The 12 M. abscessus strains used in this study were collected between July 2009 and June 2011 from the Clinical Microbiology Laboratory of University of Malaya Medical Centre (UMMC), Kuala Lumpur, Malaysia. PubMed  1. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. Methods of Classifying and Identifying Microorganisms. In the SILVA taxonomic framework, taxa names are adapted from widely accepted sources, and discrepancies are resolved with the overall aim of making taxonomy consistent with phylogeny. 1992, 42: 240-245. The distinction between prokaryotes and eukaryotes introduce in the 1960s. PubMed  Kanehisa M, Goto S: KEGG: Kyoto Encyclopedia of Genes and Genomes. Little R, Dennis PP: Regulation of RNA Polymerase Synthesis Conditional Lethal Amber Mutations In The β Subunit Gene. J Clin Microbiol. It generates trees called cladograms, which are groups of organisms that include an ancestor species and its descendants. For MSA with noisy columns, H increases. PubMed Google Scholar. Thus, it is better than the latter for the identification of closely-related mycobacterial species [23]. Tuberculosis. Antonie van Leeuwenhoek. Taking the clustering of strains based on the 50 median-ranked genes as the gold standard, we subsequently deduced the set of minimum median-ranked genes needed to recover the same clustering by iteratively reducing the number of median-ranked genes used and checking the resulting change in clustering topology. Here we describe AMPHORA2, an automated phylogenomic inference tool that can be used for high-throughput, high-quality genome tree reconstruction and metagenomic phylotyping. After Darwin published his theory of evolution in the 1800s, scientists looked for a way to classify organisms that showed phylogeny. 1999, 49: 1493-1511. nov., designation of Mycobacterium abscessus subsp. statement and All isolates were kept in Middlebrook 7H9 broth with 15% glycerol, at -80°C, until required for further testing. Inaoka T, Matsumura Y, Tsuchido T: SodA and manganese are essential for resistance to oxidative stress in growing and sporulating cells of Bacillus subtilis. Based on the set of 50 median-ranked genes, a benchmark phylogeny was obtained, and we used its topology to infer subspecies classification. This approach has been extremely successful in revealing the overwhelming diversity of microbial life , but it also has some limitations due to quantitative errors: The polymerase chain reaction (PCR) step introduces amplification bias, and it generates chimeric and otherwise erroneous molecules that hamper phylogenetic analysis (8, 9). Gram stain and motility were included in the best‐fit model but were not significant. Edgar RC: MUSCLE: multiple sequence alignment with high accuracy and high throughput. Walker GC: Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. All computation was done using an in-house R script [59] (available upon request). On aligning the sequences of the 210 orthologous genes identified with BLASTClust, we found the MSA produced using MAFFT [30] to be more robust than that produced using MUSCLE [31]. Despite the elucidation of several of these habitats, many remain to be identified. Under the umbrella of the Genomic Encyclopedia of Bacteria and Archaea (GEBA), the DOE JGI is sequencing thousands of bacterial and archaeal genomes from diverse branches of the Tree of Life. Abbreviation: SM: supermatrix; PG: phylogenomics; SNP: single-nucleotide polymorphism. J Clin Microbiol. 10.1128/JCM.00753-12. Am Rev Respir Dis. Leao SC, Tortoli E, Euzeby JP, Garcia MJ: Proposal that Mycobacterium massiliense and Mycobacterium bolletii be united and reclassified as Mycobacterium abscessus subsp. Of the 28 additional M. abscessus strains, this phylogenomics analysis revealed 19 strains as M. abscessus sensu stricto and 9 strains as M. massiliense, supported by 97% to 98% bootstrap support. However, in many phylogenetic and phylogenomic studies, M. abscessus is clearly separated into three subgroups [16–18]. Adékambi T, Reynaud-Gaubert M, Greub G, Gevaudan MJ, La Scola B, Raoult D, Drancourt M: Amoebal Coculture of “Mycobacterium massiliense” sp. The taxonomic classification system (also called the Linnaean system after its inventor, Carl Linnaeus, a Swedish botanist, zoologist, and physician) uses a hierarchical model. Additional support for the reliability of the subspecies classification came from the analysis of erm41 and ITS gene sequences, single nucleotide polymorphisms (SNPs)-based classification and strain clustering demonstrated by a