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

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.

Keywords: Listeria monocytogenes; biofilm; gene expression; metabolism monocin; sublethal antibiotic concentrations.

FIGURE 1

Listeria monocytogenes EGD genes differentially expressed when exposed to ampicillin, tetracycline, gentamicin, or co-trimoxazole. (A) Functional categories of differentially expressed genes in response to the four antibiotics. Genes that passed the statistical filtering (p < 0.05, q < 0.05 and a twofold cut-off) are shown as percentage of genes in each functional category up-regulated (blue) or down-regulated (red), respectively, when comparing antibiotic-exposed L. monocytogenes EGD with MilliQ control. The list of genes included in each functional category was based on cluster of orthologous groups (COGs) of L. monocytogenes EGD-e genes (http://www.ncbi.nlm.nih.gov/sutils/coxik.cgi?gi=204%25253e). Asterisk indicate that the functional category was overrepresented in a hypergeometric distribution test (p = 0.01). (B) Venn diagram with up-regulated genes showing antibiotic-specific and common genes significantly differentially expressed genes. (C) Venn diagram with down-regulated genes showing antibiotic-specific and common genes significantly differentially expressed genes. Ampicillin (AMP), tetracycline (TET), gentamicin (GEN), and co-trimoxazole (SXT).

FIGURE 2

Killing of L. monocytogenes EGD by (A) gentamicin, (B) co-trimoxazole, (C) tetracycline, and (D) ampicillin. An early stationary phase culture (16 h at 37°C) was diluted to OD₆₀₀ = 0.1 and exposed to either MilliQ or different concentrations of the four antibiotics. The concentrations are given relative to the concentration of antibiotic used for the transcriptomic analysis (1X), i.e., 50X (for gentamicin and co-trimoxacole), 100X (for all four antibiotics), and 1000X (for ampicillin and tetracycline). The experiment was performed with three biological replicates and error bar are standard deviation.

FIGURE 3

Killing of L. monocytogenes wild type EGD (■, black full line), Δlmo1634 (▲, black broken line), Δlmo1179 (●, gray full line) and Δlmo1634/Δlmo1179 (●, gray broken line) mutants with ampicillin (A), tetracycline (B), co-trimoxazole (C), or gentamicin (D) at 37°C. An early stationary phase culture (16 h) was diluted to OD₆₀₀ = 0.4 and exposed to 3 μg/ml ampicillin (A), 3.5 μg/ml tetracycline (B), 10 μg/ml co-trimoxazole (C), or 30 μg/ml gentamicin (D). The experiment was performed with three biological replicates and error bar are standard deviation.

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 OD_{600 nm}). 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.

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.

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.