Limits...
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.

Show MeSH

Related in: MedlinePlus

Perturbation of HSR and protein degradation shifted the IE region. (A) Fast ribosome turnover, and thus IE can be initiated by challenging bacteria with numerous antibiotics that stimulate HSR. HSR and ribosome degradation can be perturbed using temperature, chemical inhibitors, and knockouts. (B) IE is initiated with various antibiotics. Tetracycline (Tet) and chloramphenicol (Cm), which to do not induce HSR, did not lead to IE. In contrast, tobramycin (Tob), gentamicin, (Gen), nourseothricin (Nou), neomycin (Neo), puromycin (Pur), streptomycin (Str), and kanamycin (Kan), which induce HSR, resulted in IE. See sample results of Str, Pur, and Tet in Supplementary Figure S4. Dark gray regions indicate that populations exhibited IE. Light gray regions indicate that both populations went extinct. (C) Inhibition of serine proteases (Ser Inh, 10 μg/ml) shifted the bifurcation region to higher concentrations of kanamycin (Kan+Ser Inh, 12–15 μg/ml). Similarly, treatment with cysteine protease inhibitors (Cys Inh, 10 μg/ml) or aspartyl protease (Asp Inh, 10 μg/ml) inhibitors shifted the IE region to 8–12 μg/ml. Heat shock at 42°C shifted the bifurcation region to lower concentrations of kanamycin (Kan+42°C, 0.5–1 μg/ml). Inhibition of all proteases reversed this effect by shifting the IE region back to higher concentrations of kanamycin (Kan+42°C+Inh, 5–6 μg/ml). Chloramphenicol coupled with heat shock led to IE (Cm+42°C, 0.5–1 μg/ml), which was abolished by the inhibition of all proteases (Cm+42°C+Inh). See additional results in Supplementary Figure S4. (D) The wild-type BW25113 strain (BW) exhibited IE between 4 and 8 μg/ml. Knockout strain Δlon shrunk the IE region to between 5 and 8 μg/ml. Knockout strain ΔclpX shrunk the IE region to 5 μg/ml. As such, the IE region was shrunk by knockout of proteases, which is consistent with our model predictions and the perturbation results in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472685&req=5

f4: Perturbation of HSR and protein degradation shifted the IE region. (A) Fast ribosome turnover, and thus IE can be initiated by challenging bacteria with numerous antibiotics that stimulate HSR. HSR and ribosome degradation can be perturbed using temperature, chemical inhibitors, and knockouts. (B) IE is initiated with various antibiotics. Tetracycline (Tet) and chloramphenicol (Cm), which to do not induce HSR, did not lead to IE. In contrast, tobramycin (Tob), gentamicin, (Gen), nourseothricin (Nou), neomycin (Neo), puromycin (Pur), streptomycin (Str), and kanamycin (Kan), which induce HSR, resulted in IE. See sample results of Str, Pur, and Tet in Supplementary Figure S4. Dark gray regions indicate that populations exhibited IE. Light gray regions indicate that both populations went extinct. (C) Inhibition of serine proteases (Ser Inh, 10 μg/ml) shifted the bifurcation region to higher concentrations of kanamycin (Kan+Ser Inh, 12–15 μg/ml). Similarly, treatment with cysteine protease inhibitors (Cys Inh, 10 μg/ml) or aspartyl protease (Asp Inh, 10 μg/ml) inhibitors shifted the IE region to 8–12 μg/ml. Heat shock at 42°C shifted the bifurcation region to lower concentrations of kanamycin (Kan+42°C, 0.5–1 μg/ml). Inhibition of all proteases reversed this effect by shifting the IE region back to higher concentrations of kanamycin (Kan+42°C+Inh, 5–6 μg/ml). Chloramphenicol coupled with heat shock led to IE (Cm+42°C, 0.5–1 μg/ml), which was abolished by the inhibition of all proteases (Cm+42°C+Inh). See additional results in Supplementary Figure S4. (D) The wild-type BW25113 strain (BW) exhibited IE between 4 and 8 μg/ml. Knockout strain Δlon shrunk the IE region to between 5 and 8 μg/ml. Knockout strain ΔclpX shrunk the IE region to 5 μg/ml. As such, the IE region was shrunk by knockout of proteases, which is consistent with our model predictions and the perturbation results in (C).

Mentions: If fast degradation of C was the major determinant for the generation of IE, then we hypothesized that other antibiotics that induce HSR would also cause IE. To test this notion, we examined bacterial response to streptomycin, puromycin, gentamicin, tobramycin, neomycin, and nourseothricin, which induce HSR, as well as tetracycline, which does not (Vanbogelen and Neidhardt, 1990; Supplementary Table S1; Supplementary Figure S1A). Similarly to kanamycin, these antibiotics also bind to C (Supplementary Table S1) and therefore can be described by our generic model (Equation 1; Figure 1A). Consistent with our hypothesis, the antibiotics that induced HSR caused IE, whereas tetracycline did not (Figure 4A and B; Supplementary Figure S4A–C).


