Metabolomics analysis of the lactobacillus plantarum ATCC 14917 response to antibiotic stress

Abstract

Background: Lactobacillus plantarum has been found to play a significant role in maintaining the balance of intestinal flora in the human gut. However, it is sensitive to commonly used antibiotics and is often incidentally killed during treatment. We attempted to identify a means to protect L. plantarum ATCC14917 from the metabolic changes caused by two commonly used antibiotics, ampicillin, and doxycycline. We examined the metabolic changes under ampicillin and doxycycline treatment and assessed the protective effects of adding key exogenous metabolites.

Results: Using metabolomics, we found that under the stress of ampicillin or doxycycline, L. plantarum ATCC14917 exhibited reduced metabolic activity, with purine metabolism a key metabolic pathway involved in this change. We then screened the key biomarkers in this metabolic pathway, guanine and adenosine diphosphate (ADP). The exogenous addition of each of these two metabolites significantly reduced the lethality of ampicillin and doxycycline on L. plantarum ATCC14917. Because purine metabolism is closely related to the production of reactive oxygen species (ROS), the results showed that the addition of guanine or ADP reduced intracellular ROS levels in L. plantarum ATCC14917. Moreover, the killing effects of ampicillin and doxycycline on L. plantarum ATCC14917 were restored by the addition of a ROS accelerator in the presence of guanine or ADP.

Conclusions: The metabolic changes of L. plantarum ATCC14917 under antibiotic treatments were determined. Moreover, the metabolome information that was elucidated can be used to help L. plantarum cope with adverse stress, which will help probiotics become less vulnerable to antibiotics during clinical treatment.

Keywords: Lactobacillus plantarum ATCC14917; Antibiotics; Metabolomics; Probiotic protection.

Fig. 1

MIC determination and determination of sublethal concentrations of ampicilin and doxycyline. A MIC of L. plantarum ATCC14917 to different types of antibiotics. B Survival of L. plantarum ATCC14917 at different concentrations of ampicillin. C Survival of L. plantarum ATCC14917 at different concentrations of doxycycline. All data are displayed as mean ± SEM. ns: non-significant differences. ***p < 0.001, determined by Student’s t test

Fig. 2

Changes in differential metabolites of L. plantarum ATCC14917 in response to ampicillin and doxycycline. A & C Heat map showing differential abundance of metabolites. Blue and yellow colors indicate lower and higher abundances of the metabolites relative to the mean level of the control group, respectively. B & D Z-score plots of changes in differential metabolites based on control. The data were respectively scaled to the mean and standard deviation of control. Each point represents one biological repeat. Different treatments are distinguished by the color. E & F Categories of differential abundance of different metabolites

Fig. 3

Enrichment of metabolic pathways in L. plantarum ATCC14917 in response to ampicillin and doxycycline. A & B Significantly enriched metabolic pathways in response to uracil treatment (p < 0.05). C & D Changes in differential metabolites involved in the significantly enriched pathways. Yellow color and blue color indicate increased and decreased metabolites, respectively, in ampicillin-treated and doxycycline-treated group. E & F: The score plot of PCA. Each dot represents one biological replica in the plot. G & H: S-plot of OPLS-DA. Triangle represents individual metabolite. Red indicates the potential biomarkers, which is greater than or equal to 0.05 and 0.5 for absolute value of covariance p [1] and correlation p (corr) [1], respectively

Fig. 4

Effect of ADP, guanine cooperate with ampicillin and doxycycline on L. plantarum ATCC14917 survival.  A and B  The killing effects of different concentrations of ADP and guanine combined with 0.156 μg/mL ampicillin. C and D The killing effects of different concentrations of ADP and guanine combined with 6.25 μg/mL doxycycline. All data are displayed as mean ± SEM. *p < 0.05 and ***p < 0.001, determined by Student’s t test

Fig. 5

Effects of guanine, ADP cooperating with ampicillin, doxycycline on ROS of L. plantarum ATCC14917. A & B Effects of guanine, ADP cooperating with ampicillin, doxycycline on ROS content. C & D Effect of guanine, ADP cooperating with ampicillin, doxycycline on L. plantarum ATCC14917 survival at different doses of trichloride. All data are displayed as mean ± SEM. **p < 0.01 and ***p < 0.001, determined by Student’s t test