Cipro floxacin stress changes key enzymes and intracellular metabolites of Lactobacillus plantarum DNZ-4

Pin Chen, Xioqin Chen, Wei Yu, Bo Zhou, Lihu Liu,Yuzhuo Yng, Peng Du, Lio Liu,*, Chun Li,*

a Key Laboratory of Dairy Science, Ministry of Education, Food Science College, Northeast Agricultural University, Harbin 150030, China

b Institute of Animal Science (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, China

c National Dairy Product Quality Supervision and Inspection Center, Harbin 150024, China

Keywords:

Cipro floxacin

Lactobacillus plantarum DNZ-4

Key enzymes

Metabolism

A B S T R A C T

Ciprofloxacin (CIP) is an antibiotic used to treat infections caused by bacteria. In this experiment, key enzymes and intracellular metabolites of Lactobacillus plantarum DNZ-4 was researched under CIP stress.The results showed that the activities of hexokinase, pyruvate kinase, β-galactosidase and Na+, K+-ATPase after 1/2 minimum bacteriostatic concentration (MIC) CIP treatment were significantly decreased (P ＜ 0.01).Gas chromatography-mass spectrometry was used to analysis the changes of main metabolites in the cells and principal component analysis and partial least square model were constructed. The results indicated that CIP could cause changes in intracellular fatty acids, carbohydrates and amino acids, and the mechanism of amino acid metabolism under CIP stress was significantly inhibited. L. plantarum DNZ-4 made stress response to CIP by regulating the ratio of saturated fatty acids and unsaturated fats. This experiment revealed the changes of growth and metabolism mechanism of L. plantarum DNZ-4 under CIP stress, which help to provide technical means for the development of effective probiotics preparation products.

1. Introduction

Lactic acid bacteria (LAB) constitute a diverse group of Grampositive, catalase-negative bacteria producing lactic acid as the main end-product of carbohydrate fermentation [1]. As traditional strains in food fermentation, especially in dairy, fermented meat and vegetable products [2], LAB can produce lactic acid to extend the shelf life of food and provide beneficial effects to human beings by improving the body’s natural defense system and regulating the gastrointestinal tract’s (GIT) micro-ecological balance [3]. As important members of LAB,Lactobacillus plantarumDNZ-4 are always found in fermented plant-based food and have many important physiological functions [4].Ciprofloxacin (CIP) is the most widely used third-generation fluoroquinolone in humans and livestock. It has a broad spectrum of antibacterial activity and is highly effective against Gram-negative and positive bacteria [5]. CIP can inhibit bacterial DNA helicase,interfere with the normal function of bacterial cell DNA, and make the replication ability of bacteria disappear quickly, thereby achieving bactericidal action [6]. Machuca et al. [7]found that under the stress of 1.0 µg/mL CIP,Escherichia colisuffered moderate DNA damage. Inappropriate dosages and frequent use of antibiotics at low mass concentrations lead to antibiotic residue problems in humans, livestock, and the environment [8,9]. When human body ingests food with antibiotic residues or improperly takes antibiotics for a long time, the intestinal flora micro-ecology will be destroyed.Carman et al. [10]found that when the CIP dose level was below the traditional threshold ADI (acceptable daily intake) of 1.5 mg/day, the ecology of the human intestinal flora was altered.

Metabolomics is the study of the global metabolite profiles of a cell under a given set of conditions [11]. In general, bacteria are able to respond to changes in external conditions in the environment by modulating the relative amount of intracellular metabolites in a matter of seconds, while metabolomics can truly reflect the physiological state of the cell system, providing a transient, comprehensive snapshot of ongoing biological processes. Metabolomics can analyze the changes of intracellular metabolites under different environmental stress by gas chromatography-mass spectrometry (GC-MS)/LC/MS method combined with multivariate statistical analysis, and comprehensively and systematically understand the changes of intracellular metabolites [12].

