|Year : 2016 | Volume
| Issue : 3 | Page : 369-374
Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates
HY Kim1, SY Lee1, BJ Kim2, YH Kook3
1 Department of Microbiology and Immunology, Seoul National University College of Medicine, Institute of Endemic Diseases, Seoul 110 799, Republic of Korea
2 Department of Microbiology and Immunology; Department of Biomedical Sciences, Seoul National University College of Medicine, Institute of Endemic Diseases; Seoul National University Medical Research Center; Cancer Research Institute, Seoul National University College of Medicine, Seoul 110 799, Republic of Korea
3 Department of Microbiology and Immunology, Seoul National University College of Medicine, Institute of Endemic Diseases; Seoul National University Medical Research Center, Seoul National University College of Medicine, Seoul 110 799, Republic of Korea
|Date of Submission||18-Apr-2015|
|Date of Acceptance||21-Apr-2016|
|Date of Web Publication||12-Aug-2016|
Y H Kook
Department of Microbiology and Immunology, Seoul National University College of Medicine, Institute of Endemic Diseases; Seoul National University Medical Research Center, Seoul National University College of Medicine, Seoul 110 799
Republic of Korea
Source of Support: None, Conflict of Interest: None
On the basis of the structural differences of erm, we used a duplex polymerase chain reaction (PCR) to differentiate Mycobacterium abscessus subsp. abscessus and subsp. massiliense isolates and to detect the point mutations of 23S rRNA gene that confer a high level of resistance to clarithromycin. Subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined.
Keywords: 23S rRNA gene, erm (41), duplex polymerase chain reaction, Mycobacterium abscessus subspecies
|How to cite this article:|
Kim H Y, Lee S Y, Kim B J, Kook Y H. Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates. Indian J Med Microbiol 2016;34:369-74
|How to cite this URL:|
Kim H Y, Lee S Y, Kim B J, Kook Y H. Allele-specific duplex polymerase chain reaction to differentiate Mycobacterium abscessus subspecies and to detect highly clarithromycin-resistant isolates. Indian J Med Microbiol [serial online] 2016 [cited 2020 Jan 29];34:369-74. Available from: http://www.ijmm.org/text.asp?2016/34/3/369/188355
| ~ Introduction|| |
Recently, there have been increasing reports on infections caused by rapidly growing mycobacteria (RGM) worldwide. Among them, Mycobacterium abscessus, a species that usually causes respiratory and soft tissue infections and disseminated diseases in both immunosuppressed and immunocompetent persons, and its closely related species occupy the highest rate of RGM infections.  Since the existence of complicated genotype populations of M. abscessus isolates has been recognised , and subsequently supported by other characteristics, some of the M. abscessus isolates have been proposed as new species such as Mycobacterium massiliense and Mycobacterium bolletii. Soon after that, a large portion of the M. abscessus was revealed to be M. massiliense and reports of M. massiliense infections in many countries have increased. , Meanwhile, the nomenclature of these very closely related species has suffered confusions. Being more often called as the M. abscessus group; currently, the species is known as M. abscessus, subsp. abscessus, subspecies massiliense and subspecies bolletii.
Although their classification or nomenclature can be changed, one thing should not be ignored. That is, the differentiation of M. abscessus subsp. massiliense from subsp. abscessus.
Two subspecies that account for most of the M. abscessus group isolates has important clinical implications in antibiotic treatment. Although M. abscessus subsp. abscessus and subsp. massiliense are very similar in many phenotypic and genotypic aspects, they show marked difference in susceptibility to clarithromycin. ,, M. abscessus subsp. massiliense isolates are usually either markedly susceptible or highly resistant to clarithromycin.
