Interaction between rpsL and gyrA mutations affects the fitness and dual resistance of Mycobacterium tuberculosis clinical isolates against streptomycin and fluoroquinolones

Figures & data

Figure 1 Functional domain and conservation of rpsL (A) and gyrA (B) mutation sites in Mycobacterium spp.

Table 1 Mutation types of single- and dual-resistant Mycobacterium smegmatis mutants under the selection of antibiotics

Resistant type	Screening concentration (mg/L)	Number of isolates	Mutation types
STR-R	16	60	rpsL: K43R(28), K43M(7), K43N(4), K43T(2), K88E(14),K88R(4),NM(1)
FLQ-R	10	89	gyrA: D94A(19), D94Y(9), A90V(10), G88A(2), D94N(1), D94G(1), NM(47)
STR-FLQ-R	10	90	K43R: A90V(16), D94G(2), NM(4) K43M: D94G(14), D94A(1), D94Y(1), D94N(1), D89N(1), NM(2) K43N: D94G(9), D94N(1), NM(5) K43T: D94G(1), D94N(1), A90V(2), NM(5) K88R: A90V(2), NM(10) K88E: A90V(2), NM(10)

Abbreviations: FLQ, fluoroquinolones; STR, streptomycin.

Figure 2 Relative fitness of Mycobacterium smegmatis mutants.

Notes: (A) Relative fitness of STR-resistant M. smegmatis. (B) Relative fitness of FLQ-resistant M. smegmatis. (C) Relative fitness of STR and FLQ dual-resistant M. smegmatis. Gray bar is for the STR-resistant M. smegmatis, turquoise is for FLQ-resistant, and purple is for the STR–FLQ dual-resistant M. smegmatis.
Abbreviations: FLQ, fluoroquinolones; STR, streptomycin.

Figure 3 Frequency of dual-mutation of rpsL and gyrA found in clinical Mycobacterium tuberculosis isolates.

Notes: (A) Mutation combinations of clinical M. tuberculosis isolates from Sichuan. (B) Mutation combinations of 3,056 M. tuberculosis genome sequence in database.
Abbreviation: MTB, Mycobacterium tuberculosis.

Table 2 Gene mutations in clinical STR- and FLQ-resistant Mycobacterium tuberculosis isolates

Mutation type	rpsL	gyr-A	Number of isolates
rpsL(K43R)/gyrA	aag®agg K43R	gac®ggc D94G	37
	aag®agg K43R	gcg®gtg A90V	24
	aag®agg K43R	gac®gcc D94A	8
	aag®agg K43R	tcg®ccg S91P	9
	aag®agg K43R	gac®tac D94Y	10
	aag®agg K43R	gac®aac D94N	4
	aag®agg K43R	gac®aac D94H	2
rpsL(K88R)/gyrA	aag®agg K88R	gac®ggc D94G	11
	aag®agg K88R	gcg®gtg A90V	7
	aag®agg K88R	gac®gcc D94A	5
	aag®agg K88R	tcg®ccg S91P	1
	aag®agg K88R	gac®tac D94Y	1
	aag®agg K88R	gac®aac D94N	2
rpsL(K88T)/gyrA	aag®acg K88T	gac®ggc D94G	1
Single mutation	aag®agg K43R	NM	13
	aag®agg K88R	NM	3
	aag®atg K88M	NM	1
	NM	gac®ggc D94G	23
	NM	gcg®gtg A90V	10
	NM	gac®gcc D94A	3
	NM	tcg®ccg S91P	2
	NM	gac®tac D94Y	4
	NM	gac®aac D94N	6
	NM	ggc®tgc G88C	1
No mutation	NM	NM	25

Note: NM, no mutation detected.

Figure S1 Model of mutations appearing in rpsL and gyrA in this study.

Notes: (A) Structure of Escherichia coli rpsL (Protein Data Bank accession number 5U4I). The position of K43 residue (yellow) and K88 residue (red) screened in this study was highly conserved between E. coli and Mycobacterium tuberculosis. (B) M. tuberculosis gyrA (Protein Data Bank accession number 3IFZ). The position of G88 residue (yellow), A90 residue (red), and D94 residue (cyan) screened in present study is shown. Amino acids were placed in the cartoon structure in dots format using the PyMOL Viewer.

Figure S2 The alignment of rpsL in M. tuberculosis and E. coli.

Table S1 Epistasis (ε) in mutants resistant to STR and FLQ

Sample	STR-FLQ resistance		ε	SD of ε
	rpsL SNP	gyrA SNP
Ms_SF16R12	K43R	A90V	-0.057	0.021
Ms_SF16R14	K43R	D94G	0.021	0.020
Ms_SF16R210	K43N	D94N	-0.143	0.080
Ms_SF16R22	K43N	D94G	-0.127	0.076
Ms_SF16R262	K43M	D94A	-0.114	0.052
Ms_SF16R265	K43M	D94Y	-0.222	0.054
Ms_SF16R260	K43M	D94N	-0.106	0.052
Ms_SF16R261	K43M	D94G	-0.352	0.049
Ms_SF16R18	K43T	D94N	-0.027	0.015
Ms_SF16R19	K43T	A90V	-0.027	0.015
Ms SF16R13	K43T	D94G	-0.055	0.014
Ms_SF16R204	K88R	A90V	0.224	0.283
Ms_SF16R217	K88E	A90V	-0.072	0.038

Abbreviations: FLQ, fluoroquinolones; STR, streptomycin.