When antibiotics act on pathogenic bacteria, it also produces stress on probiotics colonized in the intestines. However, most scholars’ research is limited to the resistance mechanism of LAB to quinolones [13-15]. The effect of LAB on the changes of intracellular metabolites under quinolones stress has not been studied. Therefore,the aim of this experiment was to explore the influence of CIP stress on the growth and metabolic mechanism ofL. plantarumDNZ-4, and to consider whether it have the ability to actively respond to adverse stress under appropriate dose of CIP. The research may help to provide technical means for the development of effective probiotics preparation products.

2. Materials and methods

2.1 Bacterial strains and cultivation

L. plantarumDNZ-4 was provided from Northeast Agricultural University. 50% (V/V) glycerol with bacteria stock was stored at–20 °C, and the strain was incubated overnight in MRS broth at 37 °C for activation. Repeated the above incubation for three times to ensure the viability of the bacteria and make the number of live bacteria to reach 1 × 109CFU/mL.

2.2 Determination of the MIC of CIP for L. plantarum DNZ-4

The MIC of the strain was determined by using a conventional tube dilution method [16]. The above-activatedL. plantarumDNZ-4 was added to the MRS medium at inoculation amount of 2% (V/V), cultured at 37 °C for 12 h, centrifuged sample at 8 000 ×gfor 10 min, discard supernatant and adjusted to OD600nmof 0.8 with physiological saline. The above bacterial suspension was diluted 1:100 with MRS broth. CIP of different quality was added into the bacterial suspension, which made the drug concentrations of the first to eleventh tubes 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0.125 µg/mL,respectively. The MRS broth without bacterial suspensions were utilized as positive controls and negative controls were performed without CIP. The inboculated dilution tube was cultured in a 37 °C incubator for 20 h. MIC was the minimum concentration when no obvious growth of bacteria was observed during the culture time, and the experiment was repeated for 3 times.

2.3 Determination of growth curve of L. plantarum DNZ-4 under different concentrations of CIP

The activated culturedL. plantarumDNZ-4 was inoculated into MRS liquid medium at a 2% inoculation amount, wherein the control group contained no CIP, and the drug group was added with 1/40 MIC, 1/8 MIC and 1/2 MIC CIP, and then were placed in a 37 °C incubator. The bacterial liquid was taken out every 2 h, and the absorbance of the bacterial liquid at 600 nm was measured by an ultraviolet spectrophotometer.

2.4 Treatment of L. plantarum DNZ-4 with CIP stress

L. plantarumDNZ-4 grown to the end of the logarithm were collected by centrifugation (4 000 ×g, 10 min), washed twice with PBS, adjusted the concentration of bacteria to reached approximately 1 × 109CFU/mL and were subsequently resuspended in an equal volume of MRS liquid medium containing 1/2 MIC, 1/8 MIC and 1/40 MIC CIP, and no CIP addition used as the control group. The bacteria were collected by centrifugation after 12 h of treatment.

2.5 Scanning electron microscopy (SEM)

The bacterial solution treated with different concentrations of CIP for 12 h was centrifuged at 3 000 ×gfor 10 min at 4 °C, and the supernatant was discarded to collect the bacterial pellet. The collected bacterial pellet was placed in a refrigerator at 4 °C and fixed with 2.5% glutaraldehyde (pH 6.8) for 1.5 h. After washing three times with 0.1 mol/L PBS (pH 6.8), it was dehydrated with 50% , 70% , 90% ethanol for 10 min separately, following treated with 100% ethanol and tert-butanol (1:1,V/V) mixture and pure t-butanol for 15 min separately. The sample was frozen in a refrigerator at -20 °C for 30 min and then dried in an ES-2030 (HITACHI) type freeze dryer for 4 h. The processed samples were placed in the sample box for examination after the adhesion and coating. The changes of the morphology of the cells before and after the CIP stress were observed under the field emission scanning electron microscope [17].

