Prevalence and molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to fluoroquinolones in Chongqing

doi:10.3969/j.issn.1000-6621.2018.10.006

Abstract

Abstract:

Objective To analyze the molecular characteristics of fluoroquinolones (FQs) resistance-associated gene mutations and the correlation with the different genotypes among the MDR-TB strains in Chongqing. Methods Two hundred and twenty-nine MDR-TB clinical strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, and gatifloxacin using microplate alamar blue assay (MABA). PCR and sequencing were conducted for gyrA and gyrB genes conferring FQs resistance. The Beijing genotypes were identified with real-time polymerase chain reaction (PCR) melting curve assay. Results Among the 229 MDR-TB strains, 94 strains (41.0%, 94/229) were resistant to any FQ. The prevalence of FQs resistance in Chongqing were highest for ofloxacin (41.0%, 94/229), intermediate for levofloxacin (31.4%,72/229) and moxifloxacin (30.6%, 70/229), and lowest for gatifloxacin (20.1%, 46/229).The 94 strains resistant to FQs contained 81 gyrA mutation strains (86.2%, 81/94), and the highest mutation frequency was at codon 94 (60.6%,57/94). Mutations with gyrB were observed in 10 FQ-resistant strains (10.6%, 10/94). Seven strains with double mutations in gyrA, and 2 of the 9 strains with Asp94Gly and Ala504Thr/Ala504Val double mutations in gyrA and gyrB respectively, showed high-level FQ resistance. The proportion of Beijing genotype strains was 85.1% (80/94) among FQ-resistant strains in Chongqing. Of these, 60.0% (48/80) were modern-Beijing sublineage strains. Conclusion Modern-Beijing sublineage strains accounts for the majority of FQ-resistant strains and the resistant related mutations mainly occur in gyrA gene.

Key words: Mycobacterium tuberculosis, Tuberculosis, multidrug resistant fluoroquinolone, DNA mutational analysis, Genotype, Small-area analysis

Yan HU,Jie LIU,Jing SHEN,Da-mian ZHU,Xin FENG,Lin CHEN,Jian ZHAN,Xi-chao OU,Yang ZHOU,Yan-lin ZHAO. Prevalence and molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates resistant to fluoroquinolones in Chongqing[J]. Chinese Journal of Antituberculosis, 2018, 40(10): 1060-1065. doi: 10.3969/j.issn.1000-6621.2018.10.006

Figures/Tables 4

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基因	引物或探针	序列(5'-3')	浓度(μmol/L)
Rv2952	探针_W	ROX-TGGGAAGTACCCTCCTGGGC-BHQ2	0.20
	引物_F	CATGCGGCGATCTCATTGTT	0.02
	引物_R	GCACTGTTACCCGCACTTTC	0.40
mutT2	探针_W	FAM-AGAACTGGGACTCGAGGTCGC-BHQ1	0.20
	引物_F	GAACTTCCCGGCGGTAAGGTC	0.02
	引物_R	CCGTTCAACGCAATATCGTC	0.40

药物	不同MIC值(μg/ml)的菌株数											临界浓度 (μg/ml)	耐药菌株[株数(率,%)]
药物	≤0.31	0.063	0.125	0.25	0.5	1	2	4	8	16	32	临界浓度 (μg/ml)	初治 (84株)	复治 (145株)	合计 (229株)	χ²值	P值
Ofx	0	1	11	52	47	12	12	34	44	10	6	2.0	29(34.5)^abc	65(44.8)^ghi	94(41.0)	2.334	0.127
Lfx	0	4	48	57	10	10	28	44	17	8	3	2.0	22(26.2)^de	50(34.5)^jk	72(31.4)	1.697	0.193
Mfx	8	46	54	16	35	23	27	15	4	1	0	0.5	22(26.2)^f	48(33.1)^l	70(30.6)	1.198	0.274
Gfx	56	49	21	26	31	31	5	8	2	0	0	0.5	15(17.9)	31(21.4)	46(20.1)	0.411	0.521

