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The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

Tan C, Smith RP, Srimani JK, Riccione KA, Prasada S, Kuehn M, You L - Mol. Syst. Biol. (2012)

Bottom Line: The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density.A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response.Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, USA.

ABSTRACT
The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

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Kanamycin, but not chloramphenicol, caused band-pass response with periodic treatment. (A) Microscope images of bacterial growth in the flow system. At the 8th hour, bacterial densities increased with increasing pulse periods of chloramphenicol (10 μg/ml). In contrast, bacterial densities peaked at an intermediate pulse period (120 min) of kanamycin (10 μg/ml). (B) Bacteria exhibited ‘low-pass' response in varying pulse periods of chloramphenicol. The number of bacteria increased with increasing pulse periods. (C) Bacteria exhibited ‘band-pass' response in varying pulse periods of kanamycin. The number of bacteria increased significantly with the pulse period of 120 min (green line). Each error bar indicates the standard deviation of five microscope images. Representative time series were obtained from two experiments. See Supplementary Figure S7 for additional data. Source data is available for this figure in the Supplementary Information.
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f7: Kanamycin, but not chloramphenicol, caused band-pass response with periodic treatment. (A) Microscope images of bacterial growth in the flow system. At the 8th hour, bacterial densities increased with increasing pulse periods of chloramphenicol (10 μg/ml). In contrast, bacterial densities peaked at an intermediate pulse period (120 min) of kanamycin (10 μg/ml). (B) Bacteria exhibited ‘low-pass' response in varying pulse periods of chloramphenicol. The number of bacteria increased with increasing pulse periods. (C) Bacteria exhibited ‘band-pass' response in varying pulse periods of kanamycin. The number of bacteria increased significantly with the pulse period of 120 min (green line). Each error bar indicates the standard deviation of five microscope images. Representative time series were obtained from two experiments. See Supplementary Figure S7 for additional data. Source data is available for this figure in the Supplementary Information.

Mentions: To test model predictions, we tracked bacterial growth in response to periodic antibiotic treatment using the flow system. Consistent with our batch-culture experiments, bacterial cultures initiated from low density did not grow when treated with a constant, high concentration of either chloramphenicol or kanamycin (10 μg/ml) (Figure 7A–C). When challenged with 10 μg/ml chloramphenicol, bacterial densities increased with increasing pulse periods of 40, 120, 240, and 480 min (Figure 7A and B). In contrast, when challenged with pulses of either 10 μg/ml kanamycin or 80 μg/ml puromycin (a concentration that caused IE), bacterial densities peaked at an intermediate period of 120 min (Figures 7A and C; Supplementary Figure S7C). When challenged with 20 μg/ml kanamycin (a concentration that did not cause IE), bacteria did not grow at all pulse periods examined (Supplementary Figure S7A), likely because bacteria recovered much slower from the treatment (Supplementary Figure S6B, basal τlag=30). When we treated cells with chloramphenicol (10 μg/ml) and heat shock (42°C), bacterial density peaked at intermediate pulse period (120 min, Supplementary Figure S7D). As such, exogenously applied heat shock coupled with chloramphenicol treatment was able to generate both IE (Figure 4C; Supplementary Figure S4E) and band-pass behavior. For treatment with both antibiotics, we note that the lower densities at the pulse period of 40 min were not due to the loss of bacteria from the cell chamber (Supplementary Figure S7B). Our results demonstrate the drastic qualitative changes in treatment efficacy due to both IE and the recovery kinetics, which are critical factors that have been neglected in previous studies (Udekwu et al, 2009).


The inoculum effect and band-pass bacterial response to periodic antibiotic treatment.

Tan C, Smith RP, Srimani JK, Riccione KA, Prasada S, Kuehn M, You L - Mol. Syst. Biol. (2012)

Kanamycin, but not chloramphenicol, caused band-pass response with periodic treatment. (A) Microscope images of bacterial growth in the flow system. At the 8th hour, bacterial densities increased with increasing pulse periods of chloramphenicol (10 μg/ml). In contrast, bacterial densities peaked at an intermediate pulse period (120 min) of kanamycin (10 μg/ml). (B) Bacteria exhibited ‘low-pass' response in varying pulse periods of chloramphenicol. The number of bacteria increased with increasing pulse periods. (C) Bacteria exhibited ‘band-pass' response in varying pulse periods of kanamycin. The number of bacteria increased significantly with the pulse period of 120 min (green line). Each error bar indicates the standard deviation of five microscope images. Representative time series were obtained from two experiments. See Supplementary Figure S7 for additional data. Source data is available for this figure in the Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472685&req=5

f7: Kanamycin, but not chloramphenicol, caused band-pass response with periodic treatment. (A) Microscope images of bacterial growth in the flow system. At the 8th hour, bacterial densities increased with increasing pulse periods of chloramphenicol (10 μg/ml). In contrast, bacterial densities peaked at an intermediate pulse period (120 min) of kanamycin (10 μg/ml). (B) Bacteria exhibited ‘low-pass' response in varying pulse periods of chloramphenicol. The number of bacteria increased with increasing pulse periods. (C) Bacteria exhibited ‘band-pass' response in varying pulse periods of kanamycin. The number of bacteria increased significantly with the pulse period of 120 min (green line). Each error bar indicates the standard deviation of five microscope images. Representative time series were obtained from two experiments. See Supplementary Figure S7 for additional data. Source data is available for this figure in the Supplementary Information.
Mentions: To test model predictions, we tracked bacterial growth in response to periodic antibiotic treatment using the flow system. Consistent with our batch-culture experiments, bacterial cultures initiated from low density did not grow when treated with a constant, high concentration of either chloramphenicol or kanamycin (10 μg/ml) (Figure 7A–C). When challenged with 10 μg/ml chloramphenicol, bacterial densities increased with increasing pulse periods of 40, 120, 240, and 480 min (Figure 7A and B). In contrast, when challenged with pulses of either 10 μg/ml kanamycin or 80 μg/ml puromycin (a concentration that caused IE), bacterial densities peaked at an intermediate period of 120 min (Figures 7A and C; Supplementary Figure S7C). When challenged with 20 μg/ml kanamycin (a concentration that did not cause IE), bacteria did not grow at all pulse periods examined (Supplementary Figure S7A), likely because bacteria recovered much slower from the treatment (Supplementary Figure S6B, basal τlag=30). When we treated cells with chloramphenicol (10 μg/ml) and heat shock (42°C), bacterial density peaked at intermediate pulse period (120 min, Supplementary Figure S7D). As such, exogenously applied heat shock coupled with chloramphenicol treatment was able to generate both IE (Figure 4C; Supplementary Figure S4E) and band-pass behavior. For treatment with both antibiotics, we note that the lower densities at the pulse period of 40 min were not due to the loss of bacteria from the cell chamber (Supplementary Figure S7B). Our results demonstrate the drastic qualitative changes in treatment efficacy due to both IE and the recovery kinetics, which are critical factors that have been neglected in previous studies (Udekwu et al, 2009).

Bottom Line: The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density.A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response.Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC, USA.

ABSTRACT
The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes.

Show MeSH
Related in: MedlinePlus