Ketamine intervention limits pathogen expansion in vitro

Ketamine intervention limits pathogen expansion in vitro Abstract Ketamine is one of several clinically important drugs whose therapeutic efficacy is due in part to their ability to act upon ion channels prevalent in nearly all biological systems. In studying eukaryotic and prokaryotic organisms in vitro, we show that ketamine short-circuits the growth and spatial expansion of three microorganisms, Stachybotrys chartarum, Staphylococcus epidermidis and Borrelia burgdorferi, at doses efficient at reducing depression-like behaviors in mouse models of clinical depression. Although our findings do not reveal the mechanism(s) by which ketamine mediates its antifungal and antibacterial effects, we hypothesize that a function of L-glutamate signal transduction is associated with the ability of ketamine to limit pathogen expansion. In general, our findings illustrate the functional similarities between fungal, bacterial and human ion channels, and suggest that ketamine or its metabolites not only act in neurons, as previously thought, but also in microbial communities colonizing human body surfaces. antibacterial effects, antidepressant drug, antifungal effects, dissociative anesthetic, glutamate receptors INTRODUCTION Ketamine is a dissociative anesthetic commonly used in pediatric surgery with a relatively wide therapeutic range (Craven 2007). Ketamine also has rapid antidepressant actions in patients with treatment-resistant depression (Berman et al.2000; Liebrenz et al.2007; Aaan het Rot et al.2012; Scheuing et al.2015) and in patients with suicidal ideation (Ballard et al.2014). Animal and human studies suggest that ketamine acts by blocking N-methyl-D-aspartate (NMDA) receptor subunit proteins, which are activated by the excitatory neurotransmitter L-glutamate (Krystal, Sanacora and Duman 2013; Musazzi et al.2013). L-glutamate signaling pathways are found in almost all synapses where they play key roles in both the strengthening and elimination of neural-circuit output (Chen, Tracy and Nam 2007; Noh et al.2010). As such, NMDA receptors are critically involved in brain development and function, including long-term potentiation for sequence learning and prediction (Tajima et al.2016). The actions of ketamine also extend to neural circuits of the zebrafish (Danio rerio) where it acts on L-glutamate signaling pathways to elicit aberrant behaviors that are qualitatively similar to those seen in humans and rodents treated with phencyclidine, another potent NMDA receptor antagonist drug (Riehl et al.2011; Zakhary et al.2011; De Campos, Bruni and De Martinis 2015; Pittman and Hylton 2015). This suggests that L-glutamate and its ionotropic membrane-bound receptors are ancient and widespread molecules with many vertebrate species displaying their excitatory signaling code. A deep homology in basic L-glutamate signaling structure and underlying functional system is also seen in higher plant tissues, especially in the germinating seeds of the radish plant (Raphanus sativus), where homologous full-length cDNA clones encoding a putative L-glutamate receptor are localized to plasma membranes (Kang et al.2006). This would imply that constituent L-glutamate and NMDA gene clusters have expanded throughout eukaryote evolution from an ancestral, equivalent DNA cluster network, long before the origin and speciation of the vertebrate brain. Against this background, we show here that ketamine has antifungal properties, an effect that was serendipitously found while cleaning and disinfecting our cell and tissue culture room of Stachybotrys chartarum, a fungus associated with idiopathic pulmonary hemorrhage (Barnes et al.2002). As this initial finding was derived from a semi-naturalistic setting, we further tested the ability of ketamine to prevent the growth of a potentially invasive bacterium (e.g. Staphylococcus epidermidis), under controlled, experimental conditions. Again, we show here that ketamine can also act as an antibacterial-like agent by inhibiting, at least in our current experiments, microbes that elicit symptoms of inflammation and infection in high-risk patients. Notably, we also find that ketamine prevents the in vitro growth of the tick-borne spirochete Borrelia burgdorferi, a bacterium that causes an expanding erythematous rash, persistent fever, fatigue, headache, myalgias and arthralgias unless treated with antibiotics (Bratton et al.2008; Borchers et al.2015). In general, our comparative experiments illustrate additional dimensions of ketamine's actions on underlying signaling networks of the amino acid L-glutamate. MATERIALS AND METHODS In vitro growth of radish seeds and ketamine treatment Radish seeds were obtained from American Seeds (Plantation Products, MA) and placed in triplicates in closed 24-well cell culture clusters (Corning Incorporated, Corning, NY). The cell culture clusters were placed in an incubator contaminated with Stachybotrys chartarum at 37°C for either 24 h, 48 h or 7 days (radish seeds usually germinate between 5 and 10 days) under a standard 12 h light:dark condition. Seeds were placed onto moist filtered