Potential for Adaptation Overrides Cost of Resistance

Figures & data

Figure 1. Testing the evolvability of different antibiotic resistances.

First, polymorphic populations are created by mixing, in a 1:1 ratio, clones with two different antibiotic resistance mutations. All clones are isogenic, except for this different resistance mutation and a neutral fluorescent marker. 16 replicate populations are followed for each competition, where half of these replicates have one of the resistances linked with one of the neutral markers, while the other is linked to the other marker. Three different competitive scenarios were studied, between Rifampicin mutations (circles) and between Rifampicin and Streptomycin (squares). Next, populations are passaged in rich media in 96-well plates, organized in a checkered layout and separated by control wells (with media but without bacteria). Every 24 h, all populations are passaged serially into a new 96-well plate with fresh media, during 30 days. The frequencies of the neutral markers (and hence the resistance alleles) are analyzed to unravel possible differences in the evolutionary parameters according to the resistance background.

Figure 2. High evolvability of costly H526Y^Rif allows its long-term maintenance.

(A) Long-term dynamics, for 30 days (280 generations) of evolution in 15 replicates of a population composed of resistance strain H562Y^Rif and resistance strain H526D^Rif. Shown are the dynamics for the H526Y^Rif background. The slope of the black line represents the initial difference in fitness between the resistances. (B) Whisker-box shows the relative fitness differences inferred for new beneficial mutations between the two resistant backgrounds, with H526Y^Rif background as a reference. (C) Whisker-box shows the relative differences in time of appearance inferred for new beneficial mutations between the two resistance backgrounds.

Figure 3. Different evolvabilities between Rifampicin resistance alleles.

(A) Long-term dynamics, for 30 days (280 generations) of evolution in 16 replicates of a population composed of resistance strain H562Y^Rif and resistance strain S531F^Rif. Shown are the dynamics for the H526Y^Rif background. The slope of the black line represents the initial difference in fitness between the resistances. (B) Whisker-box shows the relative fitness differences inferred for new beneficial mutations between the two resistant backgrounds, with H526Y^Rif background as a reference. (C) Whisker-box shows the relative differences in time of appearance inferred for new beneficial mutations between the two resistance backgrounds.

Figure 4. Differences in evolvability between Rifampicin and Streptomycin resistance alleles.

(A) Long-term dynamics, for 30 days (280 generations) of evolution in 14 replicates of a population composed of resistance strain K43T^Str and resistance strain S531F^Rif. Shown are the dynamics for the K43T background. The slope of the black line represents the initial difference in fitness between the resistances. (B) Whisker-box shows the relative fitness differences inferred for new beneficial mutations between the two resistant backgrounds, with K43T^Str background as a reference. (C) Whisker-box shows the relative differences in time of appearance inferred for new beneficial mutations between the two resistance backgrounds.

Figure 5. Differences in selective effects of evolved clones from the competition between H526Y^Rif and S531F^Rif.

(A) Fitness effects of selected clones evolved from the H526Y^Rif background, when in competition with their ancestor (light bars) or against their other resistant competitor (S531F^Rif, dark bars). (B) Fitness effects of selected clones evolved from the S531F^Rif background, when in competition with their ancestor (light bars) or against their other resistant competitor (H526Y^Rif, dark bars).

Table 1. Evolutionary parameters estimated for the competition between strains H526Y and H526D.

		H526D (R1)		H526Y (R2)
	Initial H526Y frequency	W	T	W	T
Population 1	0.538	1.576	11	1.552	15
Population 2	0.382	1.293	11	1.263	22
Population 3	0.488	1.387	41	1.348	51
Population 4	0.468	1.483	26	1.44	32
Population 5	0.413	1.421	21	1.409	30
Population 6	0.476	1.474	31	1.478	42
Population 7	0.513	1.218	11	1.19	19
Population 8	0.493	1.776	18	1.767	23
Population 9	0.557			1.017	3
Population 10	0.461			1.028	29
Population 11	0.581			1.023	3
Population 12	0.58	1.002	46	1.02	3
Population 13	0.523			1.023	14
Population 14	0.55	1.001	38	1.028	23
Population 15	0.59	1.668	37	1.671	45

W stands for the fitness of the emerging haplotype and T its time of appearance. Initial Freq stands for the inferred initial frequency of the H526Y background. In the cases where only one of the backgrounds has acquired a mutation, the inferred parameters are in bold for the background where no mutation was inferred.

Table 2. Evolutionary parameters estimated for the competition between strains H526Y and S531F.

