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      Comparative Evaluation of Band-Based Genotyping Methods for Mycobacterium intracellulare and Its Application for Epidemiological Analysis

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          Abstract

          Mycobacterium intracellulare is a leading cause of nontuberculous mycobacterial pulmonary disease, with a rapidly increasing prevalence worldwide. This bacterium, commonly distributed in soil and water, is known to be transmitted through the environment rather than between people. Therefore, it is imperative to establish distinguishable genotyping methods to understand the clinical outcome, disease relapses, and epidemiology. Therefore, in this study, representative band-based genotyping methods were performed using M. intracellualre clinical isolates, and their Hunter–Gaston discriminatory index (HGDI) was 0.947, 0.994, and 1 for variable number tandem repetition (VNTR), VNTR-mycobacterial interspersed repetitive units, pulsed field gel electrophoresis, and repetitive sequence based-PCR, respectively. Although VNTR showed relatively low HGDI, co-infection with other M. intracellualre strains could be determined by loci showing allele diversity from 0 to 0.69. Additionally, genetic distance of clinical isolates from Gyeongnam/Korea, and other regions/countries were visualized by minimum spanning tree (MST) using the globally available VNTR profiles. The results of MST revealed that M. intracellulare isolated from patients in Gyeongnam/Korea had specific VNTR genotypes, which may be evidence of the geographic distribution of M. intracellulare specific genotypes. The comparative results of genotyping techniques and geographical characteristics in this study may provide fundamental information for the epidemiology of M. intracellulare.

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          Most cited references32

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          Poppr: an R package for genetic analysis of populations with clonal, partially clonal, and/or sexual reproduction

          Many microbial, fungal, or oomcyete populations violate assumptions for population genetic analysis because these populations are clonal, admixed, partially clonal, and/or sexual. Furthermore, few tools exist that are specifically designed for analyzing data from clonal populations, making analysis difficult and haphazard. We developed the R package poppr providing unique tools for analysis of data from admixed, clonal, mixed, and/or sexual populations. Currently, poppr can be used for dominant/codominant and haploid/diploid genetic data. Data can be imported from several formats including GenAlEx formatted text files and can be analyzed on a user-defined hierarchy that includes unlimited levels of subpopulation structure and clone censoring. New functions include calculation of Bruvo’s distance for microsatellites, batch-analysis of the index of association with several indices of genotypic diversity, and graphing including dendrograms with bootstrap support and minimum spanning networks. While functions for genotypic diversity and clone censoring are specific for clonal populations, several functions found in poppr are also valuable to analysis of any populations. A manual with documentation and examples is provided. Poppr is open source and major releases are available on CRAN: http://cran.r-project.org/package=poppr. More supporting documentation and tutorials can be found under ‘resources’ at: http://grunwaldlab.cgrb.oregonstate.edu/.
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            Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

            Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.
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              Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity.

              An index of discrimination for typing methods is described, based on the probability of two unrelated strains being characterized as the same type. This index may be used to compare typing methods and select the most discriminatory system.
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                Author and article information

                Journal
                Microorganisms
                Microorganisms
                microorganisms
                Microorganisms
                MDPI
                2076-2607
                28 August 2020
                September 2020
                : 8
                : 9
                : 1315
                Affiliations
                [1 ]Department of Microbiology, College of Medicine, Gyeongsang National University, Jinju 52727, Korea; jung20787@ 123456gmail.com (J.-I.S.); hjh8824@ 123456gmail.com (J.-H.H.); hanwa5@ 123456nate.com (D.-H.L.); lg80204594@ 123456gmail.com (J.-G.C.); kmin2514@ 123456gmail.com (K.-M.K.); mjung@ 123456gnu.ac.kr (M.J.); scbaik@ 123456gnu.ac.kr (S.-C.B.); wklee@ 123456gnu.ac.kr (W.K.L.); kangssi@ 123456gnu.ac.kr (H.-L.K.)
                [2 ]Department of Convergence Medical Sciences, Institute of Health Sciences, Gyeongsang National University, Jinju 52727, Korea
                [3 ]Department of Internal Medicine, Gyeongsang National University Hospital, College of Medicine, Gyeongsang National University, Jinju 52727, Korea; juny2278@ 123456naver.com (S.J.L.); dr202202@ 123456naver.com (Y.Y.J.); ljd8611@ 123456nate.com (J.D.L.)
                Author notes
                [* ]Correspondence: mkshin@ 123456gnu.ac.kr (M.-K.S.); chareok-sa@ 123456daum.net (J.-W.Y.); Tel.: +82-55-772-8081 (M.-K.S.); +82-55-750-9783 (J.-W.Y.)
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0001-7805-1172
                https://orcid.org/0000-0001-8124-8945
                https://orcid.org/0000-0003-1782-5351
                https://orcid.org/0000-0002-2137-3848
                Article
                microorganisms-08-01315
                10.3390/microorganisms8091315
                7564390
                32872369
                663b3b34-12bc-424d-a66d-8d021a828652
                © 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/).

                History
                : 21 July 2020
                : 27 August 2020
                Categories
                Article

                mycobacterium intracellulare,molecular epidemiology,pulsed-field gel electrophoresis (pfge),variable number tandem repeat (vntr),mycobacterial interspersed repetitive units (miru),repetitive sequence based-pcr (rep-pcr)

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