However, M. abscessus subsp. abscessus isolates show diverse patterns such as susceptible, intermediate, resistant, or highly resistant. This unique difference was proven to have originated from the structural difference of the erythromycin ribosome methyltransferase gene (erm). , Basically, the clarithromycin resistance is known to be related to erm, that is, one of the genes related to macrolide resistance, and to the 23S rRNA gene.  The erm of M. abscessus subsp. massiliense has defects such as a frame shift mutation and large deletion, which cannot make a functional erm(41), whereas subsp. abscessus has truncated erm as in M. tuberculosis. Hence, clarithromycin resistance due to erm cannot happen in subsp. massiliense. This is also a good reason to be differentiated from subsp. abscessus. However, the other mechanism, the mutations of the 23S rRNA gene, can result in the subsp. massiliense being highly resistant to clarithromycin. According to the American Thoracic Society Documents,  multidrug regimens including clarithromycin may cause symptomatic improvement and disease regression of M. abscessus infection, which is very difficult to treat due to its resistance to many antibiotics. Although the therapeutic efficiency of M. abscessus was considerably variable,  a significant difference in antibiotic treatment efficacy between the two subspecies infection was reported.  The response rates to clarithromycin-based antibiotic therapy were higher in patients with M. massiliense lung disease than in those with M. abscessus lung disease. This shows the importance of identifying members of the M. abscessus group to the subspecies level based on recent nomenclature. In other words, because of the antibiotic susceptibility and therapeutic efficacy, the differentiation of M. abscessus subsp. massiliense from subsp. abscessus is essentially needed.
Based on the findings that highly clarithromycin-resistant strains have specific mutations in the 23S rRNA gene,  and the difference in erm gene structures exists between subsp. abscessus and subsp. massiliense,, we introduce a new duplex polymerase chain reaction (PCR) targeting those two genes to rapidly and simply differentiate M. abscessus subsp. massiliense and subsp. abscessus, as well as to detect highly resistant (≥256 μg/ml) strains to clarithromycin without culture-based susceptibility testing.
| ~ Methods|| |
Bacteria and DNA extraction
Eighty-one clinical isolates of M. abscessus (39 subsp. abscessus, 2 subsp. bolletii and 40 subsp. massiliense strains) were used in this blind PCR test. These isolates were previously identified by sequence analysis of rpoB and hsp65. Their minimum inhibitory concentrations (MICs) to clarithromycin, which had been previously determined,  were compared with the results of erm and the 23S rRNA gene allele-specific duplex PCR. In addition, reference strains such as M. abscessus (ATCC 19977), M. bolletii (KCTC 19281), M. massiliense (CIP 108297), M. chelonae (clinical isolate), M. immunogenum (ATCC 700505), M. porcinum (KCTC 9517), M. mageritense (ATCC 700351) and M. tuberculosis (ATCC 27294) were used as negative controls for PCR. These had been purchased and kept in LN2 tank of our laboratory.  RGM was cultivated on Ogawa media or blood agar plates at 37°C (or 30°C for M. immunogenum) under 5% CO 2 for 4 days. Total DNA were extracted from cultured colonies using the bead beater-phenol extraction method  and were used as templates for PCR.
