1. 서울대학교 의과대학 검사의학교실1, 분당서울대학교병원 진단검사의학과2
대한임상미생물학회 15차 학술대회
(Thu.) 올림픽파크텔
Determination of clarithromycin susceptibility in M. abscessus sensu stricto and M. massiliense by erm(41)and rrl pyrosequencing
Yun Ji Hong1*, Myoung Shin Kim2, Taek Soo Kim1,2, Kyoung Un Park1,2, Junghan Song1,2, Eui-Chong Kim1 
1Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul;
2Department of Laboratory Medicine, Seoul National University Bundang Hospital, Seongnam, Korea
홍윤지1*, 김명신2, 김택수1,2, 박경운1,2, 송정한1,2, 김의종1
 서울대학교 의과대학 검사의학교실1, 분당서울대학교병원 진단검사의학과2

2. Mycobacteria Aerobic, nonmotile, acid-fast bacilli
Mycobacterium tuberculosis complex
M. tuberculosis
M. bovis
M. bovis Bacille Calmette-Guérin strain
M. caprae
M. pinnipedii
M. africanum
M. microtii
M. canettii
Nontuberculous mycobacteria
Not well understood
Antigens associated with virulence
Immune response to infection
Category
Slowly growing mycobacteria
(including MTBC)
Rapidly growing mycobacteria

3. Rapidly growing mycobacteria (RGM)
Environmental isolates
Clinical manifestation
Skin and soft tissue infection
Chronic pulmonary disease
Patient with cystic fibrosis or bronchiectasis
Endocarditis involving a prosthetic valve
Most common human pathogens are
M. abscessus
M. chelonae
M. fortuitum

4. Mycobacterium abscessus
Nomenclature
M. abscessus (separated in 1992)
Continuous change in the name
Genotype analysis (housekeeping gene, e.g. rpoB and hsp65)
heterogeneous population
Nearly half of RGM initially identified as M. abscessus: actually M. massiliense
M. abscessus sensu lato in 2004
M. abscessus Group I: M. abscessus sensu stricto
M. abscessus Group II: M. bolletii & M. massiliense
Microbiol Immunol 2010 Jun;54(6):
Antimicrob Agents Chemother 2011 Feb;55(2):
J Antimicrob Chemother 2012 Apr;67(4):810-8.

5. Treatment of mycobacterial disease
Reputation for resistance
Intrinsically resistant to the classical antituberculous drugs
Resistant to most of the antibiotics that are currently available
Clarithromycin
Adhesion to the vicinity of peptidyltransferase center
Inhibition of protein synthesis
Henry’s clinical diagnosis and management by laboratory methods, 22nd ed. Philadelphia Elsevier Saunders, pp.1153.

6. Antimycobial susceptibility testing of RGM
Conventional methods (CLSI M24-A2)
Standard broth microdilution method (M07)

7. Induction of the synthesis of a methyltransferase
Inducible resistance
Association with inducible resistance to macrolides
Susceptible after three days of in vitro incubation
Resistant in extended incubation (14 days) or preincubation with clarithromycin
Newly updated CLSI M24-A2 (2011)
Induction of the synthesis of a methyltransferase

8. Macrolide resistance 1. erm(41)
Post-transcriptional methylation of the 23S rRNA
Erythromycin ribosome methyltransferase gene (erm)
95% of macrolide resistance
Significance of erm(41) according to the species
Polymorphism in erm(41) (T or C)
M. abscessus and M. bolletii strains having C28
No inducible resistance to clarithromycin
Low MIC
J Antimicrob Chemother 2012 Apr;67(4):810-8.

9. Macrolide resistance 2.rrl (large subunit of ribosomal RNA)
Mutation of rrl gene at A2058
Encoding the peptidyltransferase domain of the 23S rRNA
rrl mutation
A base change at position 2058 or 2059 (E. coli numbering)
A2058G, A2058C, A2059G
Infrequently emerged during chemotherapy
Highly resistant M. massiliense isolates always had a point mutation
Microbiol Immunol 2010 Jun;54(6):

10. Real-time PCR & melting curve analysis
Methods
3-channel multiplex real-time PCR and melting curve analysis
Simultaneous detection and identification of MTBC and nontuberculous mycobacteria
J Clin Microbiol 2011 Nov;49(11):

11. Real-time PCR & melting curve analysis
Methods
Real-time PCR and melting curve analysis
Identification of M. abscessus and M. massiliense
Target gene
Primer sequence
Amplicon
erm(41)
erm F: 5’-GACCGGGGCCTTCTTCGTGAT-3’
673 bp: M. abscessus, M. bolletii
397 bp: M. massiliense
erm R: 5’-GACTTCCCCGCACCGATTCC-3’
Melting peak
Amplification curve

12. Pyrosequencing Pyrosequencing of erm(41) gene
Methods
Pyrosequencing
Pyrosequencing
Sanger Sequencing
Sequencing by SYNTHESIS
Sequencing by TERMINATION
Template amplification
Analysis by real-time monitoring of the DNA
Purification Termination reaction
Capillary electrophoresis bp
500-1,000 bp
Pyrosequencing of erm(41) gene
T28 or C28
Reference seq.: GACGCCAGCGG
Pyrosequencing of rrl gene
Point mutation at A2058 or A2059
Reference seq.: GGAAGGTTAAGA
CCACTGGCGTC (reverse primer) GACGCCAGTGG
GGAAGGTTAAGA
CCGCTGGCGTC (reverse primer) GACGCCAGCGG
Clin Chim Acta 2006;363:83-94

13. Proportion of each species (SNUBH)
Results
Proportion of each species (SNUBH)
NTM 1680
M. abscessus type I & II (19.7 %)
M. abscessus type II
(M. massiliense)
195 (58.9 %)
M. abscessus type I
136 (41.1 %)
J Clin Microbiol 2011 Nov;49(11):

14. M. abscessus susceptibility test (SNUBH)
Results
M. abscessus susceptibility test (SNUBH)

15. M. massiliense susceptibility test (SNUBH)
Results
M. massiliense susceptibility test (SNUBH)

16. Sensitivity after prolonged incubation (14 days)
Summary & Conclusion
M. abscessus
Reputation of being the most virulent and chemotherapy-resistant member of the RGM group
Sensitivity after prolonged incubation (14 days)
Inducible resistance
Molecular identification of M. abscessus
Species-specific ID within the M. abscessus
Fully concordant with clarithromycin resistance when it is associated with detection of rrl mutations
Helpful in therapeutic implication using molecular test
Thank you.