Rapid Species Identification Within the Mycobacterium
Rapid Species Identification Within the Mycobacterium
Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonaeabscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.
The Mycobacterium chelonaeabscessus group of rapidly growing mycobacteria consists of Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum. M abscessus was first isolated from a knee abscess in 1953 and causes pulmonary disease and wound infections. M chelonae, first described in 1972, is rare in pulmonary disease but common in cutaneous and catheter-related infections. M immunogenum was identified only recently in metalworking fluid, and, like M abscessus and M chelonae, infections often are nosocomial or related to immunodeficiency.
The different species within the M chelonaeabscessus group are biochemically similar and often are not distinguished in the clinical laboratory. However, there is less than 70% genomic homology among these species. Furthermore, each species is unique in antibiotic resistance, and M abscessus and M chelonae probably are the most antibiotic-resistant species of the pathogenic rapidly growing mycobacteria, with therapy limited to only a few agents. For treatment, tobramycin is the preferred aminoglycoside for M chelonae and amikacin for M abscessus, but the key difference is that M chelonae is resistant to cefoxitin, whereas M abscessus is often susceptible. In addition, M chelonae seems to be more susceptible to the newer antimicrobials linezolid and gatifloxacin than M abscessus. The need for appropriate therapy and epidemiologic surveillance has led to several techniques for differentiation.
Previous methods that distinguish species within the M chelonaeabscessus group include phenotyping tests such as sodium chloride tolerance and citrate utilization, high-performance liquid chromatography of mycolic acids, and multilocus enzyme electrophoresis. These tests require special expertise and equipment or take weeks to complete. Previous genotyping methods include polymerase chain reaction (PCR)restriction fragment length polymorphism analysis, randomly amplified polymorphic DNA, oligonucleotide probes, and DNA sequencing.
Regions often used for mycobacteria species identification include the 16S ribosomal RNA (rRNA) gene, the 16S-23S spacer region, the β subunit of RNA polymerase (rpoB), and the 65-kd heat shock protein (hsp) gene. The M chelonaeabscessus group is very conserved throughout the 16S rRNA gene and the 16S-23S spacer region. In fact, M abscessus and M chelonae are identical in sequence throughout the proximal 500 bases of the 16S rRNA gene that often is used for species identification of mycobacteria. We sought a simple method to differentiate M abscessus and M chelonae when 16S sequencing identifies the M chelonaeabscessus group. We chose to analyze the hsp65 gene because of significant sequence variation between species, along with sequence conservation within species.
Rapid-cycle PCR with high-resolution melting curve analysis is a new technique with advantages over restriction analysis, probe hybridization, and DNA sequencing. PCR and high-resolution melting curve analysis, when combined, can be completed in less than 20 minutes, considerably faster than other methods. Melting curve analysis, like sequencing, can detect unexpected base changes. Finally, melting curve analysis is a closed-tube method with no exposure of amplicon to the laboratory environment.
Polymerase chain reaction (PCR) amplification of the heat shock protein 65 (hsp65) gene followed by high-resolution melting analysis with LCGreen I (Idaho Technology, Salt Lake City, UT) was used to differentiate the mycobacteria species Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum in less than 20 minutes. A 105-base-pair amplicon that clustered the different species by predicted melting temperature was found from available GenBank hsp65 sequences. We identified 24 clinical isolates within the M chelonaeabscessus group by proximal 16S ribosomal RNA and hsp65 gene sequencing. Rapid-cycle PCR followed by high-resolution melting analysis clustered these samples into the following groups: M abscessus, 12; M abscessus sequence variant, 2; M chelonae, 7; unexpected M chelonae sequence variant, 1; and M immunogenum, 2. The M chelonae variant had a single base change not found in reported GenBank sequences. Advantages of the method include speed, low risk of amplicon contamination (closed-tube), and no need for separation steps (sequencing, electrophoresis, high-performance liquid chromatography) or real-time monitoring.
The Mycobacterium chelonaeabscessus group of rapidly growing mycobacteria consists of Mycobacterium chelonae, Mycobacterium abscessus, and Mycobacterium immunogenum. M abscessus was first isolated from a knee abscess in 1953 and causes pulmonary disease and wound infections. M chelonae, first described in 1972, is rare in pulmonary disease but common in cutaneous and catheter-related infections. M immunogenum was identified only recently in metalworking fluid, and, like M abscessus and M chelonae, infections often are nosocomial or related to immunodeficiency.
The different species within the M chelonaeabscessus group are biochemically similar and often are not distinguished in the clinical laboratory. However, there is less than 70% genomic homology among these species. Furthermore, each species is unique in antibiotic resistance, and M abscessus and M chelonae probably are the most antibiotic-resistant species of the pathogenic rapidly growing mycobacteria, with therapy limited to only a few agents. For treatment, tobramycin is the preferred aminoglycoside for M chelonae and amikacin for M abscessus, but the key difference is that M chelonae is resistant to cefoxitin, whereas M abscessus is often susceptible. In addition, M chelonae seems to be more susceptible to the newer antimicrobials linezolid and gatifloxacin than M abscessus. The need for appropriate therapy and epidemiologic surveillance has led to several techniques for differentiation.
Previous methods that distinguish species within the M chelonaeabscessus group include phenotyping tests such as sodium chloride tolerance and citrate utilization, high-performance liquid chromatography of mycolic acids, and multilocus enzyme electrophoresis. These tests require special expertise and equipment or take weeks to complete. Previous genotyping methods include polymerase chain reaction (PCR)restriction fragment length polymorphism analysis, randomly amplified polymorphic DNA, oligonucleotide probes, and DNA sequencing.
Regions often used for mycobacteria species identification include the 16S ribosomal RNA (rRNA) gene, the 16S-23S spacer region, the β subunit of RNA polymerase (rpoB), and the 65-kd heat shock protein (hsp) gene. The M chelonaeabscessus group is very conserved throughout the 16S rRNA gene and the 16S-23S spacer region. In fact, M abscessus and M chelonae are identical in sequence throughout the proximal 500 bases of the 16S rRNA gene that often is used for species identification of mycobacteria. We sought a simple method to differentiate M abscessus and M chelonae when 16S sequencing identifies the M chelonaeabscessus group. We chose to analyze the hsp65 gene because of significant sequence variation between species, along with sequence conservation within species.
Rapid-cycle PCR with high-resolution melting curve analysis is a new technique with advantages over restriction analysis, probe hybridization, and DNA sequencing. PCR and high-resolution melting curve analysis, when combined, can be completed in less than 20 minutes, considerably faster than other methods. Melting curve analysis, like sequencing, can detect unexpected base changes. Finally, melting curve analysis is a closed-tube method with no exposure of amplicon to the laboratory environment.
