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Using a sequential regimen to eliminate bacteria at sublethal antibiotic dosages.

Fuentes-Hernandez A, Plucain J, Gori F, Pena-Miller R, Reding C, Jansen G, Schulenburg H, Gudelj I, Beardmore R - PLoS Biol. (2015)

Bottom Line: Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics.Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse.These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.

View Article: PubMed Central - PubMed

Affiliation: Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, México.

ABSTRACT
We need to find ways of enhancing the potency of existing antibiotics, and, with this in mind, we begin with an unusual question: how low can antibiotic dosages be and yet bacterial clearance still be observed? Seeking to optimise the simultaneous use of two antibiotics, we use the minimal dose at which clearance is observed in an in vitro experimental model of antibiotic treatment as a criterion to distinguish the best and worst treatments of a bacterium, Escherichia coli. Our aim is to compare a combination treatment consisting of two synergistic antibiotics to so-called sequential treatments in which the choice of antibiotic to administer can change with each round of treatment. Using mathematical predictions validated by the E. coli treatment model, we show that clearance of the bacterium can be achieved using sequential treatments at antibiotic dosages so low that the equivalent two-drug combination treatments are ineffective. Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics. Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse. However, dual resistance due to the pump means that the antibiotics must be carefully deployed and not all sublethal sequential treatments succeed. A screen of 136 96-h-long sequential treatments determined five of these that could clear the bacterium at sublethal dosages in all replicate populations, even though none had done so by 24 h. These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.

No MeSH data available.


Related in: MedlinePlus

Some examples of successful sequential treatments at IC70 dosages.(A) This Manhattan plot at IC70 shows the mean total optical densities observed during eight seasons of treatment (Σ0D(T) on the vertical axis, vertical lines are SE, and n = 3). Note the 16 treatments marked with a red or black square: they had among the lowest final densities of the treatments trialled. After this, the 16 treatments were replicated; a red square shows that a zero cell density was observed in all three initial and subsequent replicates of that treatment, and black squares show a zero population density was observed in some, but not all, replicates. (B) The no-drug, ERY, and DOX monotherapies and the 50/50 combination treatment all produce recovering mean population densities at IC70 doses. These four unsuccessful treatments are shown next to the optical density dynamics of three replicates of a successful “red square” treatment from (A) (treatment C in panel C). The three replicates (shown as grey lines with blue [DOX] and green [ERY] circles) indicate parallel dynamics and fluctuating decay towards zero (bars are SE of optical density at 96 h, n = 3). (C) Season-by-season mean densities of all the successful (red square) treatments from (A); note how two achieve high densities early during treatment. Fig S15 in S1 Text, section 3, shows colony-forming units for replicates of these treatments. (S1 Data contains the data used in this figure.)
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pbio.1002104.g002: Some examples of successful sequential treatments at IC70 dosages.(A) This Manhattan plot at IC70 shows the mean total optical densities observed during eight seasons of treatment (Σ0D(T) on the vertical axis, vertical lines are SE, and n = 3). Note the 16 treatments marked with a red or black square: they had among the lowest final densities of the treatments trialled. After this, the 16 treatments were replicated; a red square shows that a zero cell density was observed in all three initial and subsequent replicates of that treatment, and black squares show a zero population density was observed in some, but not all, replicates. (B) The no-drug, ERY, and DOX monotherapies and the 50/50 combination treatment all produce recovering mean population densities at IC70 doses. These four unsuccessful treatments are shown next to the optical density dynamics of three replicates of a successful “red square” treatment from (A) (treatment C in panel C). The three replicates (shown as grey lines with blue [DOX] and green [ERY] circles) indicate parallel dynamics and fluctuating decay towards zero (bars are SE of optical density at 96 h, n = 3). (C) Season-by-season mean densities of all the successful (red square) treatments from (A); note how two achieve high densities early during treatment. Fig S15 in S1 Text, section 3, shows colony-forming units for replicates of these treatments. (S1 Data contains the data used in this figure.)

Mentions: After increasing dosages to their IC70 values, the following evidence of bacterial clearance by 96 h was observed. Sixteen sequential treatments that produced some of the lowest population densities after 96 h of treatment (treatments marked with boxes in Fig. 2A) were examined, and, using spot tests, we could isolate no live cells for five of these treatments in all three replicates. The 11 remaining treatments lead to a zero cell count in some replicates but not in all (Fig S15 in S1 Text, section 3). We then replicated all 16 treatments an additional three times, and the five previously successful treatments again produced a zero cell count by 96 h, although the remaining 11 treatments showed substantial between-replicate variability in their population dynamics (Fig S15). By contrast, Fig. 2B shows that the 50/50 combination treatment (with a greater inhibition than IC70 due to the synergy) and both monotherapies yielded recovering (i.e., increasing) mean population densities beyond 48 h at these dosages. (In addition, we recall that twice IC95 combinations of these drugs can fail in this treatment model too [16].) However, these observations serve to illustrate that appropriately optimised, sequential therapies at IC70 can clear a bacterium even when synergistic combination treatments with greater one-season inhibition do not.