2.6 Determination of key enzymes in L. plantarum DNZ-4 metabolism under CIP stress

The test samples prepared were used for the determination of pyruvate kinase, hexokinase, andβ-galactosidase, Na+, K+-ATPase activity, and the protein concentration was determined by Coomassie blue staining [18]. The enzyme activities of the four enzymes were determined according to the instructions of the corresponding kits supplied by Beijing Suolaibao Technology Co., Ltd.

2.7 Extraction and derivatization of small molecular metabolites

The bacteria were collected by centrifugation (3 000 ×g,10 min). Added appropriate amount of MilliQ water to the bottom cells after washing with PBS, centrifuge at 3 000 ×gfor 5 min,discarded the upper layer of MilliQ water, and retained the bottom cells after washing with PBS. The washed cells were placed in liquid nitrogen and quenched at –80 °C for 5 min; 50 mg of the cells was ground with liquid nitrogen and placed in a 1.5 mL centrifuge tube for the extraction of small molecule metabolites. 3 parallel samples were prepared for each sampling point. 1 mL of pre-cooled methanol/water extract (1:1,V/V) was added and mixed evenly for 1 min. The mixture were subjected to 5 freeze-thaw cycles using liquid nitrogen. 300 µL of the supernatant of the small molecule metabolite extract was placed in a 1.5 mL centrifuge tube, 10 µL of the internal standard mixture was added and then dried by a vacuum freeze dryer. 50 µL of methoxybammonium hydrochloride/pyridine solution(20 mg/mL) was added after lyophilization and placed it in a 40 °C water bath for 80 min; then 80 µL of MSTFA (N-methyl-N-(trimethylsilyl) tri fluoroacetamide) was added, and reacted in a 40 °C water bath for 80 min. The derivatized sample was centrifuged at 5 000 ×gfor 5 min, and 100 µL of the supernatant was taken into the sample bottle.

2.8 Detection of intracellular metabolites by GC-MS

2.8.1 Gas chromatographic conditions

Samples were separated using an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm × 0.25 μm) gas chromatography system. Temperature programmed: initial temperature 50 °C; held for 3 min, heated up to 220 °C at 10 °C/min for 3 min; finally heated up to 250 °C at 15 °C/min for 10 min. The carrier gas was helium and the flow rate was 1.0 mL/min.

2.8.2 Mass spectrometry

Mass spectrometry was performed using an Agilent 6890N/5975B gas chromatography-mass spectrometer. The injection inlet temperature was 250 °C, with a transfer line temperature of 260 °C and an ion source temperature of 230 °C. A full scan mode with a scanning range ofm/z50 to 600 was used.

2.9 Multivariate statistical analysis

The peak area and retention time were extracted using the MSD ChemStation software. A curve was drawn to calculate the content of medium and long chain fatty acids in the sample. The data of the medium and long chain fatty acids content in the sample was exported to SIMCA-P software 14.1 (Umetrics, Umea, Sweden) for multivariate statistical analysis. Principal component analysis (PCA)and partial least squares-discriminant analysis (PLS-DA) were applied to compare the different metabolites of the samples.

3. Results and discussion

3.1 Determination of growth curve of L. plantarum DNZ-4

In order to measure the antibiotic effects onL. plantarumDNZ-4, it is critical to use levels that are below the MIC of CIP,otherwise, excessive doses will lead to the death. As shown in Fig. 1,the minimum inhibitory concentration ofL. plantarumDNZ-4 to CIP was determined by double-tube dilution method which was 4 µg/mL. Therefore 1/2 MIC, 1/8 MIC and 1/40 MIC concentrations were chosen to deal withL. plantarumDNZ-4 in order to determine the effect of the CIP at a range of concentrations.

Fig. 1 Schematic diagram of minimum inhibitory concentration of L. plantarum DNZ-4 (µg/mL) determination.

