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      Crosstalk Between Culturomics and Microbial Profiling of Egyptian Mongoose ( Herpestes ichneumon) Gut Microbiome

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          Abstract

          Recently, we unveiled taxonomical and functional differences in Egyptian mongoose ( Herpestes ichneumon) 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 Firmicutes 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.

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          Most cited references 47

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          The rebirth of culture in microbiology through the example of culturomics to study human gut microbiota.

          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.
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            Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA.

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            We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16S rRNA genes from bacteria. Their specificity and efficacy were tested systematically with a bacterial species and environmental samples. They were found to be more useful for 16S rRNA gene amplification in ecological and systematic studies than PCR amplimers that are currently more generally used.
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              Using ecological diversity measures with bacterial communities.

              Abstract There are many ecological diversity measures, but their suitability for use with highly diverse bacterial communities is unclear and seldom considered. We assessed a range of species richness and evenness/dominance indices, and the use of species abundance models using samples of bacteria from zinc-contaminated and control soils. Bacteria were assigned to operational taxonomic units (OTUs) using amplified ribosomal DNA restriction analysis of 236 clones from each soil. The reduced diversity apparent in the contaminated soil was reflected by the diversity indices to varying degrees. The number of clones analysed and the weighting given to rare vs. abundant OTUs are the most important considerations when selecting measures. Our preferences, arrived at using theory and practical experience, include: the log series index alpha; the Q statistic (but only if coverage is 50% or more); the Berger-Parker and Simpson's indices, although their ecological relevance may be limited; and, unexpectedly, the Shannon-Wiener and Shannon evenness indices, even though their meanings may not be clear and their values inaccurate when coverage is low. For extrapolation, the equation for the log series distribution seems the best for extrapolating from OTU accumulation curves while non-parametric methods, such as Chao 1, show promise for estimating total OTU richness. Due to a preponderance of single-occurrence OTUs, none of the five species abundance models fit the OTU abundance distribution of the control soil, but both the log and log normal models fit the less diverse contaminated soil. Species abundance models are useful, irrespective of coverage, because they address the whole distribution of a sample, aiding comparison by revealing overall trends as well as specific changes in particular abundance classes.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                27 May 2020
                June 2020
                : 8
                : 6
                Affiliations
                [1 ]National Institute for Agrarian and Veterinary Research (INIAV, IP), Wildlife, Hunting and Biodiversity R&D Unit, 2780-157 Oeiras, Portugal; andre.c.pereira94@ 123456gmail.com
                [2 ]Centre for Ecology, Evolution and Environmental Changes (cE3c), Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal
                [3 ]Biosystems & Integrative Sciences Institute (BioISI), Faculdade de Ciências da Universidade de Lisboa, 1749-016 Lisboa, Portugal
                [4 ]Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal; victor.bandeira@ 123456ua.pt (V.B.); cfonseca@ 123456ua.pt (C.F.)
                Author notes
                [* ]Correspondence: monica.cunha@ 123456iniav.pt ; Tel.: +351-214-403-500
                Article
                microorganisms-08-00808
                10.3390/microorganisms8060808
                7355707
                32471180
                © 2020 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

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