variable number tandem repeat (VNTR) assay and a multilocus sequence analysis (MLSA). As anticipated, of the 12 M. abscessus isolates analysed, only M24 is classified as M. bolletii, supported with 100% bootstrap support. Phylogenetic methods are powerful and widely used tools for reconstructing the ancestral history of pathogen populations. The phylogenetic tree constructed using the minimal set of three genes clearly showed three major groups corresponding to the three M. abscessus subspecies (Figure 5). The current classification of Bacteria and Archaea is based on an operational-based model, the so-called polyphasic approach, comprised of phenotypic, chemotaxonomic and genotypic data, as well as phylogenetic information. Tan, J.L., Khang, T.F., Ngeow, Y.F. Landan G, Graur D: Heads or tails: A simple reliability check for multiple sequence alignments. Recently, Wong et al. Am J Respir Crit Care Med. 1972, 73: 55-70. xy Lipoxygenases (EC. 10.1093/molbev/msr121. By using this systematic approach, we selected 50 median-ranked genes which would be the most phylogenetically optimal for the purpose of subspecies phylogeny inference. Geographic proximity can support mass-effect models where microbes from one site are transferred to another via migration processes. Bacteria: (d) Median phylogenetic breadth of bacterial pathogen; (e) PubMed records for bacterial pathogen; and (f) Significant categorical predictors. Unable to display preview. The pan-bacterial 16S rRNA gene, which is generally considered the best target for bacterial identification and phylogenetic analysis, was not used in this study as it has been reported to lack discriminatory power for M. abscessus subspecies [48]. CAS  California Privacy Statement, The Sphingobacteria (FCB group) includes Bacteroidetes, Calditrichaeota, Chlorobi, candidate phylum... Microgenomates. et al. 10.1099/00207713-49-4-1493. Cite this article. In this study, we also assessed a set of genes commonly used by other workers for the subspecies classification of M. abscessus (rpoB, hsp65, recA, secA, sodA, erm41 and ITS) and found inconsistent classification for certain strains with recA, sodA, erm41 and ITS genes. As different species and their subgroups are known to differ in their pathogenicity and susceptibility to antibiotics [4, 5], their rapid and accurate identification is important for making therapeutic decisions in patient management. The taxonomic name "Bacteria" refers only to … A data mining approach was used to rank and select informative genes based on the relative entropy metric for the construction of a phylogenetic tree. It is based on the evolution of life and shows the genetic relationships among organisms. i Phylogenetic trees generated from the concatenation of the selected genes, constructed with the maximum likelihood method with boostrap values at important branches (1,000 bootstrap replicates). Next, we removed all MSA with alignment gaps greater than 5% of the size of the alignment block. The Maximum Parsimony algorithm tree of OmpR was constructed using max-mini branch and bound model. (A) hsp65-based tree (Uniform rate), (B) rpoB-based tree (Gamma rate), (C) secA-based tree (Gamma rate), (D) recA-based tree (Gamma rate), (E) sodA-based tree (Uniform rate), (F) supermatrix tree (Gamma rate). 1999, 181: 1939-1943. IEEE Xplore, delivering full text access to the world's highest quality technical literature in engineering and technology. If these possible clades, candidate phyla , are included, the number of phyla is 52 or higher. J Bacteriol. The presences of similarities between microorganisms have derived by numerical taxonomic methods based on a range of present-day observable characteristics (phonetics), usual morphological and physiological test characters. 10.1099/ijs.0.009126-0. The set of median-ranked genes has good coverage of all important functional classes compared to the top and bottom-ranked genes, so using their concatenation for phylogenetic inference potentially avoids systematic biases that can be caused by having a concentration of genes in the ranked list for some particular functional class. BMC Genomics 14, 879 (2013). 2006, 56: 133-143. To quantify the phylogenetic signal of the ith gene (g Phylogenetic approaches to microbial community classification. 10.1128/JCM.00319-08. Given a list of genes ranked from high to low entropy, we propose that a subset of genes in the neighbourhood of the median of this ranked list would be the most phylogenetically optimal for the purpose of subspecies phylogeny inference. It was classified as M. massiliense in the current study and also in two recent publications [17, 18] using our M. abscessus genome sequences for comparative analyses. None of the additional 28 strains were identified as M. bolletii. 