title: Improved identification of rapidly growing mycobacteria by a 16S-23S internal transcribed spacer region PCR and capillary gel electrophoresis.
abstract: The identification of rapidly growing mycobacteria (RGM) remains problematic because of evolving taxonomy, limitations of current phenotypic methods and absence of a universal gene target for reliable speciation. This study evaluated a novel method of identification of RGM by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) followed by resolution of amplified fragments by capillary gel electrophoresis (CGE). Nineteen American Type Culture Collection (ATCC) Mycobacterium strains and 178 clinical isolates of RGM (12 species) were studied. All RGM ATCC strains generated unique electropherograms with no overlap with slowly growing mycobacteria species, including M. tuberculosis. A total of 47 electropherograms for the 178 clinical isolates were observed allowing the speciation of 175/178 (98.3%) isolates, including the differentiation of the closely related species, M. massiliense (M. abscessus subspecies bolletii) and M. abscessus (M. abscessus sensu stricto). ITS fragment size ranged from 332 to 534 bp and 33.7% of clinical isolates generated electropherograms with two distinct peaks, while the remainder where characterized with a single peak. Unique peaks (fragment lengths) were identified for 11/12 (92%) RGM species with only M. moriokaense having an indistinguishable electropherogram from a rarely encountered CGE subtype of M. fortuitum. We conclude that amplification of the 16S-23S ITS gene region followed by resolution of fragments by CGE is a simple, rapid, accurate and reproducible method for species identification and characterization of the RGM.
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Gray
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.
authors.forename: Fanrong
authors.initials: F
authors.lastname: Kong
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Jelfs
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia; Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, New South Wales, Australia; Marie Bashir Institute for Emerging Infections and Biosecurity, Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Vitali
authors.initials: V
authors.lastname: Sintchenko
authors.affiliation: Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead, New South Wales, Australia; Centre for Infectious Diseases and Microbiology - Public Health, Westmead Hospital, Westmead, New South Wales, Australia; Marie Bashir Institute for Emerging Infections and Biosecurity, Sydney Medical School, The University of Sydney, Sydney, New South Wales, Australia.
authors.forename: Sharon C-A
authors.initials: SC
authors.lastname: Chen
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods / Q000592:standards*; D021903:DNA, Ribosomal Spacer / Q000235:genetics*; D019075:Electrophoresis, Capillary; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D009170:Nontuberculous Mycobacteria / Q000145:classification / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000382:microbiology
chemical_list: D021903:DNA, Ribosomal Spacer; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S