It can be seen from Fig. 2 that the growth curve of 1/40 MIC group showed no difference with the control strain, while the other two concentrations groups resulted in significantly different growth patterns. The reason may be that CIP entered the bacterial cells, induced double-strand breaks by interfering with gyrase and topoisomerase, inhibited DNA re-ligation, induced DNA damage,and finally initiated cell death [19]. In addition, CIP can stimulate the oxidation of NADH through an electron transport chain that depended on the TCA cycle. Excessive activation of the electron transport chain stimulated the formation of superoxide, which can damage the iron-sulfur cluster. The lethal dose of hydroxyl radicals generated by the fenton reaction destroyed the bacterial DNA, proteins and lipids, eventually led to cell death [20].However, the mechanism for inhibitingL. plantarumDNZ-4 growth is unknown. Smith et al. [21]studied the bactericidal effect of CIP 0–2.0 µg/mL onStaphylococcus aureus, and found that the number of viable bacteria ofS. aureusdecreased with the increase of CIP concentration, which is similar to the conclusion that the degree of inhibition of the cells found in this experiment is positively correlated with the concentration of CIP.

Fig. 2 Growth curve of L. plantarum DNZ-4 under different concentrations of CIP.

3.2 SEM to observe the effect of CIP stress on the morphology of L. plantarum DNZ-4

It can be seen from Fig. 3 that the morphology of the cells is significantly different under different concentrations of CIP. The appearance ofL. plantarumDNZ-4 which had not been treated with CIP was a typical rod-shaped straight line, the ends were arranged in a circular arc shape, and the cells were full and free from defects.After treatment with 1/40 MIC CIP, the cells showed depression,dryness, and collapse, and the arrangement was disordered. The surface of a few cells showed rupture and the contents of the cells leaked. After treatment with 1/8 MIC CIP, the number of cells with cell surface rupture was increased. After treatment with 1/2 MIC CIP,the morphology ofL. plantarumDNZ-4 cells was the most damaged.The length of the bacterial cells was shortened to the greatest extent,and most of the bacterial cells were ruptured and the contents of the cells were leaked and led to cell death. Studies have shown that CIP will destroy the bacterial surface and change the morphology of the bacteria [18]. Weigelt et al. [11]found that after exposure ofE. colito CIP or norfloxacin with a concentration of 1–50 µg/mL, the membrane of the cell wall fibrillated within 30 min, and then the cell wall of the bacteria ruptured.

Fig. 3 SEM images of L. plantarum DNZ-4 under different CIP stress treatment. (a) No treatment with CIP; (b) treatment with 1/40 MIC CIP; (c) treatment with 1/8 MIC CIP; (d) treatment with 1 /2 MIC CIP.

