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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

The rate of adaptation has a complex relationship with antibiotic dose.(A) Using IC50 dosages, growth rate adaptation (denoted α when defined in [14]) is greater for the 50/50 combination than for the EDEDEDED (“ERY—DOX”) and DEDEDEDE (“DOX—ERY”) sequential treatments. Adaptation can also be faster in the absence, rather than in the presence, of antibiotics. The right-hand plot shows each replicate separately (as a dot), indicating treatment clusters as coloured regions using the convex hulls of the datasets for each treatment type (whether no drug or single drug monotherapies, the 50/50 two-drug combination, or a sequential treatment). This shows sequential treatments minimise both final growth rate after eight seasons and the rate of adaptation. (B) Differences in the rate of adaptation in (A) are not accounted for by the duplication of the acrRAB operon, and sequential treatments do not prevent pump duplications. These coverage plots from sequenced populations at 24 h and 96 h show that both the combination (50/50) and sequential treatments (ERY/DOX) lead to the duplication of a genomic region from 273 Kb to 686 Kb that contains acrRAB. Left: the duplication was absent from all treatments after 24 h. Right: the duplication (the dark sector) is present in both the 50/50 combination and ERY—DOX sequential treatments after 96 h, but not in the no-drug control. Single nucleotide polymorphisms (SNPs) are highlighted as arrowheads next to the treatment in which they were observed. (S1 Data contains the data used in this figure.)
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pbio.1002104.g003: The rate of adaptation has a complex relationship with antibiotic dose.(A) Using IC50 dosages, growth rate adaptation (denoted α when defined in [14]) is greater for the 50/50 combination than for the EDEDEDED (“ERY—DOX”) and DEDEDEDE (“DOX—ERY”) sequential treatments. Adaptation can also be faster in the absence, rather than in the presence, of antibiotics. The right-hand plot shows each replicate separately (as a dot), indicating treatment clusters as coloured regions using the convex hulls of the datasets for each treatment type (whether no drug or single drug monotherapies, the 50/50 two-drug combination, or a sequential treatment). This shows sequential treatments minimise both final growth rate after eight seasons and the rate of adaptation. (B) Differences in the rate of adaptation in (A) are not accounted for by the duplication of the acrRAB operon, and sequential treatments do not prevent pump duplications. These coverage plots from sequenced populations at 24 h and 96 h show that both the combination (50/50) and sequential treatments (ERY/DOX) lead to the duplication of a genomic region from 273 Kb to 686 Kb that contains acrRAB. Left: the duplication was absent from all treatments after 24 h. Right: the duplication (the dark sector) is present in both the 50/50 combination and ERY—DOX sequential treatments after 96 h, but not in the no-drug control. Single nucleotide polymorphisms (SNPs) are highlighted as arrowheads next to the treatment in which they were observed. (S1 Data contains the data used in this figure.)

Mentions: In order to determine genetic changes due to the differential stresses found in drug-free conditions and in the sequential and combination treatments, two treatments at IC50 that produced comparable densities at 96 h were subjected to a whole-genome sequencing analysis and compared to the drug-free populations (S1 Text, section 4). Writing “E” for a season of ERY and “D” for DOX, when metagenomes from the EDEDEDED and 50/50 combination treatments were sequenced, known resistance mutations were observed in both. Fig. 3B highlights a 412 Kb genomic region containing the acrRAB operon whose duplication was observed more frequently in both the combination and sequential treatments at 96 h (namely, eight seasons) than at 24 h (or two seasons; Fisher exact test for both, p = 0.05; Fig S17 in S1 Text, section 4). Treating sequentially does not, therefore, avert selection for duplications of the acrRAB operon.


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)

The rate of adaptation has a complex relationship with antibiotic dose.(A) Using IC50 dosages, growth rate adaptation (denoted α when defined in [14]) is greater for the 50/50 combination than for the EDEDEDED (“ERY—DOX”) and DEDEDEDE (“DOX—ERY”) sequential treatments. Adaptation can also be faster in the absence, rather than in the presence, of antibiotics. The right-hand plot shows each replicate separately (as a dot), indicating treatment clusters as coloured regions using the convex hulls of the datasets for each treatment type (whether no drug or single drug monotherapies, the 50/50 two-drug combination, or a sequential treatment). This shows sequential treatments minimise both final growth rate after eight seasons and the rate of adaptation. (B) Differences in the rate of adaptation in (A) are not accounted for by the duplication of the acrRAB operon, and sequential treatments do not prevent pump duplications. These coverage plots from sequenced populations at 24 h and 96 h show that both the combination (50/50) and sequential treatments (ERY/DOX) lead to the duplication of a genomic region from 273 Kb to 686 Kb that contains acrRAB. Left: the duplication was absent from all treatments after 24 h. Right: the duplication (the dark sector) is present in both the 50/50 combination and ERY—DOX sequential treatments after 96 h, but not in the no-drug control. Single nucleotide polymorphisms (SNPs) are highlighted as arrowheads next to the treatment in which they were observed. (S1 Data contains the data used in this figure.)
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4390231&req=5

pbio.1002104.g003: The rate of adaptation has a complex relationship with antibiotic dose.(A) Using IC50 dosages, growth rate adaptation (denoted α when defined in [14]) is greater for the 50/50 combination than for the EDEDEDED (“ERY—DOX”) and DEDEDEDE (“DOX—ERY”) sequential treatments. Adaptation can also be faster in the absence, rather than in the presence, of antibiotics. The right-hand plot shows each replicate separately (as a dot), indicating treatment clusters as coloured regions using the convex hulls of the datasets for each treatment type (whether no drug or single drug monotherapies, the 50/50 two-drug combination, or a sequential treatment). This shows sequential treatments minimise both final growth rate after eight seasons and the rate of adaptation. (B) Differences in the rate of adaptation in (A) are not accounted for by the duplication of the acrRAB operon, and sequential treatments do not prevent pump duplications. These coverage plots from sequenced populations at 24 h and 96 h show that both the combination (50/50) and sequential treatments (ERY/DOX) lead to the duplication of a genomic region from 273 Kb to 686 Kb that contains acrRAB. Left: the duplication was absent from all treatments after 24 h. Right: the duplication (the dark sector) is present in both the 50/50 combination and ERY—DOX sequential treatments after 96 h, but not in the no-drug control. Single nucleotide polymorphisms (SNPs) are highlighted as arrowheads next to the treatment in which they were observed. (S1 Data contains the data used in this figure.)
Mentions: In order to determine genetic changes due to the differential stresses found in drug-free conditions and in the sequential and combination treatments, two treatments at IC50 that produced comparable densities at 96 h were subjected to a whole-genome sequencing analysis and compared to the drug-free populations (S1 Text, section 4). Writing “E” for a season of ERY and “D” for DOX, when metagenomes from the EDEDEDED and 50/50 combination treatments were sequenced, known resistance mutations were observed in both. Fig. 3B highlights a 412 Kb genomic region containing the acrRAB operon whose duplication was observed more frequently in both the combination and sequential treatments at 96 h (namely, eight seasons) than at 24 h (or two seasons; Fisher exact test for both, p = 0.05; Fig S17 in S1 Text, section 4). Treating sequentially does not, therefore, avert selection for duplications of the acrRAB operon.

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