10.1111/j.1574-6968.2001.tb10597.x. Top ten group features. The late 19th century was the beginning of bacterial taxonomy and classification of bacteria were classified on the basis of phenotypic markers. 2008, 46: 3384-3390. PubMed  10.1093/clind/16.4.463. [32]. Qamra R, Mande SC, Coates ARM, Henderson B: The unusual chaperonins of Mycobacterium tuberculosis. 10.1128/JCM.00037-09. BMC Genomics For example, the strains from 6G0125S to 4S0726RB were grouped into the M. abscessus sensu stricto cluster, whereas the strains from 47J26 to 5S0422 were likely to be M. massiliense. There are mainly three approaches, phylogenetic, Adansonian and molecular. These LUCAs eventually evolved into three different cell types, each representing a domain. [32] showed that a variable-number tandem-repeat (VNTR) typing assay for the M. abscessus species seemed to give good differentiation of M. abscessus strains. Fifty genes - 25 to the left and right (in alternating order) of the list of median-ranked genes, were concatenated and used to reconstruct the phylogenetic tree of the 12 isolate strains. PubMed Central  2005, 55: 293-302. J Clin Microbiol. PubMed  (nt) is the row marginal frequency for state nt, and p This is seen in the superior robustness of our supermatrix trees whether they are based on the concatenation of five classical genes, core genome SNPs or our three gene markers. ) gene, we used the relative entropy metric for a gene’s MSA: where s is the number of sequences, L is the length of the MSA, p 2007, 24: 2433-2442. Mycobacterium abscessus is a rapidly growing mycobacterium that is often associated with human infections. Am Rev Respir Dis. 2001, 197: 151-157. J Clin Microbiol. The addition of 28 strains into this study did not change the classification of the 12 strains from our initial analysis. 2009, 59: 2429-2436. (1990). 2012, 50: 3084-3088. Phylogenetic Classification. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O: The RAST Server: rapid annotations using subsystems technology. (nt) is the column marginal frequency for state nt. These include (a) phylogenetic, (b) Adansonian, and (c) genetic classifica-tions, which are discussed below: Phylogenetic classification. Two additional genes, erm41 and 16S-23S rRNA ITS with known features for subspecies identification were used to further support the classification results. Clin Microbiol. 2009, 47: 2596-2600. 2011, 55: 775-10.1128/AAC.00861-10. nov. from the Sputum of a Patient with Hemoptoic Pneumonia. PubMed Central  To exclude co-optimality of MSA as a potential source of alignment error, we further performed the Heads or Tails (HoT) analysis [58] on the identified orthologs. 2009, 24: 332-340. PubMed Central  All target genes for analysis were extracted from the assembled genomes of the 12 M. abscessus strains. The earliest Eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of Archaea e. Molecular biology, life-history, and fossil record evidence support the reunification of bacteria as Prokarya while subdividing Eukarya into uniquely defined subtaxa: Protoctista, Animalia, Fungi, and Plantae. nat.) Diagnostics using such DNA-based tools, such as polymerase chain reaction, are increasingly popular due to their specificity and speed, compared to culture-based methods. Therefore, an 8-week microcosm amendment of the weekly addition of 13 C-glucose was performed on an Ultisol to link the phylogenetic classification of microbial taxa with their activation via the use of exogenous labile carbon (C). M93, M94 and M152 are classified as M. abscessus sensu stricto and others as M. massiliense. massiliense. Molecular phylogenetic methods and concepts constituted a paradigm apart from classical evolutionary biology. An analysis of 154 patients. In this study, we sequenced the genomes of 12 M. abscessus strains and used phylogenomic analysis to perform subspecies classification. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG: Clustal W and Clustal X version 2.0. Describe the different types of bacteria 3. Polyphasic Bacterial Taxonomy • More data will become available, more bacteria will be identified, there will be more information, and software development will need to address the combination and linking of the different databases. As shown in (B), the concatenation of three genes is sufficient to construct a phylogenetic tree showing subspecies classification similar to that in the supermatrix tree of fifty genes. to species status: Mycobacterium abscessus comb, nov. Int J Syst Bacteriol. Delsuc F, Brinkmann H, Philippe H: Phylogenomics and the reconstruction of the tree of life. 10.1099/00207713-36-3-458. Google Scholar. nov. Int J Syst Evol Microbiol. We made the method practical by discovering a three-member minimal gene set that could return the same subspecies classification obtained using 50 median-ranked genes. In this work, we have presented a general approach for the development of phylogeny-informed proteomic assays for the diagnostic classification of bacterial pathogens with reticulated phylogenies, using the B. cepacia complex (Bcc) as an example. We would like to thank Hien Fuh Ng and Yan Ling Wong for helping us to design primers and optimizing the PCR for the 3 new gene markers in this project. Google Scholar. 10.1093/oxfordjournals.molbev.a026338. Phylogenetic classification system is based on the evolutionary ancestry. 1990, 215: 403-10. Multiple alignments of erm41 and 16S-23S rRNA ITS gene sequences. nov., two new rapidly growing species related to Mycobacterium smegmatis and associated with human wound infections: a cooperative study from the International Working Group on Mycobacterial Taxonomy. The recent introduction of next-generation sequencing has made it possible to use whole- genome sequence information for subspecies classification via phylogenomic clustering. Classification of strains according to different methods. Mol Biol Evol. Martins, E. P. & Hansen, T. F. Phylogenies and the comparative method: a general approach to incorporating phylogenetic information into the analysis of interspecific data. 1999, 37: 1714-1720. Zhang ZG, Ye ZQ, Yu L, Shi P: Phylogenomic reconstruction of lactic acid bacteria: an update. 10.1038/nrg1603. The purpose of this study is to test the phylogenetic informativeness of five classical housekeeping genes: rpoB, hsp65, secA, recA and sodA that are commonly used in M. abscessus subspecies classification. The classification for our 12 strains was similar to those obtained with the rpoB, hsp65 and secA genes. Burns DN, Wallace RJ, Schultz ME, Zhang YS, Zubairi SQ, Pang YJ, Gibert CL, Brown BA, Noel ES, Gordin FM: Nosocomial outbreak of respiratory tract colonization with Mycobacterium fortuitum: demonstration of the usefulness of pulsed-field gel electrophoresis in an epidemiologic investigation. At the end of our study, many M. abscessus genome sequences have been deposited in NCBI Genbank database. 2000, 38: 301-308. Consider the cladogram in Figure 6. Any set of two or more OTUs that constitutes a clade in the tree can also be represented as a feature, with the relative abundance of this group expressed as the sum of the frequencies of its constituent OTUs. Le´vy-Fre´bault V, Grimont F, Grimont PAD, David HL: Deoxyribonucleic acid relatedness study of the Mycobacterium fortuitum-Mycobacterium chelonae complex. 10.1093/bioinformatics/btm404. To study these relationships biologists rely on comparative genomics, as physiology and comparative anatomy are not possible methods. Rooney AP, Price NP, Ehrhardt C, Swezey JL, Bannan JD: Phylogeny and molecular taxonomy of the Bacillus subtilis species complex and description of Bacillus subtilis subsp. nov. and recognition of Mycobacterium porcinum from human clinical isolates. Phylogenetic trees were reconstructed from both data sets of aligned protein sequences and from an equivalent selection of 16S rRNA sequences by applying distance matrix and maximum parsimony methods. A potential drawback of this method is that the optimality of phylogenetic signal in the genes used, as quantified using some suitable metric, is largely unknown because there is no systematic procedure to find suitable candidates from a population of genes. This classification was in accordance with and supported by the analysis of hsp65 and rpoB gene sequences (Additional file 1: Figure S2). CAS  10.1111/j.1348-0421.2010.00221.x. The trees were constructed using the maximum likelihood method, with 1000 bootstrap replicates. Answer: D. A bacterial species differs from a species of eukaryotic organisms in that a bacterial species The phylogenetic classification of bacteria is based on. Figure 2: A mass spectrum of an E. colisample. 10.1099/ijs.0.63222-0. Google Scholar. 