Using a sequential regimen to eliminate bacteria at sublethal antibiotic dosages.

Fuentes-Hernandez A, Plucain J, Gori F, Pena-Miller R, Reding C, Jansen G, Schulenburg H, Gudelj I, Beardmore R - PLoS Biol. (2015)

Some examples of successful sequential treatments at IC70 dosages.(A) This Manhattan plot at IC70 shows the mean total optical densities observed during eight seasons of treatment (Σ0D(T) on the vertical axis, vertical lines are SE, and n = 3). Note the 16 treatments marked with a red or black square: they had among the lowest final densities of the treatments trialled. After this, the 16 treatments were replicated; a red square shows that a zero cell density was observed in all three initial and subsequent replicates of that treatment, and black squares show a zero population density was observed in some, but not all, replicates. (B) The no-drug, ERY, and DOX monotherapies and the 50/50 combination treatment all produce recovering mean population densities at IC70 doses. These four unsuccessful treatments are shown next to the optical density dynamics of three replicates of a successful “red square” treatment from (A) (treatment C in panel C). The three replicates (shown as grey lines with blue [DOX] and green [ERY] circles) indicate parallel dynamics and fluctuating decay towards zero (bars are SE of optical density at 96 h, n = 3). (C) Season-by-season mean densities of all the successful (red square) treatments from (A); note how two achieve high densities early during treatment. Fig S15 in S1 Text, section 3, shows colony-forming units for replicates of these treatments. (S1 Data contains the data used in this figure.)
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pbio.1002104.g002: Some examples of successful sequential treatments at IC70 dosages.(A) This Manhattan plot at IC70 shows the mean total optical densities observed during eight seasons of treatment (Σ0D(T) on the vertical axis, vertical lines are SE, and n = 3). Note the 16 treatments marked with a red or black square: they had among the lowest final densities of the treatments trialled. After this, the 16 treatments were replicated; a red square shows that a zero cell density was observed in all three initial and subsequent replicates of that treatment, and black squares show a zero population density was observed in some, but not all, replicates. (B) The no-drug, ERY, and DOX monotherapies and the 50/50 combination treatment all produce recovering mean population densities at IC70 doses. These four unsuccessful treatments are shown next to the optical density dynamics of three replicates of a successful “red square” treatment from (A) (treatment C in panel C). The three replicates (shown as grey lines with blue [DOX] and green [ERY] circles) indicate parallel dynamics and fluctuating decay towards zero (bars are SE of optical density at 96 h, n = 3). (C) Season-by-season mean densities of all the successful (red square) treatments from (A); note how two achieve high densities early during treatment. Fig S15 in S1 Text, section 3, shows colony-forming units for replicates of these treatments. (S1 Data contains the data used in this figure.)
Mentions: After increasing dosages to their IC70 values, the following evidence of bacterial clearance by 96 h was observed. Sixteen sequential treatments that produced some of the lowest population densities after 96 h of treatment (treatments marked with boxes in Fig. 2A) were examined, and, using spot tests, we could isolate no live cells for five of these treatments in all three replicates. The 11 remaining treatments lead to a zero cell count in some replicates but not in all (Fig S15 in S1 Text, section 3). We then replicated all 16 treatments an additional three times, and the five previously successful treatments again produced a zero cell count by 96 h, although the remaining 11 treatments showed substantial between-replicate variability in their population dynamics (Fig S15). By contrast, Fig. 2B shows that the 50/50 combination treatment (with a greater inhibition than IC70 due to the synergy) and both monotherapies yielded recovering (i.e., increasing) mean population densities beyond 48 h at these dosages. (In addition, we recall that twice IC95 combinations of these drugs can fail in this treatment model too [16].) However, these observations serve to illustrate that appropriately optimised, sequential therapies at IC70 can clear a bacterium even when synergistic combination treatments with greater one-season inhibition do not.

Bottom Line: Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics.Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse.These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.

View Article: PubMed Central - PubMed

Affiliation: Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, México.

ABSTRACT
We need to find ways of enhancing the potency of existing antibiotics, and, with this in mind, we begin with an unusual question: how low can antibiotic dosages be and yet bacterial clearance still be observed? Seeking to optimise the simultaneous use of two antibiotics, we use the minimal dose at which clearance is observed in an in vitro experimental model of antibiotic treatment as a criterion to distinguish the best and worst treatments of a bacterium, Escherichia coli. Our aim is to compare a combination treatment consisting of two synergistic antibiotics to so-called sequential treatments in which the choice of antibiotic to administer can change with each round of treatment. Using mathematical predictions validated by the E. coli treatment model, we show that clearance of the bacterium can be achieved using sequential treatments at antibiotic dosages so low that the equivalent two-drug combination treatments are ineffective. Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics. Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse. However, dual resistance due to the pump means that the antibiotics must be carefully deployed and not all sublethal sequential treatments succeed. A screen of 136 96-h-long sequential treatments determined five of these that could clear the bacterium at sublethal dosages in all replicate populations, even though none had done so by 24 h. These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.

No MeSH data available.


Related in: MedlinePlus