3.3 Determination of key enzymes in L. plantarum DNZ-4 metabolism under CIP stress

Fluoroquinolones stress can exert adverse effects on microorganisms. Under quinolone stress conditions, microorganisms altered the metabolism and energy flux by altering the synthesis of enzymes and metabolites, as well as changing the growth ratio by adjusting the metabolic pathway under the new environment [22].It can be seen from Fig. 4 that the enzyme activity of hexokinase decreased significantly after treated with 1/2 MIC CIP (P＜ 0.01),which was (3.29 ± 0.63) U/mg prot. After treated with 1/40 MIC,1/8 MIC CIP, the enzyme activity inL. plantarumDNZ-4 metabolism was not significantly decreased (P＞ 0.05). Compared with the control group, the activities of pyruvate kinase,β-galactosidase and Na+,K+-ATPase ofL. plantarumDNZ-4 decreased significantly after 1/40 MIC,1/8 MIC, 1/2 MIC CIP stress. The degree of decline in enzyme activity was positively correlated with the concentrations of CIP.This result indicated that pyruvate kinase,β-galactosidase and Na+,K+-ATPase are key metabolic enzymes for the ofL. plantarumDNZ-4 under CIP stress. The activity of the key enzymes of cell glucose metabolism decreased after CIP treatment, inducing the bacteria to reduce the glucose utilization rate and inhibit the aerobic metabolism in the cells, thereby saved the glucose metabolism,energy flux and the carbon source metabolism to adapt the new environment, which was beneficial to the growth of the bacteria [23].The decrease ofβ-galactosidase in the bacteria might be the overall inhibitory effect of CIP on the expression of the enzyme. The cause might be thatL. plantarumDNZ-4 responded by slowing down metabolism under stress conditions [16]. Meanwhile, theβ-galactosidase activity decreased under the stress of CIP, indicating that the hydrolysis of polysaccharide, glycoprotein and galactolipid terminal galactose residues was inhibited during cell wall component metabolism and cell wall degradation of damaged cells [24]. Na+,K+-ATPase is a biofilm enzyme system widely distributed in living organisms. It maintains membrane potential on both sides of the cell membrane, regulates cell osmotic pressure, and provides power for nutrient absorption, and plays an extremely important role in maintaining normal physiological functions of cells. CIP attacked the cell membrane ofL. plantarumDNZ-4, altered membrane integrity and fluidity, which in turn affected enzyme activity [25]. Na+,K+-ATPase leaked from plasma membrane due to the damage of cell membrane, hexokinase, pyruvate kinase,β-galactosidase which were located in cytosol [26]. It can be hypothesized that impaired cell membrane integrity caused by CIP stress may also be responsible for the change of the above enzyme activity.

Fig. 4 Determination of key enzymes.

3.4 Metabolomics analysis

3.4.1 Detection and qualitative analysis of intracellular metabolites under CIP stress

LAB undergo significant metabolic fluctuations under environmental stress conditions. Due to environmental stress, cells reduce their metabolic activity, thereby reducing energy and proton motility, and altering cell growth and viability [27,28]. In general, the cells respond to environmental stress by selectively utilizing other carbon sources to replace pyruvate, activating free amino acids in the proteolytic system and/or catabolic cells. In order to investigate the effects of CIP stress on the physiological metabolism ofL. plantarumDNZ-4, the intracellular fatty acid, carbohydrate and amino acid metabolism ofL. plantarumDNZ-4 with and without CIP stress were extracted and compared.

A total of 29 fatty acids, 8 carbohydrates and 19 amino acid metabolites were detected by the GC-MS in the experiment.Multivariate statistical analysis of the test data was performed by unsupervised PCA and supervised PLS. The statistical data of the two models were established as shown in Table 1.1. hexanoic acid, 2. dodecanoic acid, 3. myristic acid, 4. myristoleic acid, 5. pentadecanoic acid, 6.cis-10-pentadecenoic acid, 7.palmitic acid, 8. palmitoleic acid, 9. heptadecanoic acid, 10.cis-10-heptadecenoic acid, 11. stearic acid, 12. oleic acid, 13. linoleic acid,14.γ-linolenic acid, 15.α-linolenic acid, 16. arachidic acid, 17.cis-11-eicosenoic acid, 18.cis-11,14-eicosadienoic acid, 19. heneicosanoic acid,20.cis-8,11,14-eicosatrienoic acid, 21. arachidonic acid, 22.cis-11,14,17-eicosatrienoic acid, 23.cis-5,8,11,14,17-eicosapentaenoic acid, 24. behenic acid, 25. erucic acid, 26. tricosanoic acid, 27. tetracosanoic acid, 28. nervonic acid, 29.cis-4,7,10,13,16,19-docosahexaenoic acid, 30. ribose, 31. xylitol,32. galactose, 33. fructose, 34. lactose, 35. myo-inositol, 36. glucose,37. trehalose, 38. glycine, 39. alanine, 40. leucine, 41. isoleucine, 42.valine, 43. proline, 44. threonine, 45. serine, 46. glutamine, 47. glutamate,48. methionine, 49. phenylalanine, 50. tryptophan, 51. asparagine, 52. tyrosine,53. aspartate, 54. lysine, 55. arginine, 56. histidine; same as Fig. 8.