Allele-specific duplex polymerase chain reaction
The following primer pairs were used on the basis of previously reported erm,, and 23S rRNA gene, , respectively: Primers ermF2 (5'-GAC CGG GGC CTT CTT CGT GAT C-3') and ermR2 (5'- GAC TTC CCC GCA CCG ATT CCA-3') for the erm gene [Figure 1]a, and primers crF1 (5'- TAC GCG CGG CAG GAC GAS-3'), crF2 (5'- ACG CGC GGC AGG ACG AAS-3') and crR (5'- AAA CCC AGC TCG CGT GCC-3') for the 23S rRNA gene [Figure 1]b. The ermF2-ermR2 primer set differentiates subsp. massiliense from the subsp. abscessus and subsp. bolletii. With this primer set, a 673-bp erm DNA can be amplified from subsp. abscessus and subsp. bolletii, whereas 397-bp DNA can be amplified from subsp. massiliense, but nothing from other mycobacteria. In addition, with the crF1 (or crF2)-crR primer set 546 (or 545)-bp 23S rRNA, a gene fragment produced, indicating that the strain has mutations related to clarithromycin resistance; i.e., A2059C or A2059G of 23S rRNA gene [Figure 1]b and c. As a result, the identification of M. abscessus subsp. abscessus (and bolletii) and subsp. massiliense, as well as the determination of their clarithromycin resistance, can be promptly achieved by interpreting four different band patterns. The template DNA (ca. 50 ng) and each erm primer (ermF2, ermR2) and crR, 10 pmol; crF1 and crF2, 5 pmol were added to a PCR mixture tube (AccuPower™ HotStart PCR PreMix; Bioneer, Daejeon, Korea) that contained 1 unit of HotStart DNA polymerase, 250 μM of deoxynucleotide triphosphate, 1.5 mM of MgCl2 and gel loading dye. The final volume was then adjusted to 20 μl with distilled water, after which the reaction mixture was amplified using a model 9700 Thermocycler (Perkin-Elmer Cetus). PCR products were detected by a 3% agarose gel electrophoresis.
|Figure 1: Primers used for the differential amplification of erm (41) (a) and 23S rRNA gene (b) from Mycobacterium abscessus subsp. abscessus, massiliense, and bolletii DNA. Because of the different size of the erm gene between subsp. abscessus (and bolletii; dark black) and subsp. massiliense (light black), the erm polymerase chain reaction produces 673 and 397-bp DNA, separately. The 23 rRNA gene polymerase chain reaction produces 545/546-bp DNA only in resistant strains (c) (S: Susceptible; R: Resistant)|
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| ~ Results|| |
As expected, newly developed allele-specific erm and 23S rRNA gene duplex PCR produced three different DNA bands from the DNAs of clinical isolates [Figure 2]. M. abscessus subsp. abscessus and subsp. bolletii showing 673 bp amplicon, and subsp. massiliense showing 397 bp amplicon could be easily distinguished. Although the purpose of this duplex PCR is to differentiate the subspecies of M. abscessus, other mycobacteria were also tested at the same time. However, no PCR product (neither 673 nor 397 bp) from other mycobacteria was detected [Figure 2]a.
|Figure 2: Amplification of erm and 23S rRNA gene by duplex polymerase chain reaction to differentiate and to detect highly resistant Mycobacterium abscessus subspecies strains to clarithromycin. (a) Mycobacterium abscessus subsp. abscessus and bolletii were identified with 673 bp from massiliense showing 397 bp erm amplicon, (b and c) including clinical isolates of massiliense or abscessus. The highly resistant isolates to clarithromycin showed 545 (or 546) bp 23S rRNA amplicons. a: M, 100-bp ladder marker; 1, Mycobacterium abscessus; 2, Mycobacterium bolletii; 3, Mycobacterium massiliense; 4, Mycobacterium chelonae; 5, Mycobacterium immunogenum; 6, Mycobacterium porcinum; 7, Mycobacterium mageritense; 8, Mycobacterium tuberculosis|
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All of the tested clinical isolates could also be easily identified as either M. abscessus subsp. abscessus or subsp. massiliense. In addition, clarithromycin resistance of all clinical isolates could be easily determined by the presence of 545 (or 546) bp 23S rRNA gene amplicons [Figure 2]b and c]. The clarithromycin MICs of M. abscessus strains, which had been determined in a previous study,  were compared to the results of the duplex PCR [Table 1].
|Table 1: Comparison of the clarithromycin susceptibility of Mycobacterium abscessus isolates to the results of duplex polymerase chain reaction |
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All the tested M. abscessus isolates were differentiated into two groups showing either 673 bp or 397 bp DNA. Forty-one isolates yielded a 673-bp band, which could be subsp. abscessus and subsp. bolletii. The other forty strains that yielded 397-bp DNA were subsp. massiliense. An additional 545/546 bp band, which stands for high resistance to clarithromycin, was found in two strains of the subsp. M. abscessus and five strains of subsp. massiliense identified [Table 1]. These were highly resistant (MIC > 256 μg/ml) strains having point mutations of adenine at the 2058 th or 2059 th nucleotide in the 23S rRNA gene. , Compared to the MIC, the sensitivity and specificity of the duplex PCR to differentiate two subspecies and to detect highly resistant strains were 100% in M. abscessus subspecies.
