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Molecular Identification of Strains within the Mycobacterium abscessus Complex and Determination of Resistance to Macrolides and Aminoglycosides Cover

Molecular Identification of Strains within the Mycobacterium abscessus Complex and Determination of Resistance to Macrolides and Aminoglycosides

Open Access
|Dec 2023

Figures & Tables

Fig. 1.

Mycobacterium abscessus subsp. abscessus. Photography of an auramine-stained acid-fast bacilli smear from liquid BBL culture. The visible bacilli forming cord-like structures.
Mycobacterium abscessus subsp. abscessus. Photography of an auramine-stained acid-fast bacilli smear from liquid BBL culture. The visible bacilli forming cord-like structures.

Fig. 2.

Mycobacterium abscessus subsp. abscessus. Photography of smear stained by Ziehl-Neelsen method for acid-fast bacilli (AFB). Counterstaining creates a compelling visual contrast of red acid-fast bacilli.
Mycobacterium abscessus subsp. abscessus. Photography of smear stained by Ziehl-Neelsen method for acid-fast bacilli (AFB). Counterstaining creates a compelling visual contrast of red acid-fast bacilli.

Fig. 3.

Growth of Mycobacterium abscessus after 7 days of incubation at 37°C in aerobic condition.
a) cream-colored, waxy colonies on Löwenstein-Jensen medium
b) mycobacterial colonies on non-selective media – Tryptic Soy Agar
c) mycobacterial colonies on Columbia agar with 5% of sheep blood.
Growth of Mycobacterium abscessus after 7 days of incubation at 37°C in aerobic condition. a) cream-colored, waxy colonies on Löwenstein-Jensen medium b) mycobacterial colonies on non-selective media – Tryptic Soy Agar c) mycobacterial colonies on Columbia agar with 5% of sheep blood.

Fig. 4.

Culture of rapidly growing non-tuberculous mycobacteria on selective NTM Elite agar (bioMérieux, France), after 5 days of incubation at 37°C under aerobic conditions.
a) colonies of Mycobacterium fortuitum subsp. fortuitum ATCC® 6841™, b) rough colonies of Mycobacterium abscessus subsp. abscessus.
Culture of rapidly growing non-tuberculous mycobacteria on selective NTM Elite agar (bioMérieux, France), after 5 days of incubation at 37°C under aerobic conditions. a) colonies of Mycobacterium fortuitum subsp. fortuitum ATCC® 6841™, b) rough colonies of Mycobacterium abscessus subsp. abscessus.

Fig. 5.

Prevalence of non-tuberculosis mycobacteria species isolated from patients in the Malopolska region of Poland, etween 2018–2022. Each year, the most prevalent species were Mycobacterium kansasii and Mycobacterium avium. Occurrence of Mycobacterium abscessus varies between the years and amounted to three strains in 2018, one in 2020, and eight in 2021.
Prevalence of non-tuberculosis mycobacteria species isolated from patients in the Malopolska region of Poland, etween 2018–2022. Each year, the most prevalent species were Mycobacterium kansasii and Mycobacterium avium. Occurrence of Mycobacterium abscessus varies between the years and amounted to three strains in 2018, one in 2020, and eight in 2021.

Fig. 6.

Analysis of the banding patterns received for 12 clinical strains (strips 1-12) with the use of GenoType NTM DR (Hain Lifescience GmbH, Germany). The strips coated with specific probes are complementary to selectively amplified nucleic acid sequences, that enable species determination as well as point mutations referring to resistance to aminoglycosides and macrolides.
Analysis of the banding patterns received for 12 clinical strains (strips 1-12) with the use of GenoType NTM DR (Hain Lifescience GmbH, Germany). The strips coated with specific probes are complementary to selectively amplified nucleic acid sequences, that enable species determination as well as point mutations referring to resistance to aminoglycosides and macrolides.

Fig. 7.