papers, and the cell culture clusters were kept moist for the duration of the experiments or until leaves appeared. Under this experimental protocol, ∼90% of all seeds germinated. Ketamine hydrochloride (100 mg/ml; AmTech Group Inc. distributed by Phoenix Scientific, MO) was applied at different drug concentrations (ranging from 6 μl to 50 μl) per well cell culture cluster. The number of seedlings was adjusted to 3 plants per well. After 24 h of ketamine exposure, seeds were washed (3X) in distilled water (dH2O) and allowed to sow for at least 5–7 days in a sterile incubator at 37°C. To remove moisture slowly while at the same time maintaining as much of the original shape and texture as possible, germinating radish material exposed to either ketamine or dH20 was dried in an oven at 100°C for 12 h. After this incubation phase, radish plants were allowed to cool (in sealed plastic bags to prevent moisture) at room temperature for 20 min. Then, dried plant material was weighed and the number of leaves counted with the aid of bright-field light microscopy. All experiments were performed during the lights on period. Quantification of mycotoxin molds infecting radish seeds was recorded by an investigator unaware of the experimental conditions with the aid of bright-field light microscopy. In vitro growth of bacterial cultures and ketamine treatment Bacterial cultures and conditions for measuring the effects of ketamine on bacterial growth kinetics were established as previously described by us (Pavia, Pierre and Nowakowski 2000) with slight modifications. In brief, an ATCC-derived isolate of Staphylococcus epidermidis was purchased from Chrisope Technologies (Lake Charles, LA) as a culti-loop. It was initially cultured in BBL trypticase soy broth (Becton Dickinson and Co., Cockeysville, MD), then sub-cultured onto tryptic soy agar and incubated aerobically at 37°C. Cultures were either used immediately in subsequent experiments, or were stored at 4°C or at –70°C until further use. For the St. epidermidis experiments, a known quantity (0.5 μl) of bacteria that had been diluted (4.95 ml) in phosphate-buffered saline (PBS) to a desire concentration was added to sterile small snap-cap tubes containing known concentrations of ketamine (ranging from 6 to 50 μl). Controls contained St. epidermidis mixed with the PBS diluent alone. These mixtures were incubated for 24 h at 37°C. At the end of the incubation period, bacterial numbers were estimated using a colony counter (Redco Science Inc.) and a No. 2, 4 Digits Tally Register (Compass, Taiwan) under blinded outcome conditions (i.e. without knowledge of control or experimental conditions) or based on spectrophotometric measurements (see below). For the static and dynamic (i.e. viability) ketamine–St. epidermidis experiments, we followed a previously published procedure from our group (Kim et al.2004) with slight modifications. In brief, for the static experiments, ketamine (50 μl) or PBS (50 μl) was incubated at 37°C for 4 h on standard 2 ml LB agar plates (n = 3/group) to allow the incorporation and diffusion of the above solutions into specific sites of the LB agar plates. After 4 h of incubation, the LB agar plates were removed and allowed to dry for 2 h at 37°C. A 50 μl aliquot of St. epidermidis was spread directly onto the LB agar plate and then incubated overnight at 37°C. Bacterial growth was visualized directly on the LB agar plate and photographs were then taken as representative of the biomass results. For the dynamic (i.e. viability) experiments, bacteria solutions were standardized (∼ 1.0 × 108 cells/ml) to an absorbance value between 0.1 and 0.2 at 625 nm. To each aliquot, ketamine (50 μl) or PBS (50 μl) was added. All solutions were then incubated at 37°C with constant shaking (200 rpm) for 24 h and the absorbance at 625 nm read for each aliquot (n = 3/group). For the ketamine–Borrelia burgdorferi experiments, mixed cultures of late log-phase B. burgdorferi (strain 297) and ketamine hydrochloride were established in sterile 1 cc screw-cap tubes (Nunc vials, Thermo Fisher Scientific, Suzhou, Jiangsu, P. R. China). Each tube contained 30 μl of B. burgdorferi suspension (final concentration, 5 × 107B. burgdorferi/ml) and 290 μl of Barbour Stoenner Kelly (BSK) medium. Ketamine or vehicle (PBS) was added to designated tubes in 10 μl volumes (n = 3 per condition). This yielded a final concentration of 1:10. From these suspensions, 2-fold dilutions were made using BSK medium until an endpoint dilution was reached in which there was no inhibition of bacterial growth. Therefore, each tube contained approximately 1.5 × 106B. burgdorferi in a final volume of 330 μl. Cultures were kept airtight by screwing the caps tightly followed by incubation at 35°C. After incubating the cultures for 24 and 48 h, the number of B. burgdorferi in each separate tube was counted microscopically as previously described (Pavia, Wormser and Norman 1997). The percentage inhibition of growth was calculated as follows: [1 – (number of motile B. burgdorferi + ketamine/number of motile B. burgdorferi + saline] × 100. Statistical analysis Data are reported as means ± SEM. One-way ANOVA followed by Mann–Whitney rank sum test and Student's t-tests were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Statistically significant differences were defined as P ≤ 0.05. RESULTS Ketamine intervention prevents the growth of fungi Indoor building materials, including air-conditioners, are often heavily colonized by fungi and bacteria with the mold Stachybotrys chartarum being a prominent resident of ongoing dampness or water damage. Our cell and tissue culture room, unfortunately, was colonized by the aforementioned fungus, which was seen on seeds of the radish plant (Raphanus sativus; Fig. 1A and C). Interestingly, radish seeds treated with 24 μl ketamine for 24 h showed little or no signs of colonization as observed by bright-field light microscopy (Fig. 1B and D). This finding was further demonstrated by quantifying the number of mycotoxin molds on seeds colonized by S. chartarum. The majority of untreated seeds showed a significant number of mycotoxin molds (≥200) on their surface shell as compared with seeds treated with ketamine (≤25; unpaired Student's t test, P ≤ 0.05). Remarkably, nearly 80% of all seeds treated with ketamine were completely devoid of mycotoxin molds. Figure 1. View largeDownload slide Ketamine intervention limits fungal growth and expansion in radish seeds. Ketamine at a concentration of 24 μl dissolved in 12 ml dH20 was applied to radish seeds (n = 3 seeds/well) for 24 h. Untreated (A) and treated (B) radish seeds were then visualized by bright- eld light microscopy and the number of myocotoxin molds (arrows) counted for each well. Ketamine exposure (D) also limits S. chartarum growth in germinating radish plants relative to untreated germinating plants (C; arrow). Similar results were obtained with ketamine concentrations of 12 μl dissolved in 12 ml dH20 and 6 μl dissolved in 12 ml dH20 (data not shown). Scale bar = 0.5 cm for A and B; 1 cm for C and D. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 1. View largeDownload slide Ketamine intervention limits fungal growth and expansion in radish seeds. Ketamine at a concentration of 24 μl dissolved in 12 ml dH20 was applied to radish seeds (n = 3 seeds/well) for 24 h. Untreated (A) and treated (B) radish seeds were then visualized by bright- eld light microscopy and the number of myocotoxin molds (arrows) counted for each well. Ketamine exposure (D) also limits S. chartarum growth in germinating radish plants relative to untreated germinating plants (C; arrow). Similar results were obtained with ketamine concentrations of 12 μl dissolved in 12 ml dH20 and 6 μl dissolved in 12 ml dH20 (data not shown). Scale bar = 0.5 cm for A and B; 1 cm for C and D. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Radish seeds were evaluated for the development of morphological untoward signs potentially associated with ketamine treatment as this drug is thought to cause neuronal death and weakening of neural circuits in children and infant mice undergoing surgery (Wang et al.2014; Disma et al.2016). Treatment of ketamine beginning on the day radish seeds (n = 27) were placed to sow did not prevent normal plant development as germination was not significantly different from seeds (n = 27) not treated with the aforementioned NMDA receptor antagonist drug (data not shown). More specifically, the shapes and sizes of plant organs such as leaves and stems as well as the coloring of the leaves did not vary as a function of ketamine treatment when compared to untreated radish seeds. In addition, dry weight of 1-week germinating radish plants did not differ markedly between untreated (mean ± SEM = 0.0784 g ± 0.02) vs. ketamine-treated groups (mean ± SEM = 0.1477 g ± 0.22; Mann–Whitney rank sum test, P ≥ 0.05), thus suggesting that ketamine treatment preserves normal seed development without inducing apparent cell toxicity. Although these data do not identify a set of mechanistic events that are specifically associated with the putative antifungal properties of ketamine, they nevertheless provide fundamental insight into additional dimensions of psychotropic drugs that act at evolutionarily conserved gated ion channels. Ketamine intervention prevents the growth of bacteria Bacteria possess several classes of gated ion channels, such as calcium-gated potassium channels and ionotropic glutamate receptor channels that are structurally similar to those found in vertebrate neurons (Prindle et al.2015). Therefore, we wondered whether the actions of ketamine also extended to Staphylococcus epidermidis, a gram-positive bacterium that is part of the normal human flora, primarily on the skin surface. In this context, this particular bacterium is commonly found in catheter-associated infections and in blood cultures from neutropenic patients (Adam, Baillie and Douglas 2002). Treatment with 50 μl ketamine for 24 h resulted in a significant diminution in the number of bacterial colonies growing on standard LB agar plates (Fig. 2A and B). Indeed, both visual inspection and St. epidermidis quantification confirmed this observation. That is, our experimental protocol generated a population of untreated