Molecular characterization of mutations associated with resistance to second line drugs in <i>Mycobacterium tuberculosis</i> patients from Casablanca, Morocco

Momen, Ghizlane; Aainouss, Achraf; Lamaammal, Abdelmajid; Chettioui, Fouad; Blaghen, Mohamed; Messoudi, Malika; Belghmi, Khalid; Mouslim, Jamal; El Mzibri, Mohammed; El Messaoudi, My Driss; Khyatti, Meriem; Chaoui, Imane

doi:10.1590/S1678-9946202163019

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Original Article • Rev. Inst. Med. trop. S. Paulo 63 • 2021 • https://doi.org/10.1590/S1678-9946202163019 copy

Molecular characterization of mutations associated with resistance to second line drugs in Mycobacterium tuberculosis patients from Casablanca, Morocco

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

The emergence and spread of extensively drug-resistant tuberculosis (XDR-TB) is a serious threat to global health. Therefore, its rapid diagnosis is crucial. The present study aimed to characterize mutations conferring resistance to second line drugs (SLDs) within multidrug Mycobacterium tuberculosis (MDR-MTB) isolates and to estimate the occurrence of XDR-TB in Casablanca, Morocco. A panel of 200 MDR-TB isolates was collected at the Pasteur Institute between 2015-2018. Samples were subjected to drug susceptibility testing to Ofloxacin (OFX), Kanamycin (KAN) and Amikacin (AMK). The mutational status of gyrA, gyrB, rrs, tlyA and eis was assessed by sequencing these target genes. Drug susceptibility testing for SLDs showed that among the 200 MDR strains, 20% were resistant to OFX, 2.5% to KAN and 1.5% to AMK. Overall, 14.5% of MDR strains harbored mutations in gyrA, gyrB, rrs and tlyA genes. From the 40 OFX^R isolates, 67.5% had mutations in QRDR of gyrA and gyrB genes, the most frequent one being Ala90Val in gyrA gene. Of note, none of the isolates harbored simultaneously mutations in gyrA and gyrB genes. In eight out of the 200 MDR-TB isolates resistant either to KAN or AMK, only 25% had A1401G or Lys89Glu change in rrs and tlyA genes respectively. This study is very informative and provides data on the alarming rate of fluoroquinolone resistance which warrants the need to implement appropriate drug regimens to prevent the emergence and spread of more severe forms of Mycobacterium tuberculosis drug resistance.

KEYWORDS:
Mycobacterium tuberculosis ; XDR-TB; DNA sequencing; Drug susceptibility testing; Morocco

Gene	Primer	Sequence 5′ to 3′	Fragment lenght (bp)	Annealing temperature (°C)
gyrA	gyrA For	5′-TGACATCGAGCAGGAGATGC-3′	320	59
gyrA	gyrA Rev	5′-GGGCTTCGGTGTACCTCATC-3′
gyrB	gyrB For	5′-GTGGAAATATGTTGGCCGTC-3′	413	58
gyrB	gyrA Rev	5′-GTCGTTGTGAACAACGCTGTG-3
rrs	rrs For	5′-GTAATCGCAGATCAGCAACG-3′	216	58
rrs	rrs Rev	5′-GTGATCCAGCCGCACCTT-3′
Eis promotor	eis p For	5′-AAATTCGTCGCTGATTCTCG-3′	387	56
Eis promotor	eis p Rev	5′-CGCGACGAAACTGAGACC-3′
tlyA	tlyA For	5′-GTCTCTGGCCGAACTCGAAG-3′	1,000	52
tlyA	tlyA Rev	5′-ATTGTCGCCCAATACTTTTTCTAC-3′

ATB drug	Gene	Codon/Nucleotid Position	Substitution	Amino acid change	Number of isolates	Sub-total
FQs	gyrA	Codon 90	GCG → GTG	Ala / Val	14	25
		Codon 94	GAC → GCC	Asp / Ala	4
			GAC → GGC	Asp / Gly	3
			GAC → CAC	Asp / His	4
	gyrB	Codon 472	GAC → CAC	Asp / His	3	4
	gyrB	Codon 472	GAC → AAC	Asp / Asn	1	4
Aminosides /cyclic peptides	rrs	N1401	SNP A/G	–	1	1
	tlyA	Codon 89	AAA → GAA	Lys / Glu	1	1
	eis	–	–	–	–	–

Techniques for detection of MTB		Phenotypic DST		Total
Techniques for detection of MTB		FQ^R	FQ^S	Total
Molecular drug resistance genotyping	Presence of resistance associated mutations	27	2	29
Molecular drug resistance genotyping	Absence of resistance associated mutations	13	158	171
Total		40	160	200

ATB drug	Gene	Position	Substitution	Amino acid change	Number of isolates
FQs	gyrA	Codon 95	AGC → ACC	Ser / Thr	3
Aminosides	tlyA	Position 33	A/G	–	10

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[1] Correspondence to: Chaoui Imane Centre National de l'Energie, des Sciences et Techniques Nucléaires, Département des Sciences du Vivant, Unité de Recherches Médicales et Biologiques, BP 1382, RP 10001, Rabat, Morocco. Tel: +212 537 712 031 Fax: +212 537 711 846 E-mail: im_chaoui@yahoo.fr, chaoui@cnesten.org.ma