1993, 31: 175-178. Indeed, recent data using these approaches, while meagre, support the view that speciation processes may occur at the subspecies level within ecological niches ecovars and owing to biogeography geovars. Subsequently, the species was divided into three subspecies: M. abscessus sensu stricto, M. massiliense and M. bolletii [12–14] but the most recent classification describes just two subspecies which are M. abscessus subspecies abscessus (formerly M. abscessus sensu stricto) and M. abscessus subspecies bolletii that includes the previous two subspecies of M. bolletii and M. massiliense [15]. The OmpR gene sequences were used for finding similarities and genetic distances with other related bacterial species. These include (a) phylogenetic, (b) Adansonian, and (c) genetic classifica-tions, which are discussed below: Phylogenetic classification 2011, 61: 2311-2313. Previous studies have shown that the erm41 and the 16S-23S rRNA ITS genes could be used to differentiate the subspecies of M. abscessus [12, 19]. In Wolbachia phylogenetic studies, ftsZ has been used in conjunction with other genes [35, 36]. Tsung Fei Khang or Siew Woh Choo. In biology, phenetics ( Greek: phainein - to appear) / fɪˈnɛtɪks /, also known as taximetrics, is an attempt to classify organisms based on overall similarity, usually in morphology or other observable traits, regardless of their phylogeny or evolutionary relation. Clin Infect Dis. BibTex; Full citation; Abstract. Phylogenetic methods are powerful and widely used tools for reconstructing the ancestral history of pathogen populations. Kusunoki S, Ezaki T: Proposal of Mycobacterium peregrinum sp. Microbiol Immunol. J Clin Microbiol. A multiple gene-based approach, if the genes are carefully chosen, may amplify the individual phylogenetic signals present in the genes and lead to an accurate inference of phylogenetic tree for subspecies classification [40, 41]. Bacterial taxonomy (G: taxis = arrangement or order, nomos = law or nemein = to distribute or govern), in a broader sense, consists of three separate but interrelated disciplines: classification… Existing methods (multiple-genes, MLSA) are not able to produce such benchmarks, and their resulting classification accuracy may be largely a matter of luck rather than science. Single-celled microorganisms were the first forms of life to develop on earth, approximately 3 billion�4 billion years ago. The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. We have shown how a large pool of orthologs can be identified bioinformatically from the genomes of 12 M. abscessus isolates, assembled from fragments generated using Illumina shotgun sequencing. This helps to trace their evolution. Other housekeeping genes that have been used in RGM studies, albeit at lower frequency, include the internal transcribed spacer (ITS) of the 16S-23S rRNA gene [24], the rpoB [25], sodA [26], secA [16]. Traditionally, species identification for RGM is based on biological and biochemical tests such as pigment production, 3-day arylsulfatase reaction, nitrate reduction, iron uptake, and tolerance to 5% NaCl. Nucleic Acids Res. 1997, 46: 523-536. volume 14, Article number: 879 (2013) bolletii comb. Here, we examine the utility and effectiveness of traditional alignment-based phylogenies for reconstructing the evolutionary relationships among bacterial species, but also consider the more recently developed whole-genome (as well as alignment-free) approaches. OTUs can be useful entities for classifying microbial communities, but a strict OTU-based approach cannot directly use phylogenetically cohesive subsets of lineages within an OTU, or groups of OTUs, as predictors. With the use of BLASTClust on the putative CDS from all genomes including those of the three reference strains and the three outgroup species, 210 orthologous genes were identified. Antibiotic susceptibility has also been used to assist in species or subspecies classification. (1990). The phylogenetic classification of bacteria is based on differences in ribosomal RNA or rrna sequences. Introduction. 10.1099/00221287-73-1-55. Most of these clades and groups are annotated in the guide tree based on literature surveys and occasionally based on personal communications; therefore, not all clades are available in publications. It is responsible for more than 80% of the chronic lung diseases caused by RGM, some of which require surgical resection of the infected lung for complete resolution of symptoms [8]. Part of The number of plastic-degrading microorganisms reported is rapidly increasing, making it possible to explore the conservation and distribution of presumed plastic-degrading traits across the diverse microbial tree of life. The classification results of the 12 sequenced genomes using different approaches are summarized in Figure 6. An MSA with noiseless columns would yield a zero value since the nucleotide frequencies in the columns would be degenerate, and those in the rows would be the same as the nucleotide frequencies in the MSA. nov., Mycobacterium houstonense sp. 2002, 30: 3059-3066. 