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)

Perturbation of HSR and protein degradation shifted the IE region. (A) Fast ribosome turnover, and thus IE can be initiated by challenging bacteria with numerous antibiotics that stimulate HSR. HSR and ribosome degradation can be perturbed using temperature, chemical inhibitors, and knockouts. (B) IE is initiated with various antibiotics. Tetracycline (Tet) and chloramphenicol (Cm), which to do not induce HSR, did not lead to IE. In contrast, tobramycin (Tob), gentamicin, (Gen), nourseothricin (Nou), neomycin (Neo), puromycin (Pur), streptomycin (Str), and kanamycin (Kan), which induce HSR, resulted in IE. See sample results of Str, Pur, and Tet in Supplementary Figure S4. Dark gray regions indicate that populations exhibited IE. Light gray regions indicate that both populations went extinct. (C) Inhibition of serine proteases (Ser Inh, 10 μg/ml) shifted the bifurcation region to higher concentrations of kanamycin (Kan+Ser Inh, 12–15 μg/ml). Similarly, treatment with cysteine protease inhibitors (Cys Inh, 10 μg/ml) or aspartyl protease (Asp Inh, 10 μg/ml) inhibitors shifted the IE region to 8–12 μg/ml. Heat shock at 42°C shifted the bifurcation region to lower concentrations of kanamycin (Kan+42°C, 0.5–1 μg/ml). Inhibition of all proteases reversed this effect by shifting the IE region back to higher concentrations of kanamycin (Kan+42°C+Inh, 5–6 μg/ml). Chloramphenicol coupled with heat shock led to IE (Cm+42°C, 0.5–1 μg/ml), which was abolished by the inhibition of all proteases (Cm+42°C+Inh). See additional results in Supplementary Figure S4. (D) The wild-type BW25113 strain (BW) exhibited IE between 4 and 8 μg/ml. Knockout strain Δlon shrunk the IE region to between 5 and 8 μg/ml. Knockout strain ΔclpX shrunk the IE region to 5 μg/ml. As such, the IE region was shrunk by knockout of proteases, which is consistent with our model predictions and the perturbation results in (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Perturbation of HSR and protein degradation shifted the IE region. (A) Fast ribosome turnover, and thus IE can be initiated by challenging bacteria with numerous antibiotics that stimulate HSR. HSR and ribosome degradation can be perturbed using temperature, chemical inhibitors, and knockouts. (B) IE is initiated with various antibiotics. Tetracycline (Tet) and chloramphenicol (Cm), which to do not induce HSR, did not lead to IE. In contrast, tobramycin (Tob), gentamicin, (Gen), nourseothricin (Nou), neomycin (Neo), puromycin (Pur), streptomycin (Str), and kanamycin (Kan), which induce HSR, resulted in IE. See sample results of Str, Pur, and Tet in Supplementary Figure S4. Dark gray regions indicate that populations exhibited IE. Light gray regions indicate that both populations went extinct. (C) Inhibition of serine proteases (Ser Inh, 10 μg/ml) shifted the bifurcation region to higher concentrations of kanamycin (Kan+Ser Inh, 12–15 μg/ml). Similarly, treatment with cysteine protease inhibitors (Cys Inh, 10 μg/ml) or aspartyl protease (Asp Inh, 10 μg/ml) inhibitors shifted the IE region to 8–12 μg/ml. Heat shock at 42°C shifted the bifurcation region to lower concentrations of kanamycin (Kan+42°C, 0.5–1 μg/ml). Inhibition of all proteases reversed this effect by shifting the IE region back to higher concentrations of kanamycin (Kan+42°C+Inh, 5–6 μg/ml). Chloramphenicol coupled with heat shock led to IE (Cm+42°C, 0.5–1 μg/ml), which was abolished by the inhibition of all proteases (Cm+42°C+Inh). See additional results in Supplementary Figure S4. (D) The wild-type BW25113 strain (BW) exhibited IE between 4 and 8 μg/ml. Knockout strain Δlon shrunk the IE region to between 5 and 8 μg/ml. Knockout strain ΔclpX shrunk the IE region to 5 μg/ml. As such, the IE region was shrunk by knockout of proteases, which is consistent with our model predictions and the perturbation results in (C).
Mentions: If fast degradation of C was the major determinant for the generation of IE, then we hypothesized that other antibiotics that induce HSR would also cause IE. To test this notion, we examined bacterial response to streptomycin, puromycin, gentamicin, tobramycin, neomycin, and nourseothricin, which induce HSR, as well as tetracycline, which does not (Vanbogelen and Neidhardt, 1990; Supplementary Table S1; Supplementary Figure S1A). Similarly to kanamycin, these antibiotics also bind to C (Supplementary Table S1) and therefore can be described by our generic model (Equation 1; Figure 1A). Consistent with our hypothesis, the antibiotics that induced HSR caused IE, whereas tetracycline did not (Figure 4A and B; Supplementary Figure S4A–C).

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