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Sublethal Concentrations of Antibiotics Cause Shift to Anaerobic Metabolism in Listeria monocytogenes and Induce Phenotypes Linked to Antibiotic Tolerance.

Knudsen GM, Fromberg A, Ng Y, Gram L - Front Microbiol (2016)

Bottom Line: A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance.However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested.Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
The human pathogenic bacterium Listeria monocytogenes is exposed to antibiotics both during clinical treatment and in its saprophytic lifestyle. As one of the keys to successful treatment is continued antibiotic sensitivity, the purpose of this study was to determine if exposure to sublethal antibiotic concentrations would affect the bacterial physiology and induce antibiotic tolerance. Transcriptomic analyses demonstrated that each of the four antibiotics tested caused an antibiotic-specific gene expression pattern related to mode-of-action of the particular antibiotic. All four antibiotics caused the same changes in expression of several metabolic genes indicating a shift from aerobic to anaerobic metabolism and higher ethanol production. A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance. However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested. This shift in metabolism could be a survival strategy in response to antibiotics to avoid generation of ROS production from respiration by oxidation of NADH through ethanol production. The monocin locus encoding a cryptic prophage was induced by co-trimoxazole and repressed by ampicillin and gentamicin, and this correlated with an observed antibiotic-dependent biofilm formation. A monocin mutant (ΔlmaDCBA) had increased biofilm formation when exposed to increasing concentration of co-trimoxazole similar to the wild type, but was more tolerant to killing by co-trimoxazole and ampicillin. Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

No MeSH data available.


Related in: MedlinePlus

The effect of sublethal antibiotic concentrations on biofilm formation of L. monocytogenes EGD at 37°C. (A) Biofilm formation of wild type EGD being exposed to increasing concentration of antibiotics measured as crystal violet stained biofilm and measured spectrometrically at 590 nm and calibrated to biomass (measured as planktonic cells at OD600nm). Asterisk denote p < 0.05 when comparing biofilm of the control to the antibiotic exposed biofilm. (B) Images of crystal violet stained biofilm formed by wild type EGD being exposed to increasing concentration of antibiotics. Eight technical replicates were performed at each experiment. Representative images are shown. (C) Biofilm formation of wild type (black bar) and lmaDCBA mutant (gray bar) with increasing concentration of co-trimoxazole ranging from 0.1 to 2 μg/ml co-trimoxazole. The experiment was performed with two biological replicates and error bar are standard deviation.
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Figure 4: The effect of sublethal antibiotic concentrations on biofilm formation of L. monocytogenes EGD at 37°C. (A) Biofilm formation of wild type EGD being exposed to increasing concentration of antibiotics measured as crystal violet stained biofilm and measured spectrometrically at 590 nm and calibrated to biomass (measured as planktonic cells at OD600nm). Asterisk denote p < 0.05 when comparing biofilm of the control to the antibiotic exposed biofilm. (B) Images of crystal violet stained biofilm formed by wild type EGD being exposed to increasing concentration of antibiotics. Eight technical replicates were performed at each experiment. Representative images are shown. (C) Biofilm formation of wild type (black bar) and lmaDCBA mutant (gray bar) with increasing concentration of co-trimoxazole ranging from 0.1 to 2 μg/ml co-trimoxazole. The experiment was performed with two biological replicates and error bar are standard deviation.

Mentions: Biofilm formation is affected by antibiotics in several bacteria (Bernier and Surette, 2013; Nguyen et al., 2014), and we investigated if the four antibiotics affected biofilm formation in L. monocytogenes EGD. We calibrated all biofilm to biomass measured as planktonic cells (OD600) to ensure that effects on growth/maximum cell density were not affecting the results. Sublethal concentrations of tetracycline did not affect the biofilm formation/biomass (p = 0.107; Figures 4A,B). Increasing co-trimoxazole concentrations significantly increased the biofilm formation per biomass at 0.5 μg/ml (p = 0.001) whereas ampicillin and gentamicin significantly reduced the biofilm formation per biomass (p = 0.012 and 0.008, respectively). These changes in biofilm formation correlated with expression of the cryptic prophage locus including lmaDCBA operon as co-trimoxazole increased expression of the monocin locus and ampicillin and gentamicin repressed the expression of the locus as measured by RNA seq and qRT-PCR of planktonic cells. The monocin locus encodes a cryptic prophage that produces a listeriolytic tail and we hypothesized that increased expression of the monocin locus of planktonic cells would increase monocin production and cell lysis thus increasing biofilm formation. We attempted to determine the level of monocins in sterile supernatant but no plaques were observed in a standard phage-plaque assay indicating that the monocin concentration in the supernatant was too low to kill indicator strains (data not shown).


Sublethal Concentrations of Antibiotics Cause Shift to Anaerobic Metabolism in Listeria monocytogenes and Induce Phenotypes Linked to Antibiotic Tolerance.

