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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

Killing of L. monocytogenes wild type EGD (■) and the ΔlmaDCBA (▲) mutant with co-trimoxazole (A), ampicillin (B), and tetracycline (C) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 10 μg/ml co-trimoxazole (A), 3 μg/ml ampicillin, (B) and 3.5 μg/ml tetracycline (C). The experiment was performed with two biological replicates, except ampicillin that was performed with three biological replicates, and error bar are standard deviation.
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Figure 5: Killing of L. monocytogenes wild type EGD (■) and the ΔlmaDCBA (▲) mutant with co-trimoxazole (A), ampicillin (B), and tetracycline (C) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 10 μg/ml co-trimoxazole (A), 3 μg/ml ampicillin, (B) and 3.5 μg/ml tetracycline (C). The experiment was performed with two biological replicates, except ampicillin that was performed with three biological replicates, and error bar are standard deviation.

Mentions: To investigate the presumed correlation between monocin expression of planktonic cells and the antibiotic-dependent biofilm formation, we constructed a ΔlmaDCBA mutant. In a killing assay, the ΔlmaDCBA mutant was significantly (p < 0.046) more tolerant by killing of co-trimoxazole at 12 h and afterward (Figure 5A) as well as killing by ampicillin after 48 and 72 h (p = 0.009 and p = 0.031, respectively; Figure 5B) but not when treated with tetracycline (p-values ranging from 0.055 to 0.61; Figure 5C) indicating a role of monocin in antibiotic killing of planktonic cells. Secondly, we investigated the role of monocin in biofilm formation when exposed to increasing concentration of co-trimoxazole and, consistent with increased survival during killing with co-trimoxazole, the ΔlmaDCBA mutant grew with higher concentration of co-trimoxazole than the wild type (Supplementary Figure S5A). Similar to the wild type having increased biofilm formation per biomass with increasing growth-inhibiting co-trimoxazole concentration, the ΔlmaDCBA mutant also had increased biofilm formation (Supplementary Figure S5B) and biofilm formation per biomass (Figure 4C) indicating that that the cryptic prophage, monocin, is not involved in co-trimoxazole-dependent biofilm formation (Figure 4C).


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)

Killing of L. monocytogenes wild type EGD (■) and the ΔlmaDCBA (▲) mutant with co-trimoxazole (A), ampicillin (B), and tetracycline (C) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 10 μg/ml co-trimoxazole (A), 3 μg/ml ampicillin, (B) and 3.5 μg/ml tetracycline (C). The experiment was performed with two biological replicates, except ampicillin that was performed with three 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 5: Killing of L. monocytogenes wild type EGD (■) and the ΔlmaDCBA (▲) mutant with co-trimoxazole (A), ampicillin (B), and tetracycline (C) at 37°C. An early stationary phase culture (16 h) was diluted to OD600 = 0.4 and exposed to 10 μg/ml co-trimoxazole (A), 3 μg/ml ampicillin, (B) and 3.5 μg/ml tetracycline (C). The experiment was performed with two biological replicates, except ampicillin that was performed with three biological replicates, and error bar are standard deviation.
Mentions: To investigate the presumed correlation between monocin expression of planktonic cells and the antibiotic-dependent biofilm formation, we constructed a ΔlmaDCBA mutant. In a killing assay, the ΔlmaDCBA mutant was significantly (p < 0.046) more tolerant by killing of co-trimoxazole at 12 h and afterward (Figure 5A) as well as killing by ampicillin after 48 and 72 h (p = 0.009 and p = 0.031, respectively; Figure 5B) but not when treated with tetracycline (p-values ranging from 0.055 to 0.61; Figure 5C) indicating a role of monocin in antibiotic killing of planktonic cells. Secondly, we investigated the role of monocin in biofilm formation when exposed to increasing concentration of co-trimoxazole and, consistent with increased survival during killing with co-trimoxazole, the ΔlmaDCBA mutant grew with higher concentration of co-trimoxazole than the wild type (Supplementary Figure S5A). Similar to the wild type having increased biofilm formation per biomass with increasing growth-inhibiting co-trimoxazole concentration, the ΔlmaDCBA mutant also had increased biofilm formation (Supplementary Figure S5B) and biofilm formation per biomass (Figure 4C) indicating that that the cryptic prophage, monocin, is not involved in co-trimoxazole-dependent biofilm formation (Figure 4C).

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