10.1099/ijs.0.02743-0. 2004, 32: 1792-1797. nov. and Mycobacterium brisbanense sp. nov. and Mycobacterium aubagnense sp. Additionally, it was identified as M. abscessus subsp. Summary: With the explosive growth of bacterial and archaeal sequence data, large-scale phylogenetic analyses present both opportunities and challenges. Since both taxonomy and species are dynamic entities, changes are rapid and supplemental resources are required to stay up-to-date. 1998, 52: 453-90. Ringuet H, Akoua-Koffi C, Honore S, Varnerot A, Vincent V, Berche P, Gaillard JL, Pierre-Audigier C: hsp65 Sequencing for Identification of Rapidly Growing Mycobacteria. 10.1146/annurev.micro.52.1.453. The orthologs among the putative open reading frames (ORFs) were identified using BLASTClust in a standalone BLAST program [47]. With DNA-DNA hybridization studies, however, it became recognized as a distinct species [10, 11]. 1997, 47: 535-540. Although 16S phylogeny is arguably excellent for classification of Bacteria and Archaea from the Domain level down to the family or genus, it lacks resolution below that level. The resulting SNP-based tree showed that the classification of the 12 M. abscessus strains is similar to the classification obtained from the phylogenomics tree generated using our systemic data mining approach. More extensive sampling of M. abscessus populations from different geographical locations and clinical settings would help to resolve current taxonomic uncertainties. Syst Biol. Google Scholar. © 2021 BioMed Central Ltd unless otherwise stated. Microbial phylogenetics is the study of the manner in which various groups of microorganisms are genetically related. Of the 12 strains we isolated, M139 had the most ambiguous taxonomic identity. abscessus in the MALDI Biotyper system (Bruker, Germany) that uses the MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry to identify organisms by their molecular (protein) fingerprints. Schinsky MF, Morey RE, Steigerwalt AG, Douglas MP, Wilson RW, Floyd MM, Butler WR, Daneshvar MI, Brown-Elliott BA, Wallace RJ, McNeil MM, Brenner DJ, Brown JM: Taxonomic variation in the Mycobacterium fortuitum third biovariant complex: description of Mycobacterium boenickei sp. The Protista contains unicellular microorganisms includ-ing eukaryotes and prokaryotes. Figure \(\PageIndex{1}\): A phylogenetic tree based on rRNA data, showing the separation of bacteria, archaea, and eukaryota domains. The purpose of phylogenetic analysis is to understand the past evolutionary path of organisms. The genome information of the reference strains was extracted from the NCBI Genbank [43] database. A new method for phylogenetic classification of bacterial strains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. 2004, 54: 1653-1667. By using this website, you agree to our 10.1093/molbev/msm176. Originally proposed by Woese and Fox, the classification of ribosomal RNA genes has been the gold standard for molecular taxonomic research for decades , .The 16S small ribosomal subunit gene (16S rRNA), in particular, has been widely used to study and characterize bacterial community compositions in a variety of ecological niches including host associated … J Clin Microbiol. Felsenstein J: Inferring Phylogenies. 1.13.11.12) are a non-heme iron enzymes consisting of one polypeptide chain folded into two domains, the N-terminal domain and the catalytic moiety β-barrel domain. Roth A, Fischer M, Hamid ME, Michalke S, Ludwig W, Mauch H: Differentiation of phylogenetically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences. Griffith DE, Girard WM, Wallace RJ: Clinical feature of pulmonary disease caused by rapidly growing mycobacteria. Analyzed the data: JLT, SWC and TFK. Google Scholar. Subsequently, we chose the MAFFT-aligned MSA for downstream analysis. The PCR-Restriction Enzyme analysis (PRA) is one of the most commonly used genotypic methods. For DNA extraction, 200 μL of suspension was processed with the ZR Fungal/Bacterial DNA MiniPrepTM (Zymo Research, USA) according to the protocol provided by the manufacturer.
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