Knudsen GM, Fromberg A, Ng Y, Gram L - Front Microbiol (2016)

The effect of sublethal antibiotic concentrations on biofilm formation of L. monocytogenes EGD at 37°C. (A) Biofilm formation of wild type EGD being exposed to increasing concentration of antibiotics measured as crystal violet stained biofilm and measured spectrometrically at 590 nm and calibrated to biomass (measured as planktonic cells at OD600nm). Asterisk denote p < 0.05 when comparing biofilm of the control to the antibiotic exposed biofilm. (B) Images of crystal violet stained biofilm formed by wild type EGD being exposed to increasing concentration of antibiotics. Eight technical replicates were performed at each experiment. Representative images are shown. (C) Biofilm formation of wild type (black bar) and lmaDCBA mutant (gray bar) with increasing concentration of co-trimoxazole ranging from 0.1 to 2 μg/ml co-trimoxazole. The experiment was performed with two biological replicates and error bar are standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940397&req=5

Figure 4: The effect of sublethal antibiotic concentrations on biofilm formation of L. monocytogenes EGD at 37°C. (A) Biofilm formation of wild type EGD being exposed to increasing concentration of antibiotics measured as crystal violet stained biofilm and measured spectrometrically at 590 nm and calibrated to biomass (measured as planktonic cells at OD600nm). Asterisk denote p < 0.05 when comparing biofilm of the control to the antibiotic exposed biofilm. (B) Images of crystal violet stained biofilm formed by wild type EGD being exposed to increasing concentration of antibiotics. Eight technical replicates were performed at each experiment. Representative images are shown. (C) Biofilm formation of wild type (black bar) and lmaDCBA mutant (gray bar) with increasing concentration of co-trimoxazole ranging from 0.1 to 2 μg/ml co-trimoxazole. The experiment was performed with two biological replicates and error bar are standard deviation.
Mentions: Biofilm formation is affected by antibiotics in several bacteria (Bernier and Surette, 2013; Nguyen et al., 2014), and we investigated if the four antibiotics affected biofilm formation in L. monocytogenes EGD. We calibrated all biofilm to biomass measured as planktonic cells (OD600) to ensure that effects on growth/maximum cell density were not affecting the results. Sublethal concentrations of tetracycline did not affect the biofilm formation/biomass (p = 0.107; Figures 4A,B). Increasing co-trimoxazole concentrations significantly increased the biofilm formation per biomass at 0.5 μg/ml (p = 0.001) whereas ampicillin and gentamicin significantly reduced the biofilm formation per biomass (p = 0.012 and 0.008, respectively). These changes in biofilm formation correlated with expression of the cryptic prophage locus including lmaDCBA operon as co-trimoxazole increased expression of the monocin locus and ampicillin and gentamicin repressed the expression of the locus as measured by RNA seq and qRT-PCR of planktonic cells. The monocin locus encodes a cryptic prophage that produces a listeriolytic tail and we hypothesized that increased expression of the monocin locus of planktonic cells would increase monocin production and cell lysis thus increasing biofilm formation. We attempted to determine the level of monocins in sterile supernatant but no plaques were observed in a standard phage-plaque assay indicating that the monocin concentration in the supernatant was too low to kill indicator strains (data not shown).

Bottom Line: A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance.However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested.Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Technical University of Denmark Kongens Lyngby, Denmark.

ABSTRACT
The human pathogenic bacterium Listeria monocytogenes is exposed to antibiotics both during clinical treatment and in its saprophytic lifestyle. As one of the keys to successful treatment is continued antibiotic sensitivity, the purpose of this study was to determine if exposure to sublethal antibiotic concentrations would affect the bacterial physiology and induce antibiotic tolerance. Transcriptomic analyses demonstrated that each of the four antibiotics tested caused an antibiotic-specific gene expression pattern related to mode-of-action of the particular antibiotic. All four antibiotics caused the same changes in expression of several metabolic genes indicating a shift from aerobic to anaerobic metabolism and higher ethanol production. A mutant in the bifunctional acetaldehyde-CoA/alcohol dehydrogenase encoded by lmo1634 did not have altered antibiotic tolerance. However, a mutant in lmo1179 (eutE) encoding an aldehyde oxidoreductase where rerouting caused increased ethanol production was tolerant to three of four antibiotics tested. This shift in metabolism could be a survival strategy in response to antibiotics to avoid generation of ROS production from respiration by oxidation of NADH through ethanol production. The monocin locus encoding a cryptic prophage was induced by co-trimoxazole and repressed by ampicillin and gentamicin, and this correlated with an observed antibiotic-dependent biofilm formation. A monocin mutant (ΔlmaDCBA) had increased biofilm formation when exposed to increasing concentration of co-trimoxazole similar to the wild type, but was more tolerant to killing by co-trimoxazole and ampicillin. Thus, sublethal concentrations of antibiotics caused metabolic and physiological changes indicating that the organism is preparing to withstand lethal antibiotic concentrations.

No MeSH data available.


Related in: MedlinePlus