		S531F (R1)		H526Y (R2)
	Initial H526Y frequency	W	T	W	T
Population 1	0.369	1.459	15	1.472	17
Population 2	0.458	1.041	46	1.051	3
Population 3	0.277	1.436	15	1.45	19
Population 4	0.472	1.034	7	1.054	4
Population 5	0.286	1.529	8	1.554	12
Population 6	0.454	1.585	55	1.637	60
Population 7	0.381	1.615	36	1.655	40
Population 8	0.325	1.465	11	1.491	15
Population 9	0.473	1.199	19	1.194	14
Population 10	0.378	1.163	14	1.154	10
Population 11	0.356	1.595	17	1.601	19
Population 12	0.483	1.028	3	1.049	4
Population 13	0.37	1.344	11	1.349	12
Population 14	0.444	1.542	9	1.562	12
Population 15	0.446	1.505	16	1.514	17
Population 16	0.468	1.647	55	1.7	59

Table 3. Evolutionary parameters estimated for the competition between strains K43T and S531F.

		S531F (R1)		K43T (R2)
	Initial K43T frequency	W	T	W	T
Population 1	0.505	1.207	16	1.188	3
Population 2	0.319	1.349	20	1.359	26
Population 3	0.299	1.546	18	1.547	21
Population 4	0.349	1.26	16	1.253	18
Population 5	0.319	1.669	4	1.737	5
Population 6	0.359	1.457	4	1.474	10
Population 7	0.333	1.088	52	1.06	34
Population 8	0.256	1.325	7	1.334	12
Population 9	0.43	1.687	37	1.732	42
Population 10	0.335	1.097	3	1.09	4
Population 11	0.357	1.537	50	1.529	53
Population 12	0.388	1.16	11	1.144	6
Population 13	0.41	1.195	5	1.193	7
Population 14	0.379	1.095	43	1.058	6
Population 15	0.329	1.663	25	1.672	30

Table 4. Potential compensatory mutations identified in the genomes of the clones evolved in the competition between resistances H526Y and S531F.

Background	Genome Position	Gene(s)	Mutation	Annotation	Frequency	(%) Function
H526Y	3,438,465	rpoA	C→T	T196I	51.5	RNA polymerase, alpha subunit
	4,184,828	rpoC	T→C	S486P	25.5
	4,187,042	rpoC	C→T	R1224C	6.1	RNA polymerase, beta prime subunit
	4,184,721	rpoC	A→C	H450P	2.9
	3,797,382	waaZ	T→C	S280P	19.7	Lipopolysaccharide core biosynthesis protein
	3,946,224	yifE	G→A	G39D	12.0	Conserved protein, UPF0438 family, unknown function
	4,292,389	nrfG/gltP	A→T	Intergenic	5.0	[nrfG] Heme lyase (NrfEFG) for insertion of heme into c552, subunit NrfG/[gltP] glutamate/aspartate: proton symporte
	3,637,091	pitA	C→T	A476V	2.6	Phosphate transporter, low- affinity; tellurite importer
	374,196	mhpE/mhpT	T→C	Intergenic	2.4	4- hyroxy- 2- oxovalerate/4- hydroxy- 2- oxopentanoic acid aldolase, class I/putative 3- hydroxyphenylpropionic transporter
	3,464,375	bfr	C→T	R125C	2.0	Bacterioferritin, iron storage and detoxification protein
	1,743,180	ydhQ	T→C	S324S	3.1	Hypothetical protein
	3,898,043	yieL	C→A	T186T	2.5	Putative xylanase
S531F	4,183,521	rpoC	A→C	K50T	14.8	RNA polymerase, beta prime subunit
	4,292,389	nrfG/gltP	A→T	Intergenic	19.6	[nrfG] Heme lyase (NrfEFG) for insertion of heme into c552, subunit NrfG/[gltP] Glutamate/aspartate:proton symporte
	2,749,808	nadK/recN	δ1 bp	Intergenic	18.1	[nadK] NAD kinase/[recN] Recombination and repair protein
	1,940,499	znuA	T→C	F37L	17.7	Zinc transporter subunit: periplasmic- binding component of ABC superfamily
	3,374,976	sspA	T→C	L156P	12.2	Stringent starvation protein A
	300,420	paoB	T→A	V231E	11.8	PaoABC aldehyde oxidoreductase, FAD- containing subunit
	2,036,728	yedW	IS1 Ins	Coding	9.9	Putative DNA- binding response regulator in two- component system with
	1,877,853	yeaR	IS186 Ins	Coding	8.4	Hypothetical protein
	3,410,893	envR	IS5 Ins	Coding	6.8	DNA-binding transcriptional regulator
	2,361,326	yfaY	A→G	V110V	9.4	Hypothetical protein

Mutations were identified in the genomes of the evolved clones in comparison with a reference genome. Shown are the single nucleotide polymorphisms or IS events identified in either of the backgrounds, and the frequencies at which they were detected. Mutations that occurred between genes (intergenic) are identified as such, otherwise all remaining mutations occurred within the gene indicated. The mutations previously identified to be compensatory are identified with a gray shaded cell.

IS: Insertion sequence.