the fraction of gut microbiota required for colonization resistance remains unclear. We used culturomics to isolate a gut microbiota culture collection comprising 1,590 isolates belonging to 102 species. This culture collection represents 34.57% of the taxonomic diversity and 70% functional capacity, as estimated by metagenomic sequencing of the fecal samples used for culture. Using whole-genome sequencing, we characterized species representatives from this collection and predicted their phenotypic traits, further characterizing isolates by defining nutrient utilization profiles and short-chain fatty acid production. When screened with a coculture assay, 66 species in our culture collection inhibited Several phenotypes, particularly, growth rate, production of SCFAs, and the utilization of mannitol, sorbitol, or succinate, correlated with inhibition. We used a combinatorial community assembly approach to formulate defined bacterial mixes inhibitory to We tested 256 combinations and found that both species composition and blend size were important in inhibition. Our results show that the interaction of bacteria with one another in a mix and with other members of gut commensals must be investigated to design defined bacterial mixes for inhibiting Antibiotic treatment causes instability of gut microbiota and the loss of colonization resistance, thus allowing pathogens such as to colonize and causing recurrent infection and mortality. Although fecal microbiome transplantation has been shown to be an effective treatment for infection (CDI), a more desirable approach would be the use of a defined mix of inhibitory gut bacteria. The -inhibiting species and bacterial combinations identified herein improve the understanding of the ecological interactions controlling colonization resistance against and could aid in the design of defined bacteriotherapy as a nonantibiotic alternative against CDI.
Optimization of Culturomics Strategy in Human Fecal Samples.
Chang Yuxiao,Hou Fengyi,Pan Zhiyuan,Huang Zongyu,Han Ni,Bin Lei,Deng Huimin,Li Zhengchao,Ding Lei,Gao Hong,Zhi Fachao,Yang Ruifu,Bi Yujing
Frontiers in microbiology
Most bacteria in the human gut are difficult to culture, and culturomics has been designed to overcome this issue. Culturomics makes it possible to obtain living bacteria for further experiments, unlike metagenomics. However, culturomics is work-intensive, which prevents its wide application. In this study, we performed a 30-day continuous enrichment in blood culture bottles and cultured bacterial isolates from pre-cultures removed at different time points. We compared the bacteria isolated from the enriched culture with or without adding fresh medium after each pre-culture was removed. We also compared "experienced" colony picking (i.e., picking two to three colonies for each recognized colony type) and picking all the colonies from each plate. In total, from five fecal samples, 106 species were isolated, including three novel species and six that have not previously been isolated from the human body. Adding fresh medium to the culture increased the rate of bacterial species isolation by 22% compared with the non-supplemented culture. Picking all colonies increased the rate of bacterial isolation by only 8.5% compared with experienced colony picking. After optimization through statistical analysis and simulation, sampling aerobic and anaerobic enrichment cultures at six and seven time-points, respectively, is likely to isolate >90% of bacterial species, reducing the workload by 40%. In conclusion, an extended enrichment step ensures isolation of different bacterial species at different time-points, while adding the same quantity of fresh medium after sampling, the experienced picking and the optimized time-points favor the chance of isolating more bacterial species with less work.
Optimization and standardization of the culturomics technique for human microbiome exploration.
Diakite Ami,Dubourg Grégory,Dione Niokhor,Afouda Pamela,Bellali Sara,Ngom Issa Isaac,Valles Camille,Tall Mamadou Lamine,Lagier Jean-Christophe,Raoult Didier
Culturomics is a high-throughput culture approach that has dramatically contributed to the recent renewal of culture. While metagenomics enabled substantial advances in exploring the microbiota, culturomics significantly expanded our knowledge regarding the bacterial gut repertoire through the discovery and the description of hundreds of new taxa. While this approach relies on the variation of culture conditions and media, we have tested so far more than 300 conditions since the beginning of culturomics studies. In this context, we aimed herein to identify the most profitable conditions for optimizing culturomics approach. For this purpose, we have analysed a set of 58 culturomics conditions that were previously applied to 8 faecal specimens, enabling the isolation of 497 bacterial species. As a result, we were able to reduce the number of conditions used to isolate these 497 of more than a half (i.e. to 25 culture conditions). We have also established a list of the 16 conditions that allowed to capture 98% of the total number of species previously isolated. These data constitute a methodological starting point for culture-based microbiota studies by improving the culturomics workflow without any loss of captured bacterial diversity.
Many More Microbes in Humans: Enlarging the Microbiome Repertoire.
Lagier Jean-Christophe,Drancourt Michel,Charrel Rémi,Bittar Fadi,La Scola Bernard,Ranque Stéphane,Raoult Didier
Clinical infectious diseases : an official publication of the Infectious Diseases Society of America
The proportion of cultured microorganisms is dramatically lower than those predicted to be involved in colonization, acute, or chronic infections. We report our laboratory's contribution to promoting culture methods. As a result of using culturomics in our clinical microbiology laboratories (including amoeba co-culture and shell-vial culture) and through the use of matrix-assisted laser desorption/ionization-time-of-flight and the 16S rRNA gene for identification, we cultured 329 new bacterial species. This is also the first time that 327 of species have been isolated from humans, increasing the known human bacterial repertoire by 29%. We isolated 4 archaeal species for the first time from human, including 2 new species. Of the 100 isolates of giant viruses, we demonstrated the human pathogenicity of Mimivirus in pneumonia and Marseillevirus in diverse clinical situations. From sand flies, we isolated most of the known Phlebovirus strains that potentially cause human infections. Increasing the repertoire of human-associated microorganisms through culture will allow us to test pathogenicity models with viable microorganisms.