Table 1Main statistical data established by PCA and PLS models.

The 56 variables were subjected to dimensionality reduction using an unsupervised PCA. The clustering of each experimental group is shown in Fig. 5. The PCA model showed that the 4 groups of samples were distributed in different regions, and the discrete grouping phenomenon was obvious. The 4 parallel samples in each group were closely aggregated, indicating that there were significant differences in intracellular metabolites ofL. plantarumDNZ-4 under different concentrations of CIP. In the first principal component,the 1/40 MIC group was not significantly different from the control group, but when the concentration of CIP was 1/8 MIC and 1/40 MIC,the sample was significantly different from the former two groups.Among the second principal components, the 1/40 MIC and 1/2 MIC group was significantly separated from the control group, while the 1/8 MIC group and the control group were not significantly dispersed. The PCA load map shows the distribution of the detected intracellular metabolites, and the distribution of the variables in the load map corresponds to the sample distribution and position in the score map. As can be seen from Fig. 6, the substances causing the sample difference are glutamine, serine, threonine, myristic acid,phenylalanine, glutamic acid, aspartic acid, alanine, sinapic acid.

Fig. 5 PCA scores of L. plantarum DNZ-4 under different concentrations of CIP.

Fig. 6 PCA load diagram of L. plantarum DNZ-4 under different concentrations of CIP.

Statistical analysis of intracellular metabolites ofL. plantarumDNZ-4 with/without CIP stress was performed using supervised PLS-DA, and PCA statistical results were supplemented. As can be seen from Fig. 7, the 1/8 MIC group and the 1/2 MIC group showed significant separation from the control group, indicating that there was a significant difference in intracellular metabolism between them in the first principal component. In the second principal component,the 1/40 MIC, 1/2 MIC groups were significantly separated from the control group and the 1/8 MIC group, similar to the PCA analysis.From the PLS score map, it can be shown that the main intracellular metabolites ofL. plantarumDNZ-4 do have differences under the different concentrations of CIP. Fig. 8 shows the PLS load map. The main differential metabolic contributions obtained by PCA were verified from the PLS load map. As can be seen from Fig. 8, the metabolites with a large contribution rate were glutamine, threonine,alanine, serine, glutamic acid, aspartic acid, sinapic acid, and asparagine. Except for asparagine, the other major metabolites were analyzed in the PCA load map analysis.

Fig. 7 PLS scores of L. plantarum DNZ-4 under different concentrations of CIP.

Fig. 8 PLS load diagram of L. plantarum DNZ-4 under different concentrations of CIP.

The variable interdependent parameters (VIP) analysis under the PLS model is shown in Fig. 9. The metabolites with a VIP value ＞ 1 were intercepted and sorted according to the contribution rate of the extracted biomarkers. The ranking results were: palmitic acid, stearic acid, nervonic acid, myristic acid, glutamine,cis-10-heptadecenoic acid,cis-10-pentadecenoic acid, oleic acid, threonine, alanine,cis-11-eicosenoic acid, trehalose, glutamic acid, aspartic acid, serine.

Fig. 9 VIP diagram of L. plantarum DNZ-4 under different concentrations of CIP.