耐药表型	gyrA基因QRDR 区突变	gyrB基因QRDR 区突变	突变菌株 [株数(率,%)]	MIC (μg/ml)
耐药表型	gyrA基因QRDR 区突变	gyrB基因QRDR 区突变	突变菌株 [株数(率,%)]	Ofx	Lfx	Mfx	Gfx
FQs耐药^a(94株)	Gly88Cys		1(1.1)	8.0	4.0	1.0	0.5
	Asp89Asn		1(1.1)	4.0	2.0	2.0	0.5
	Ala90Val		16(17.0)	4.0~8.0	2.0~4.0	0.25~1.0	0.125~0.5
	Ser91Pro		2(2.1)	4.0	2.0~4.0	0.5~1.0	0.5~1.0
	Asp94Asn		7(7.4)	4.0~8.0	2.0~8.0	0.25~4.0	0.125~1.0
	Asp94His		1(1.1)	8.0	4.0	2.0	1.0
	Asp94Ala		5(5.3)	4.0	2.0~4.0	0.5~1.0	0.125~0.5
	Asp94Gly		30(31.9)	4.0~16.0	4.0~8.0	1.0~4.0	0.25~2.0
	Asp94Tyr		2(2.1)	8.0~16.0	4.0~8.0	2.0~4.0	1.0~2.0
	Asp94Ala&Ala90Val		4(4.3)	16.0~32.0	16.0~32.0	4.0~8.0	4.0~8.0
	Asp94Ala&Ser91Pro		1(1.1)	16.0	16.0	8.0	4.0
	Asp94Gly&Ala74Ser		2(2.1)	16.0	16.0	4.0~8.0	4.0
		Gly512Arg	1(1.1)	4.0	4.0	2.0	1.0
	Ala90Val	Asp461Asn	3(3.2)	16.0~32.0	8.0~32.0	2.0~4.0	1.0~4.0
	Asp94Gly	Ala504Thr	1(1.1)	16.0	16.0	8.0	4.0
	Asp94Gly	Ala504Val	1(1.1)	32.0	16.0	16.0	8.0
	Asp94Ala	Gly512Arg	1(1.1)	8.0	4.0	1.0	0.5
	Asp94Gly	Gly512Arg	1(1.1)	8.0	4.0	1.0	1.0
	Ala90Val	Gly512Arg	1(1.1)	4.0	2.0	0.25	0.5
	Asp94Asn	Val496Val	1(1.1)	16.0	8.0	2.0	1.0
	合计		82(87.2)	4.0~32.0	2.0~32.0	0.25~16.0	0.125~8.0
FQs敏感^b(135株)	Asp94Ala		3(2.2)	0.5~2.0	0.25~1.0	0.125~0.5	0.063~0.125
	Ala90Val		6(4.4)	1.0~2.0	1.0~2.0	0.25~0.5	0.125~0.25
	Gly88Ala		1(0.7)	1.0	0.5	0.5	0.25
		Arg446Leu	1(0.7)	1.0	1.0	0.5	0.25
		Asp461Asn	4(3.0)	1.0~2.0	0.5~2.0	0.25~0.5	0.125~0.5
		Gly512Arg	1(0.7)	0.125	0.125	0.063	0.031
	合计		16(11.9)	0.125~2.0	0.125~2.0	0.063~0.5	0.031~0.5

	北京型(80株)			χ²值	P值	非北京型 (14株)	χ²值	P值
	古代北京型 (32株)	现代北京型 (48株)	合计	χ²值	P值	非北京型 (14株)	χ²值	P值
gyrA基因	26(81.3)	43(89.6)	69(86.3)	0.531	0.466	12(85.7)	0.000	1.000
Ala74Ser	0(0.0)	2(4.2)	2(2.5)	-	0.514	0(0.0)	-	1.000
Gly88Cys	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149
Asp89Asn	1(3.1)	0(0.0)	1(1.3)	-	0.400	0(0.0)	-	1.000
Ala90Val	8(25.0)	13(27.1)	21(26.3)	0.043	0.836	3(21.4)	0.002	0.961
Ser91Pro	0(0.0)	2(4.2)	2(2.5)	-	0.514	1(7.1)	-	0.387
94位密码子	17(53.1)	33(68.8)	50(62.5)	2.000	0.157	7(50.0)	0.780	0.377
Asp94Asn	1(3.1)	4(8.3)	5(6.3)	0.222	0.637	3(21.4)	1.846	0.174
Asp94His	0(0.0)	1(2.1)	1(1.3)	-	1.000	0(0.0)	-	1.000
Asp94Ala	2(6.3)	9(18.8)	11(13.8)	1.585	0.208	0(0.0)	1.052	0.305
Asp94Gly	13(40.6)	19(39.6)	32(40.0)	0.009	0.926	3(21.4)	1.758	0.185
Asp94Tyr	1(3.1)	0(0.0)	1(1.3)	-	0.400	1(7.1)	-	0.277
gyrB基因	8(25.0)	0(0.0)	8(10.0)	10.700	0.001	2(14.3)	0.000	0.992
Asp461Asn	3(9.4)	0(0.0)	3(3.8)	2.439	0.118	0(0.0)	-	1.000
Gly512Arg	4(12.5)	0(0.0)	4(5.0)	3.958	0.047	0(0.0)	-	1.000
Ala504Thr	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149
Ala504Val	1(3.1)	0(0.0)	1(1.3)	-	0.400	0(0.0)	-	1.000
Val496Val	0(0.0)	0(0.0)	0(0.0)	-	-	1(7.1)	-	0.149