MIC value of selected antimycobacterial agents obtained by RAPMYCO2 plate. AN – amikacin, CIP – ciprofloxacin, DOX – doxycycline, LZD – linezolid, MXF – moxifloxacin, TGC – tigecycline, CLA – clarithromycin, IMI – imipenem
MIC value of selected antimycobacterial agents obtained by RAPMYCO2 plate. AN – amikacin, CIP – ciprofloxacin, DOX – doxycycline, LZD – linezolid, MXF – moxifloxacin, TGC – tigecycline, CLA – clarithromycin, IMI – imipenem

Results of susceptibility testing to antimycobacterial agents obtained by Sensititre™ RAPMYCO2 plates_ Interpretation criteria following CLSI_

MIC values (μg/ml) (Interpretation S/I/R)
No.SpeciesANFOXCIPDOXLZDMXFSXTIMITGCCLA after 5dCLA after 14 d
1Mabs16 (S)64 (I)> 4 (R)> 16 (R)≤ 1 (S)4 (R)4/76 (R)16 (I)MIC = 10,06 (S)0,125 (S)
2Mabs16 (S)>128 (R)> 4 (R)> 16 (R)4 (S)2 (I)> 8/152 (R)16 (I)MIC = 0,50,06 (S)> 16 (R)
3Mabs16 (S)32 (I)> 4 (R)> 16 (R)4 (S)> 8 (R)4/76 (R)16 (I)MIC = 0,50,5 (S)> 16 (R)
4Mabs16 (S)>128 (R)> 4 (R)> 16 (R)16 (I)> 8 (R)> 8/152 (R)16 (I)MIC = 20,06 (S)> 16 (R)
5Mabs16 (S)64 (I)> 4 (R)> 16 (R)16 (I)> 8 (R)> 8/152 (R)16 (I)MIC = 10,06 (S)> 16 (R)
6Mabs16 (S)64 (I)> 4 (R)> 16 (R)4 (S)> 8 (R)> 8/152 (R)16 (I)MIC = 24 (I)> 16 (R)
7Mmas-----------
8Mmas16 (S)>128 (R)> 4 (R)> 16 (R)16 (I)4 (R)4/76 (R)16 (I)MIC = 0,250,06 (S)0,125 (S)
9Mabs16 (S)>128 (R)> 4 (R)> 16 (R)16 (I)4 (R)4/76 (R)16 (I)MIC = 0,50,06 (S)0,125 (S)
10Mmas16 (S)32 (I)> 4 (R)1 (S)1 (S)2 (I)2/38 (S)16 (I)MIC = 0,50,06 (S)0,06 (S)
11Mbol4(S)32 (I)> 4 (R)> 16 (R)4 (S)> 8 (R)> 8/152 (R)16 (I)MIC = 0.250.06 (S)> 16 (R)
12Mabs4 (S)33 (I)> 4 (R)> 16 (R)8 (S)> 8 (R)> 8/152 (R)16 (I)MIC = 0.124 (I)> 16 (R)

Interpretation of susceptibility pattern of rapidly growing mycobacteria according to the CLSI recommendations_

Antimycobacterial agentsMIC breakpoints (μg/ml)
SIR
Amikacin≤ 1632≥ 64
Cefoxitin≤ 1632-64≥ 128
Doxycycline≤ 12-4≥ 32
Ciprofloxacin≤ 12≥ 4
Clarithromycin≤ 24≥ 8
Linezolid≤ 816≥ 32
Moxifloxacin≤ 12≥ 4
Trimthoprim/sulfamethoazole≤ 2/38≥ 4/76
Imipenem≤ 48-16≥ 32
Tigecycline*

Results of molecular identification of resistance mechanisms to amikacin obtained by GenoType NTM in relation to phenotypic susceptibility testing results_

No. of strainSpeciesGenoType NTM-DR resultsPhenotypic results of susceptibility to amikacin
Type of bands (WT or mutation)Molecular mechanisms of resistance (Yes/No)
1Mabsrrs rrs WTNo(S)
2Mabsrrs rrs WTNo(S)
3Mabsrrs rrs WTNo(S)
4Mabsrrs rrs WTNo(S)
5Mabsrrs rrs WTNo(S)
6Mabsrrs rrs WTNo(S)
7Mmasrrs rrs WTNont
8Mmasrrs rrs WTNo(S)
9Mabsrrs MUT1 (A1408G)Yes(S)
10Mmasrrs rrs WTNo(S)
11Mb olrrs rrs WTNo(S)
12Mabsrrs rrs WTNo(S)

