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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

Model of the central metabolism indicating the shift from acetoin to ethanol production observed by all four antibiotics. Blue indicate decreased expression of lmo1992 and alsS and red indicate increased expression of lmo1634 by all four antibiotics. Pyruvate formate-lyase encoded by pflAC (light red) and involved in degradation of pyruvate under anaerobic condition is induced by gentamicin and co-trimoxazole but not ampicillin and tetracycline whereas pyruvate dehydrogenase encoded by pdhA (light blue) involved in degradation of pyruvate under aerobic condition are down-regulated by co-trimoxazole but not any of the other antibiotics. Broken circle illustrate the microcompartment in which ethanolamine is degraded and the degradation pathway of ethanolamine to ethanol and acetyl-CoA. Neither of the four genes lmo1179, lmo1171, lmo1165, or lmo1166 (gray) are differentially expressed by any of the four antibiotics.
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Figure 6: Model of the central metabolism indicating the shift from acetoin to ethanol production observed by all four antibiotics. Blue indicate decreased expression of lmo1992 and alsS and red indicate increased expression of lmo1634 by all four antibiotics. Pyruvate formate-lyase encoded by pflAC (light red) and involved in degradation of pyruvate under anaerobic condition is induced by gentamicin and co-trimoxazole but not ampicillin and tetracycline whereas pyruvate dehydrogenase encoded by pdhA (light blue) involved in degradation of pyruvate under aerobic condition are down-regulated by co-trimoxazole but not any of the other antibiotics. Broken circle illustrate the microcompartment in which ethanolamine is degraded and the degradation pathway of ethanolamine to ethanol and acetyl-CoA. Neither of the four genes lmo1179, lmo1171, lmo1165, or lmo1166 (gray) are differentially expressed by any of the four antibiotics.

Mentions: Listeria monocytogenes responded to antibiotics by remodeling the gene expression of the central metabolism, which indicate a shift from acetoin to ethanol production driven by a shift in expression of mainly alsS, lmo1992, lmo1634, and lmo2104-5. This shift correlate with a shift from aerobic to anaerobic metabolism as L. monocytogenes under aerobic conditions converts 26% of the carbon from glucose to acetoin, while no acetoin is produced under anaerobic conditions (Romick et al., 1996). In contrast, ethanol is specifically produced under anaerobic conditions (Romick et al., 1996). This shift in metabolic gene expression was more pronounced for the bactericidal antibiotic than the bacteriostatic compounds. It was recently shown that Burkholderia thailandensis tolerate antibiotics by adapting to and inducing anaerobic nitrate respiration and the tolerance could be eliminated by oxygenating the system or with the addition of nitrate (Hemsley et al., 2014). Fumarate is the likely alternative electron acceptor in L. monocytogenes under anaerobic conditions (Müller-Herbst et al., 2014) and consistent with B. thailandensis, gentamicin and co-trimoxazole, but not ampicillin and tetracycline, induced lmo0355, encoding a fumarate reductase, more than fourfold at 3 h supporting our hypothesis with a larger metabolic shift for cell exposed to bactericidal antibiotic than to bacteriostatic. Based on the transcriptomic analysis and functional analysis of the lmo1634 gene encoding a bifunctional acetaldehyde-CoA/ADH, we speculate that this gene plays a central role in the metabolic shift (Figure 6). The bifunctional acetaldehyde-CoA/ADH Lmo1634 has 49% protein identity (E-value 0.0 over 872 amino acids) to AdhE of Escherichia coli but in L. monocytogenes EGD-e this protein is also known as LAP (Listeria adhesion protein) and promotes bacterial adhesion to enterocyte-like Caco-2 cells (Jagadeesan et al., 2010). Both lmo1634 and adhE are induced by anaerobic conditions (Leonardo et al., 1996; Burkholder et al., 2009) and in E. coli, AdhE also acts as a H2O2 scavenger under aerobic conditions and has an important role in protection against H2O2 stress (Echave et al., 2003). The role of Lmo1634 in oxidative stress in L. monocytogenes is unknown but both 3 mM ferricyanide (FeCN) and 6 mM dithiothreitol (DTT) that altered redox potential repress the expression of lmo1634 (Ignatova et al., 2013). However, a Δlmo1634 mutant was as sensitive to antibiotic as the wild type EGD whereas a mutant in lmo1179 (eutE) was highly tolerant to ampicillin, tetracycline and co-trimoxazole, but not gentamicin. lmo1179 (eutE) are located in the eut operon that involved in utilization of ethanolamine and encoded an aldehyde oxidoreductase that convert acetaldehyde to acetyl-CoA under generation of NADH from NAD+ (Garsin, 2010; Mellin et al., 2014). Ethanolamine is a degradation product of phosphatidylethanolamine that is abundant in mammalian and bacterial cell membranes (Garsin, 2010) and the eut genes involved in virulence in L. monocytogenes (Joseph et al., 2006; Mellin et al., 2014). In this study, we show that the absence of lmo1179 (eutE) cause antibiotic tolerance to three of four antibiotic tested and we speculate this tolerance is caused by rerouting of all acetaldehyde to ethanol, i.e., anaerobic metabolism and that there is redundancy among the ADHs in L. monocytogenes. However, interesting neither of the eut genes are significantly affected by any of the four antibiotics.