The contribution of culturomics to the repertoire of isolated human bacterial and archaeal species.
Bilen Melhem,Dufour Jean-Charles,Lagier Jean-Christophe,Cadoret Fréderic,Daoud Ziad,Dubourg Grégory,Raoult Didier
After a decade of research and metagenomic analyses, our knowledge of the human microbiota appears to have reached a plateau despite promising results. In many studies, culture has proven to be essential in describing new prokaryotic species and filling metagenomic gaps. In 2015, only 2172 different prokaryotic species were reported to have been isolated at least once from the human body as pathogens or commensals. In this review, we update the previous repertoire by reporting the different species isolated from the human body to date, increasing it by 28% to reach a total of 2776 species associated with human beings. They have been classified into 11 different phyla, mostly the Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. Finally, culturomics contributed up to 66.2% towards updating this repertoire by reporting 400 species, of which 288 were novel. This demonstrates the need to continue the culturing work, which seems essential in order to decipher the hidden human microbial content.
The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota.
Lagier Jean-Christophe,Hugon Perrine,Khelaifia Saber,Fournier Pierre-Edouard,La Scola Bernard,Raoult Didier
Clinical microbiology reviews
Bacterial culture was the first method used to describe the human microbiota, but this method is considered outdated by many researchers. Metagenomics studies have since been applied to clinical microbiology; however, a "dark matter" of prokaryotes, which corresponds to a hole in our knowledge and includes minority bacterial populations, is not elucidated by these studies. By replicating the natural environment, environmental microbiologists were the first to reduce the "great plate count anomaly," which corresponds to the difference between microscopic and culture counts. The revolution in bacterial identification also allowed rapid progress. 16S rRNA bacterial identification allowed the accurate identification of new species. Mass spectrometry allowed the high-throughput identification of rare species and the detection of new species. By using these methods and by increasing the number of culture conditions, culturomics allowed the extension of the known human gut repertoire to levels equivalent to those of pyrosequencing. Finally, taxonogenomics strategies became an emerging method for describing new species, associating the genome sequence of the bacteria systematically. We provide a comprehensive review on these topics, demonstrating that both empirical and hypothesis-driven approaches will enable a rapid increase in the identification of the human prokaryote repertoire.
Culturing the human microbiota and culturomics.
Lagier Jean-Christophe,Dubourg Grégory,Million Matthieu,Cadoret Frédéric,Bilen Melhem,Fenollar Florence,Levasseur Anthony,Rolain Jean-Marc,Fournier Pierre-Edouard,Raoult Didier
Nature reviews. Microbiology
The gut microbiota has an important role in the maintenance of human health and in disease pathogenesis. This importance was realized through the advent of omics technologies and their application to improve our knowledge of the gut microbial ecosystem. In particular, the use of metagenomics has revealed the diversity of the gut microbiota, but it has also highlighted that the majority of bacteria in the gut remain uncultured. Culturomics was developed to culture and identify unknown bacteria that inhabit the human gut as a part of the rebirth of culture techniques in microbiology. Consisting of multiple culture conditions combined with the rapid identification of bacteria, the culturomic approach has enabled the culture of hundreds of new microorganisms that are associated with humans, providing exciting new perspectives on host-bacteria relationships. In this Review, we discuss why and how culturomics was developed. We describe how culturomics has extended our understanding of bacterial diversity and then explore how culturomics can be applied to the study of the human microbiota and the potential implications for human health.
Culture of previously uncultured members of the human gut microbiota by culturomics.
Lagier Jean-Christophe,Khelaifia Saber,Alou Maryam Tidjani,Ndongo Sokhna,Dione Niokhor,Hugon Perrine,Caputo Aurelia,Cadoret Frédéric,Traore Sory Ibrahima,Seck El Hadji,Dubourg Gregory,Durand Guillaume,Mourembou Gaël,Guilhot Elodie,Togo Amadou,Bellali Sara,Bachar Dipankar,Cassir Nadim,Bittar Fadi,Delerce Jérémy,Mailhe Morgane,Ricaboni Davide,Bilen Melhem,Dangui Nieko Nicole Prisca Makaya,Dia Badiane Ndeye Mery,Valles Camille,Mouelhi Donia,Diop Khoudia,Million Matthieu,Musso Didier,Abrahão Jônatas,Azhar Esam Ibraheem,Bibi Fehmida,Yasir Muhammad,Diallo Aldiouma,Sokhna Cheikh,Djossou Felix,Vitton Véronique,Robert Catherine,Rolain Jean Marc,La Scola Bernard,Fournier Pierre-Edouard,Levasseur Anthony,Raoult Didier
Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.