j_pjm-2023-048_tab_007

Strip 1– a clinical strain of Mycobacterium abscessus subs. abscessus (band characteristic for M. abscessus subsp. abscessus; SP4, SP5, SP6, SP9, SP10); no point mutation was detected;
Strips 2-6, 10, 12 -–clinical strains of M. abscessus subs. abscessus (band characteristic for M. abscessus subsp. abscessus; SP4, SP5, SP6, SP9, SP10); point mutation erm(41)T28 referring to macrolide resistance;
Strips 7, 9– clinical strains of M. abscessus subs. massiliense (band characteristic for M. abscessus subsp. massiliense; SP4, SP5, SP8, SP9); point mutation erm(41)T28 no clinical implication in case of M. masiliense;
Strips 8– clinical strains of M. abscessus subs. abscessus (band characteristic for M. abscessus subsp. abscessus; SP4, SP5, SP6, SP9, SP10); point mutations were detected: erm(41)T28 and rrl MUT referring to macrolide resistance as well as point mutation A1408G referring to amikacin resistance;
Strip 11– clinical strains of M. abscessus subs. bolletii (band characteristic for M. abscessus subsp. bolletii; SP4, SP5, SP6, SP7, SP9, SP10), point mutation erm(41)T28 referring to macrolide resistance.

Interpretation of the mutation type conferring to the macrolides and aminoglicosides resistance according to the GenoType NTM-DR VER 1_0 test instructions_

Discernible phenotypic pattern resistanceTarget nucleic acid sequenceNascent mutation bandMutation
Macrolides2058–2059rrl MUT1A2058C
rrl MUT2A2058G
A2058T
rrl MUT3A2059C
rrl MUT4A2059T
Aminoglycosides1406–1409rrs MUT1A1408G
T1406A
C1409T

Results of susceptibility testing to amikacin and clarithromycin obtained by manually conducted microdilution method_ Interpretation criteria following CLSI_

No.SpeciesANInterpretation*CLA after 5 days of incubationInterpretation*CLA after 14 days of incubationInterpretation*
1Mabs16(S)0.06(S)0,5(S)
2Mabs8(S)0.06(S)> 64(R)
3Mabs16(S)0.06(S)= 32(R)
4Mabs16(S)0.06(S)= 32(R)
5Mabs16(S)0.06(S)= 32(R)
6Mabs16(S)0.06(S)= 32(R)
7Mmas
8Mmas16(S)0.06(S)= 0.125(S)
9Mabs16(S)0.06(S)= 0.125(S)
10Mmas16(S)0.06(S)= 0.06(S)
11Mbol0.5(S)0.06(S)> 64(R)
12Mabs0.250(S)4(R)= 16(R)

Results of molecular identification of resistance mechanisms to clarithromycin by GenoType NTM in relation to phenotypic susceptibility testing results_

No. of strainSpeciesGenoType NTM-DR resultsPhenotypic results of susceptibility to clarithromycin
Type of bands (WT or mutation)Molecular mechanisms of resistance (Yes/No)
1Mabserm(41) C28 rrlWTNo(S)
2Mabserm(41)T28 rrlWTYes(R)
3Mabserm(41)T28 rrlWTYes(R)
4Mabserm(41)T28 rrlWTYes(R)
5Mabserm(41)T28 rrlWTYes(R)
6Mmaserm(41)T28 rrlWTYes(R)
7Mmaserm(41)T28* rrlWTNont
8Mmaserm(41)T28* rrlWTNo(S)
9Mabserm(41)T28 rrlMUTYes(S)
10Mb olerm(41)T28* rrlWTNo(S)
11Mabserm(41)T28 rrlWTYes(R)
12Mabserm(41)T28 rrlWTYes(R)
DOI: https://doi.org/10.33073/pjm-2023-048 | Journal eISSN: 2544-4646 | Journal ISSN: 1733-1331
Language: English
Page range: 491 - 506
Submitted on: Sep 15, 2023
Accepted on: Nov 11, 2023
Published on: Dec 16, 2023
Published by: Polish Society of Microbiologists
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year

© 2023 Katarzyna Kania, Katarzyna Wόjcik, Joanna Czekajewska, Magdalena Grzesiak, Karolina Klesiewicz, published by Polish Society of Microbiologists
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.