3.4.2 Analysis of major differences in intracellular

Fluctuations in intracellular fatty acid content can affect functional properties such as fluidity of the cell membrane. Monitoring the changes of intracellular fatty acid content ofL. plantarumDNZ-4 under CIP stress is helpful to understand the functional status of the cells. As can be seen from Figs. 10a–c, compared with the control group, Under the stress of 1/2 MIC group, the content of palmitic acid,stearic acid, myristic acid and erucic acid decreased, and the content of sinapic acid was 0.5 times compared with that of the control group.The nervonic acid content was 2.43 times of that in the control group.The results showed that the content of unsaturated fatty acids other than erucic acid decreased in different degrees compared with the control group, while the content of saturated fatty acids decreased.Churkina et al. [29]studied the effects of antibiotics on the fatty acid composition ofS. aureusandE. coli, and found that sub-inhibitory doses of antibiotics could induce the appearance of unsaturated fatty acids inS. aureus. InE. coli, the content of unsaturated linear fatty acids had almost tripled. This is consistent with the experimental results. Smirnov et al. [30]also published a similar study. Under the action of antibiotics at sub-inhibitory concentrations,S. aureusformed unsaturated branched-chain fatty acids, and the content was higher than that of saturated fatty acids, which suggested that the presence of antibiotics was associated with increased bacterial lipid mobility. The above studies showed that under the action of antibiotics, bacteria can change the fluidity and permeability of the membrane by changing the content of saturated fatty acids and unsaturated fatty acids, thereby improving the function of the cells in the stress state.

Trehalose and inositol were carbohydrates with a higher contribution rate ofL. plantarumDNZ-4 under CIP stress, and the content changes are shown in the Fig. 10d. As the concentration of CIP increased, the content of intracellular trehalose and inositol increased. Compared with the control group, the trehalose content increased by 8.53% , 12.97% and 14.86% under the stress of 1/40 MIC,1/8 MIC and 1/2 MIC group, respectively, while the content of inositol increased slightly. Trehalose has a protective effect on microorganisms under environmental stress and helps maintain the normal osmotic pressure of cells. Because of its high water holding capacity, trehalose can stabilize the structure of macromolecular substances, such as biofilm, protein and nucleic acid in cells [31].Therefore, under the stress of CIP, the intracellular trehalose content ofL. plantarumDNZ-4 increased, which was beneficial to protect the stability of macromolecular structures such as intracellular proteins and nucleic acids. The increase of inositol content was a stress reaction ofL. plantarumDNZ-4 to CIP stress. Inositol can be converted into a special soluble phosphate in bacteria, which has a certain role in protecting cells from stress [32].

Fig. 10 Intracellular major differences in L. plantarum DNZ-4 under different concentrations of CIP. (a–c) changes in intracellular fatty acid content; (d) changes in intracellular carbohydrate content; (e, f) changes in intracellular amino acid content.

As can be seen from the Figs. 10e–f, except for the tendency of proline content increased, the other seven amino acid contents showed a trend of decrease. Under the treatment of 1/40 MIC, 1/8 MIC and 1/2 MIC, the content of glutamine was significantly reduced compared with the control group, which was reduced by 61.73% ,68.77% and 85.11% , respectively. In the stress of 1/40 MIC group,the content of glutamic acid did not change significantly, but it decreased significantly under 1/8 MIC and 1/2 MIC stress. The study found that under environmental stress, microorganisms responded to stress conditions by changing the amino acid content in their body [33].Banerjee et al. [34]found that the reduction of alanine, aspartic acid and glutamate metabolic pathways was the biggest factor affecting the change ofE. colimetabolic activity under CIP stress, which was similar to the results of Belenky et al. [35]. They found that the relative concentrations of alanine, aspartic acid and glutamic acid inE. coliwere generally decreased after treatment with nor floxacin. The experimental results was similar to the results of the above studies.Under the stress of CIP,L. plantarumDNZ-4 responded to the stress of CIP by changing the intracellular amino acid content.

