Ketamine used in the therapy of depressive disorders impacts protein profile, proliferation rate, and phagocytosis resistance of enterococci

The 27 studied enterococcal strains were isolated from various clinical samples as etiological agents of infection and collected in the Department of Medical Microbiology, Medical University of Gdansk (Table 1). The isolates were identified to species level by strep ID test (BioMerieux, Poland) and classified as different strains of Enterococcus faecalis by biochemical and antibiotic resistance profiles.

The growth rate was studied in Brain Heart Infusion Broth (BHI, Oxoid, England). Medium was inoculated with bacterial strains and cultured at 37°C in aerobic conditions with shaking (200 rpm). After 24 h, the fresh BHI was inoculated with 10 μL of bacterial suspension. Each isolate was then incubated at 37°C for 3 hours in two variants: with or without ketamine. The concentration of ketamine used in the experiment was 200 ng/mL – the maximal plasma concentration obtained from a patient undergoing anti-depression therapy 40 minutes after infusion [22].

The samples were then centrifuged and pellets were washed three times with saline. To measure metabolic activity of E. faecalis, 10 μM of CFDA-SE (carboxyfluorescein diacetate succinimidyl ester, Sigma-Aldrich, USA) were added to 200 μL of bacterial suspension of each strain. Mixtures were then incubated for 45 min at 37 °C and centrifuged. The pellets were collected and washed three times with PBS (phosphate-buffered saline, Sigma-Aldrich, USA). The number of bacterial cells and their fluorescence were determined using FACSVerse flow cytometer (Becton Dickinson, San Jose, CA, USA). Effect was measured as the ratio of number of the cells and green fluorescence intensity in culture with ketamine versus culture without ketamine.

For the assessment of phagocytosis, THP-1 human monocytes cell line (TIB-202™, ATCC, American Type Culture Collection) was used. The cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS) (all from Sigma-Aldrich, USA). For each phagocytosis assay, the enterococcal culture stained with CFDA-SE was used.

Each phagocytic mixture contained 0.4 mL of stained enterococci (8 × 10⁷) and 0.4 mL of monocytes (2.5 × 10⁷). The mixture was incubated for 45 min at 37 °C. Suspension of monocytes was then stained with propidium iodide (PI, Sigma Aldrich, USA) at room temperature to determine cells’ membrane permeability and to evaluate cytotoxicity of enterococci. Emitted fluorescence was determined using a FACSVerse flow cytometer. Differences in phagocytosis effectiveness between strains treated with ketamine and without ketamine were measured as the changes in green fluorescence (for CFDA-SE) and the cytotoxicity of bacterial cells (treated with ketamine vs. not treated with ketamine) was measured as the changes in red fluorescence (for PI). The red fluorescence emitted by PI-stained monocytes not exposed to bacterial suspension was considered a baseline value for monocytes’ viability.

To analyze protein profiles, BHI medium was inoculated with bacterial strains and cultured at 37°C in aerobic conditions with shaking (200 rpm). After 24 h, the fresh BHI was inoculated with 10 μL of bacterial suspension. Each isolate was then incubated at 37°C for 18 hours in two variants: with or without ketamine. The samples were centrifuged, and pellets were washed three times with saline. The bacterial pellet was applied to a metal plate and dried at room temperature. Then, 1 μL of an α-cyano-4-hydroxycinnamic acid matrix solution (HCCA, Bruker Daltonics) was applied and the sample was left to dry at room temperature. Measurement of the spectrum and comparative analysis with reference spectra of bacteria were performed using MALDI-Biotyper 3.0 (Bruker Daltonics, USA). Mass spectra were collected using FlexControl software (Bruker Daltonics, USA). Then, the spectra were baseline analyzed using mMass Software, version 5.5.0. [23].

The ratio of the number of bacteria and the intensity of fluorescence, expressed as mean fluorescence per particle (green fluorescence for CFDA-SE/FSC; red fluorescence for PI/FSC), were subjected to the analysis of variance (ANOVA) with Statistica™10 software (StatSoft, Poland). The normality of distribution was verified by χ² test, and the data were normalized by Box–Cox transformation.

Although the ketamine does not affect the formation of SCVs, Enterococcus faecalis strains show two opposite reactions when it comes to the response to ketamine. Among tested strains, there were those responding by inhibiting their metabolism as well as the strains which in the presence of ketamine boosted their metabolism considerably. In 42.3% of strains the inhibition of their metabolism, measured as CFDA-SE hydrolysis, was observed. Within this group, in 23.1% of strains also proliferation rate was lower than in culture without ketamine and susceptibility to phagocytosis mediated by THP-1 cells increased up to 1.51 times while the median cytotoxicity was usually lower.

In contrast to our expectations, the majority of strains (57.7%) increased both proliferation and cellular metabolism at tested concentration of ketamine. The increase of proliferation varied from 1.23 up to 4.5 times (median 2.45) while increase of metabolism varied from 1.24 up to 5.90 times (median 2.75) when compared to bacteria not exposed to ketamine. Correlation between induction of proliferation and metabolism rate measured by CFDA-SE hydrolysis has not been proved, but based on abovementioned criteria, we might define two groups of strains: boosted and inhibited by ketamine (Figure 1).

Fig. 1

The diverse response of strains to ketamine was also visible in protein profiles analyzed by MALDI-TOF. In detailed analysis, in the spectral profiles of studied enterococci, 15 peaks were identified with m/z value of 2211, 2633, 3420, 3670, 4440, 4780, 5360 6080, 6230, 6400, 6840, 7330, 7570, 8910 and 9540. Eight of those peaks were described previously by Stępień-Pyśniak D as characteristic for Enterococcus faecalis, while others were unique for the studied collection [24].

In both groups of strains (inhibiting and intensifying the proliferation rate) and in the abovementioned study of Stępień-Pyśniak et al. [24], relative intensity of the peak was the highest at m/z 4440 and this value remained unchanged after incubation with ketamine.

In the strains responding to the exposure to ketamine by increasing the proliferation, most of the proteins increased in amount, decreased peak intensity was noticed at m/z 5360 and 6400. In the group of strains with inhibited proliferation, the intensity of peaks was lower after incubation with ketamine, with the exception of m/z value at 4439, 5360 and 9540 (Table 2).

Not only proliferation rate and protein profiles were different in both groups. Susceptibility to phagocytosis was lower within groups “boosted” by ketamine strains. The ANOVA analysis proved decreased cytotoxicity and increased phagocytosis rates in the group of strains inhibited by ketamine. The changes of cytotoxicity and phagocytosis were not as evident in cases of strains with increased proliferation (Figure 2).

Fig. 2