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)

Model of the central metabolism indicating the shift from acetoin to ethanol production observed by all four antibiotics. Blue indicate decreased expression of lmo1992 and alsS and red indicate increased expression of lmo1634 by all four antibiotics. Pyruvate formate-lyase encoded by pflAC (light red) and involved in degradation of pyruvate under anaerobic condition is induced by gentamicin and co-trimoxazole but not ampicillin and tetracycline whereas pyruvate dehydrogenase encoded by pdhA (light blue) involved in degradation of pyruvate under aerobic condition are down-regulated by co-trimoxazole but not any of the other antibiotics. Broken circle illustrate the microcompartment in which ethanolamine is degraded and the degradation pathway of ethanolamine to ethanol and acetyl-CoA. Neither of the four genes lmo1179, lmo1171, lmo1165, or lmo1166 (gray) are differentially expressed by any of the four antibiotics.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Model of the central metabolism indicating the shift from acetoin to ethanol production observed by all four antibiotics. Blue indicate decreased expression of lmo1992 and alsS and red indicate increased expression of lmo1634 by all four antibiotics. Pyruvate formate-lyase encoded by pflAC (light red) and involved in degradation of pyruvate under anaerobic condition is induced by gentamicin and co-trimoxazole but not ampicillin and tetracycline whereas pyruvate dehydrogenase encoded by pdhA (light blue) involved in degradation of pyruvate under aerobic condition are down-regulated by co-trimoxazole but not any of the other antibiotics. Broken circle illustrate the microcompartment in which ethanolamine is degraded and the degradation pathway of ethanolamine to ethanol and acetyl-CoA. Neither of the four genes lmo1179, lmo1171, lmo1165, or lmo1166 (gray) are differentially expressed by any of the four antibiotics.
Mentions: Listeria monocytogenes responded to antibiotics by remodeling the gene expression of the central metabolism, which indicate a shift from acetoin to ethanol production driven by a shift in expression of mainly alsS, lmo1992, lmo1634, and lmo2104-5. This shift correlate with a shift from aerobic to anaerobic metabolism as L. monocytogenes under aerobic conditions converts 26% of the carbon from glucose to acetoin, while no acetoin is produced under anaerobic conditions (Romick et al., 1996). In contrast, ethanol is specifically produced under anaerobic conditions (Romick et al., 1996). This shift in metabolic gene expression was more pronounced for the bactericidal antibiotic than the bacteriostatic compounds. It was recently shown that Burkholderia thailandensis tolerate antibiotics by adapting to and inducing anaerobic nitrate respiration and the tolerance could be eliminated by oxygenating the system or with the addition of nitrate (Hemsley et al., 2014). Fumarate is the likely alternative electron acceptor in L. monocytogenes under anaerobic conditions (Müller-Herbst et al., 2014) and consistent with B. thailandensis, gentamicin and co-trimoxazole, but not ampicillin and tetracycline, induced lmo0355, encoding a fumarate reductase, more than fourfold at 3 h supporting our hypothesis with a larger metabolic shift for cell exposed to bactericidal antibiotic than to bacteriostatic. Based on the transcriptomic analysis and functional analysis of the lmo1634 gene encoding a bifunctional acetaldehyde-CoA/ADH, we speculate that this gene plays a central role in the metabolic shift (Figure 6). The bifunctional acetaldehyde-CoA/ADH Lmo1634 has 49% protein identity (E-value 0.0 over 872 amino acids) to AdhE of Escherichia coli but in L. monocytogenes EGD-e this protein is also known as LAP (Listeria adhesion protein) and promotes bacterial adhesion to enterocyte-like Caco-2 cells (Jagadeesan et al., 2010). Both lmo1634 and adhE are induced by anaerobic conditions (Leonardo et al., 1996; Burkholder et al., 2009) and in E. coli, AdhE also acts as a H2O2 scavenger under aerobic conditions and has an important role in protection against H2O2 stress (Echave et al., 2003). The role of Lmo1634 in oxidative stress in L. monocytogenes is unknown but both 3 mM ferricyanide (FeCN) and 6 mM dithiothreitol (DTT) that altered redox potential repress the expression of lmo1634 (Ignatova et al., 2013). However, a Δlmo1634 mutant was as sensitive to antibiotic as the wild type EGD whereas a mutant in lmo1179 (eutE) was highly tolerant to ampicillin, tetracycline and co-trimoxazole, but not gentamicin. lmo1179 (eutE) are located in the eut operon that involved in utilization of ethanolamine and encoded an aldehyde oxidoreductase that convert acetaldehyde to acetyl-CoA under generation of NADH from NAD+ (Garsin, 2010; Mellin et al., 2014). Ethanolamine is a degradation product of phosphatidylethanolamine that is abundant in mammalian and bacterial cell membranes (Garsin, 2010) and the eut genes involved in virulence in L. monocytogenes (Joseph et al., 2006; Mellin et al., 2014). In this study, we show that the absence of lmo1179 (eutE) cause antibiotic tolerance to three of four antibiotic tested and we speculate this tolerance is caused by rerouting of all acetaldehyde to ethanol, i.e., anaerobic metabolism and that there is redundancy among the ADHs in L. monocytogenes. However, interesting neither of the eut genes are significantly affected by any of the four antibiotics.

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