As shown in Fig. 11, these changes were not randomly distributed; instead, they occurred coordinately in distinct localized regions of the network. Characterizing the metabolic changes of LAB under antibiotic stress, and understanding how these changes affect bacterial cell viability, is of great significance for the determination of the overall change of LAB metabolism in the intestinal tract after antibiotic stress. The synthetic precursors of the 8 amino acids detected herein are derived from glycolysis or TCA cycle. Studies have shown that the metabolism of amino acid precursors affects the change in its content [36]. Pyruvic acid and oxaloacetate are precursors of alanine and aspartic acid, respectively. CIP stress caused down-regulation of proteins involved in glycolysis/gluconeogenesis,thereby inhibiting glycolysis or gluconeogenesis [37], which may cause the pyruvic acid content to drop, further causing a decrease in alanine content. As the concentration of CIP increased, the aspartate content decreased, which can affect the change in threonine content since aspartic acid is a synthetic precursor of threonine. The common precursor for the synthesis of threonine and serine is glyceraldehyde triphosphate, and the decrease in threonine and serine content may be due to the lack of sufficient precursors during amino acid synthesis.Glutamate and proline are amino acids involved in the self-protective response metabolism of cells, and their synthetic precursors areα-ketoglutaric acid that is an important intermediate in the TCA cycle. Under the action of 1/8 MIC and 1/2 MIC group, the content of glutamic acid decreased significantly. It might be that glutamate was used in the synthesis of other substances and energy to alleviate the stress of CIP. The proline content increased under different concentrations of CIP stress, which may be its stress protection effect to CIP stress, or the conversion of glutamate to proline, which may reduce the content of glutamic acid and therefor the content of proline increase. Glutamine can be formed by the reaction of glutamic acid with ammonia. Glutamine is beneficial to protein metabolism and reduces the degree of bacterial stress response. After CIP stress, the glutamine content of the bacteria decreased, which may cause CIP to destroy protein metabolism inL.plantarumDNZ-4, causing the bacteria to initiate a stress response. The content of asparagine is regulated by aspartic acid. In addition, decreased levels of intracellular amino acids and other metabolites also affected the metabolism of alanine, aspartic acid, and glutamate. From the perspective of amino acid metabolites, it can be concluded thatL. plantarumDNZ-4 affected the amino acid metabolism pathway and changed the amino acid contentin vivo, which was a stress response of the bacteria to CIP stress.

Fig. 11 Schematic showing changes of metabolite abundance mapped onto the metabolic network. Red symbols denote significant increases (P ＜ 0.05)and green symbols denote significant decreases (P ＜ 0.05), whereas navy blue symbols denote no significant changes in metabolite levels (P ＞ 0.05). PEP,phosphoenolpyruvate; OAA, oxaloacetate; α-KG, α-ketoglutarate; FA, fatty acid; Asn, asparagine; Asp, aspartic acid; Glu, glutamic acid; Gln, Glutamine; Pro,proline; Thr, threonine; Ala, Alanine; Ser, serine.

4. Conclusion

The use of global antibiotics has led to the emergence of resistant strains of LAB, but little knows about the effects of LAB metabolism under antibiotic stress. In the present study, it was found that CIP treatment had a certain degree of influence on the cell metabolism ofL. plantarumDNZ-4. The hexokinase, pyruvate kinase,β-galactosidase and Na+, K+-ATPase in the main energy metabolism pathway of the cells all showed different degrees of decline under the stress of CIP. The content of fatty acids, carbohydrates and amino acids were also changed. Unsaturated fatty acids inL. plantarumDNZ-4 was increased, and saturated fatty acids was decreased, which was beneficial to the improvement of cell functional properties such as cell membrane fluidity. The increase of specific carbohydrates such as trehalose and inositol indicated that theseL. plantarumDNZ-4 cells can resist CIP stress. They can also responded to the stressful environment of CIP by affecting the amino acid metabolism pathway. In conclusion, the results of this study will deepen our understanding of the metabolic mechanisms of how LAB response to CIP treatment, and furthermore, it may also contribute to the development of new probiotic preparation technologies against adverse antibiotic stress.

Declaration of competing interest

All authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant No. 31671874), National Key Research and Development Project (2018YFD0502404), Natural Science Foundation of Heilongjiang Province of China (Grant No.C2018022)and Academic Backbone Plan of Northeast Agricultural University(Grant No. 18XG27) and Research Fund for Key Laboratory of Dairy Science, Ministry of Education, Heilongjiang Province, China(2015KLDSOF-07), and the Project of Young Innovative Talents of Colleges and Universities (UNPYSCT-2016149).