However, thirty-nine strains of subsp. abscessus, two strains of subsp. bolletii and 35 strains of subsp. massiliense did not show a 545/546 bp band. When compared to the previously determined MICs, subsp. bolletii and eighteen susceptible subsp. abscessus strains (48.8%, MIC ≤ 2 μg/ml), four intermediate strains (9.8%, MIC = 4 μg/ml) and 15 moderate resistant strains (36.6%, MIC < 64 μg/ml) of the subsp. abscessus belonged to this group, whereas 87.5% of subsp. massiliense strains were susceptible (MIC ≤ 2 μg/ml).
| ~ Conclusion|| |
One of the most important "key" for the classification and identification of bacteria is antibiotic susceptibility. Clarithromycin resistance invariably appears among subsp. abscessus and M. massiliense isolates. Although the phenotype is very similar and classification (or nomenclature) is continuously changed, subsp. massiliense and subsp. abscessus clearly show quite different clarithromycin-resistance patterns. Much of the mechanisms of this different clarithromycin resistance were known at the gene level such as erm and 23SrRNA gene. Hence, using these genes, we can identify and differentiate these two subspecies and predict their clarithromycin susceptibility which, in turn, helps treatment choices. 
The different pattern of clarithromycin susceptibility may be due to their species-specific erm (41) sequences. Unlike subsp. massiliense, subsp. abscessus has truncated erm, which may possibly confer atypical resistance to macrolide as in M. tuberculosis. It may be plausible that intermediate and moderate resistance not observed in subsp. massiliense are related to the difference of erm (41) structure. Another pattern may only be caused by point mutations of in the 23S rRNA gene, which confers a very high MIC (>256 μg/ml) in subsp. massiliense, whereas that of subsp. abscessus can be caused either by point mutations of the 23S rRNA gene and/or by target alteration or modification  such as methylation. Although we do not have experimental evidences, we can suspect that the intermediate or moderate resistances of subsp. abscessus strains may possibly be due to the target alteration or modification. In addition, if we observe them longer period (>1 week) after 3-day culture for susceptibility testing, it is not uncommon to observe their slow growth in testing media.
Inducible resistance was already reported.  In subsp. Massiliense, we cannot see such phenomenon. Hence, if clinicians are reported susceptible subsp. massiliense, they can use clarithromycin with ease and anticipate good treatment efficacy. However, if susceptible subsp. abscessus is reported, it is better to regard it as a low-level resistant strain and consider combination therapy according to the progress of patient's condition. The significance and importance of differentiating subsp. massiliense from subsp. abscessus was clearly demonstrated in a previous clinical analysis. ,,
To meet the above clinical needs and on the basis of the structural differences of erm, we used a duplex PCR to differentiate subsp. abscessus and subsp. massiliense strains and to detect the point mutations of 23S rRNA gene. The subsp. abscessus strains that show a relatively low level of resistance cannot be detected with this PCR. Although this is the weakness of the current assay, the advantages of this duplex PCR is that subsp. massiliense strains occupying almost half of the clinical isolates can be simply identified, and their clarithromycin susceptibility can be rapidly determined without culture-based testing. This will be helpful for the diagnosis and treatment of M. abscessus group infections.
This work was supported by grant 0420120250 (2012-1252) from the SNUH Research Fund and in part by National Research Foundation of Korea (NRF) grant (Grant No., NRF-2014R1A1A2004008), Republic of Korea.
Financial support and sponsorship
SNUH Research Fund and in part by National Research Foundation of Korea.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]