rpoB -Based Identification of Nonpigmented and Late-Pigmenting Rapidly Growing Mycobacteria

Abstract

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial ␤ subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited <2% partial rpoB gene sequence divergence from 1 of 20 species under study and were regarded as correctly identified at the species level. Mycobacterium abscessus and Mycobacterium mucogenicum isolates were clearly distinguished from Mycobacterium chelonae; Mycobacterium mageritense isolates were clearly distinguished from "Mycobacterium houstonense." Four isolates were not identified at the species level because they exhibited >3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.Rapidly growing mycobacteria (RGM) that require less than 7 days to produce easily visible colonies on solid media (42) comprise 56 environmental species. Fifteen species commonly encountered in humans and animals (7, 39) belong primarily to the Mycobacterium chelonae-abscessus, Mycobacterium fortuitum, and Mycobacterium smegmatis groups (7). They are increasingly encountered in clinical microbiology laboratories (54,65,67,70,76). They are responsible for pseudo-outbreaks of health care-associated septicemia and lung disease following bronchoscopy (1, 2, 37, 69). They are also responsible for colonization of airways (8, 15); skin and soft-tissue infections characterized by slowly progressive granulomatous inflammation, lymphadenitis, disseminated infection, and chronic pulmonary disease (4,7,26,28,66,69,75). Also, communityacquired outbreaks of skin infection following liposuction (43), M. fortuitum furunculosis following footbaths (73), hypersensitivit...

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

“…Most of the infections were related to laparoscopic or arthroscopic surgeries and were caused by RGM with a Mycobacterium abscessus type 2 polymerase chain reaction (PCR) restriction-enzyme analysis (PRA)-hsp65 pattern (BstE II: 235 and 210 bp; Hae III: 200, 70, 60 and 50 bp) (Devallois et al 1997). The identification of these isolates was confirmed by rpoB sequencing (Adékambi et al 2003). All of the obtained sequences were highly similar to the rpoB sequence of Mycobacterium massiliense CCUG 48898 (GenBank accession AY593981.2) and had two base substitutions (GenBank accession EU117207) (Leão et al 2010).…”

“…The 752 bp rpoB fragment described by Adékambi et al (2003) contains multiple DpnII recognition sites and a new primer, MycoR2 (Table II), was designed for the amplification of a 221 bp fragment that contains only the DpnII site of interest. This DpnII restriction site is absent in the epidemic strain due to an A/G substitution and the corresponding amplicon was not cut with this enzyme.…”

“…The rpoB amplification was performed using primers MycoF and MycoR, as described by Adékambi et al (2003). Two primers described in previous publications (Adékambi & Drancourt 2004, Gomila et al 2007 were used for the amplification and sequencing of the 3' region of the 16S rDNA.…”

“…PRA-rpoB -For the PRA-rpoB determinations, the primers MycoF (Adékambi et al 2003) and MycoR2 (Table II) were used to amplify a 221 bp fragment of the rpoB gene. The 15 µL reactions were composed of the following reagents: 1.5 µL of 10X Buffer (150 mM TrisHCl, pH 8.75, 500 mM KCl, 1% Triton X-100 and 20 mM of MgCl 2 ), 140 µM of each dNTP, 0.4 µM of each primer, 0.3 U of Taq DNA Polymerase (RBC Biosciences Corp, Taipei, Taiwan) and 2 µL of DNA.…”

Association between 16S-23S Internal Transcribed Spacer Sequence Groups of Mycobacterium avium Complex and Pulmonary Disease

“…Persons who had a respiratory specimen that grew MAC between 1 January 1993 and 30 June 1999 and available medical records were considered for inclusion (n ϭ 600). Case subjects were considered for inclusion if they met the 1997 American Thoracic Society criteria for MAC pulmonary disease (1). The following criteria were used to exclude both potential cases and controls: (i) human immunodeficiency virus infection, (ii) cystic fibrosis, (iii) age under 18 years, (iv) recipient of organ or bone marrow transplant, (v) pulmonary alveolar proteinosis, and (iv) active tuberculosis.…”