Characterization of a Clone of Mycobacterium tuberculosis Clinical Isolates with Mutations in KatG (A110V), EthA (Q269STOP), and the inhA Promoter (−15C→T)

LETTER

Among the Mycobacterium tuberculosis clinical isolates collected during the 2003-2014 period at the French National Reference Center for Mycobacteria, 29 showed a remarkable combination of mutations in katG, ethA, and the inhA promoter. The aim of this study was to establish the epidemiologic and molecular features of these isolates.

The 29 isolates were characterized using the following approaches. Drug susceptibility testing of these isolates was performed on Lowenstein-Jensen medium with the standard proportion method (1). The genes rpoB, katG, inhA and its promoter, pncA, embB (codon 306), gyrA, gyrB, ethA, and ethR and the rrs1400 region were sequenced. Restriction fragment length polymorphism analysis of IS6110 (IS6110-RFLP), spoligotyping, and 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing were used to compare the isolates (2). Epidemiological data were available, including country of birth.

The 29 isolates, which were multidrug resistant and ethionamide resistant, shared the mutations Q269STOP in EthA, A110V in KatG, and −15C→T in the inhA promoter region (3). All the isolates showed the S531L mutation in RpoB, except one isolate with D516Y in RpoB (collected in 2008, from a patient born in the Republic of Congo), while the genes pncA, gyrA/B, ethR, and embB306 (embB with mutation in codon 306) and the rrs1400 region were wild type in all isolates. The KatG A110V mutation has been already described in isoniazid-resistant M. tuberculosis clinical isolates (4). This mutation has been previously reported to confer a marked increase of peroxidase activity on the catalase-peroxidase KatG (4). This effect could compensate for the defect of mono-oxygenase activity due to the STOP codon in EthA. The IS6110-RFLP performed for the 7 oldest isolates showed 3 different profiles. The spoligotyping and MIRU-VNTR typing carried out for the 22 most recent isolates classified them into 2 groups: (i) cluster 1 with the same MIRU-VNTR code and belonging to the T3 spoligotype with spoligotype international type (SIT) 73 (n = 16) and (ii) isolates closely related to cluster 1 but varying by 1 or 2 MIRU loci and classified as T3 variant (SIT 73) (n = 5) or unknown variant (SIT unknown) (n = 1) by spoligotyping. The countries of birth for these 29 patients are Democratic Republic of Congo (n = 11), Angola (n = 5), Republic of Congo (n = 3), Senegal (n = 2), Ivory Coast (n = 1), Cameroon (n = 1), Morocco (n = 1), Algeria (n = 1), France (n = 2), or unknown (n = 2). Apart from the country of birth, no obvious epidemiological relationship was found, except for 2 patients who were friends and for 2 others who were in close contact in a prison.

In conclusion, these M. tuberculosis clinical isolates are closely related considering the available molecular typing results and seem to originate from the Congo area, but based on the significant molecular and epidemiological variations highlighted in this study, it is likely that they do not result from the recent diffusion of a single epidemic clone. Clinicians should be aware of the spread of these multidrug-resistant isolates and should perform molecular diagnosis of resistance when facing tuberculosis cases in the Congo area or in patients coming from this area.

Nucleotide sequence accession numbers.

Sequences were deposited in GenBank under the accession numbers KC122363, HM587459, and KT067737 to KT067739.