colonies that grew exponentially (mean ± SEM = ≥4666.7 ± 210.8), and a ketamine-treated population that was limited in colony-formation, maintenance and spatial expansion (mean ± SEM = 32 ± 26.04; Mann–Whitney rank sum test, P ≤ 0.004). Figure 2. View largeDownload slide Ketamine intervention limits bacterial growth and clone expansion in vitro. Photomicrographs depict untreated bacterial colonies (A) and ketamine-treated bacterial colonies (B). Note the significant bacterial inhibition produced by 50 μl ketamine following 24 h of drug exposure. Scale bar for both photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 2. View largeDownload slide Ketamine intervention limits bacterial growth and clone expansion in vitro. Photomicrographs depict untreated bacterial colonies (A) and ketamine-treated bacterial colonies (B). Note the significant bacterial inhibition produced by 50 μl ketamine following 24 h of drug exposure. Scale bar for both photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Static experiments carried out along with dynamic (i.e. viability) experiments further indicated that ketamine arrested microbial growth after 24 h of drug treatment. To assess the ability of different concentrations of ketamine to inhibit colony-formation and clonal expansion, we treated the aforementioned bacterium with decreasing concentrations of ketamine. As expected, untreated bacterial colonies expanded exponentially with some colonies exceeding ≥5000 clones. In sharp contrast, bacterial colonies treated with 50 μl, 25 μl but not 12.5 μl ketamine resulted in a profound colony growth inhibition (≤4 clones; Fig. 3A–D). Additional confirmatory experiments further recapitulated the above findings, with the 50 μl ketamine concentration associated with the greatest inhibition of colony-formation (Fig. 4A and B). Collectively, these data suggest that ketamine has broad-spectrum activity against gram-positive pathogens such as St. epidermidis and a eukaryotic fungus (e.g. S. chartarum) implicated in building-related illness. Figure 3. View largeDownload slide Application of ketamine at various doses limits bacterial growth and clone expansion in vitro. Staphylococcus epidermidis cultures were grown on standard LB agar plates and quantification was performed on the resultant cellular biomass. Representative images of bacterial colonies: (A) control conditions (i.e. PBS-treated bacteria; ≥5000 colonies). (B) Staphylococcus epidermidis treated with 12.5 μl ketamine (≥2000 colonies). (C) Staphylococcus epidermidis treated with 25 μl ketamine (≤800 colonies). (D) Staphylococcus epidermidis treated with 50 μl ketamine (≤30 colonies). Note the significant inhibition and limited expansion of clones (in C and D) after incubation with ketamine for 24 h. Scale bar for all photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 3. View largeDownload slide Application of ketamine at various doses limits bacterial growth and clone expansion in vitro. Staphylococcus epidermidis cultures were grown on standard LB agar plates and quantification was performed on the resultant cellular biomass. Representative images of bacterial colonies: (A) control conditions (i.e. PBS-treated bacteria; ≥5000 colonies). (B) Staphylococcus epidermidis treated with 12.5 μl ketamine (≥2000 colonies). (C) Staphylococcus epidermidis treated with 25 μl ketamine (≤800 colonies). (D) Staphylococcus epidermidis treated with 50 μl ketamine (≤30 colonies). Note the significant inhibition and limited expansion of clones (in C and D) after incubation with ketamine for 24 h. Scale bar for all photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 4. View largeDownload slide Static (A) and dynamic (i.e. viability; B) experiments further demonstrate the ability of ketamine in limiting the uncontrolled expansion of St. epidermidis in vitro. A representative photomicrograph depicts bacterial colonies with and without ketamine during 24 h of exponential growth on standard LB agar plates. Note that application of 50 μl ketamine (K) to a given site of the LB agar plate successfully repels St. epidermidis invasion and colonization (A; red dotted lines). Spectrometry was used to quantify St. epidermidis depletion as a function of ketamine or PBS intervention (B). Data are means ± SEM. One-way ANOVA was used for statistical analyses, F (2, 6 = 203.2, **P ≤ 0.0001). NS = not significant. Control = absolute control: dH20 condition. These results suggest a potential clinical application for ketamine in microbiology as well as new mechanisms for the therapeutic effects of NMDA receptor antagonist drugs. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 4. View largeDownload slide Static (A) and dynamic (i.e. viability; B) experiments further demonstrate the ability of ketamine in limiting the uncontrolled expansion of St. epidermidis in vitro. A representative photomicrograph depicts bacterial colonies with and without ketamine during 24 h of exponential growth on standard LB agar plates. Note that application of 50 μl ketamine (K) to a given site of the LB agar plate successfully repels St. epidermidis invasion and colonization (A; red dotted lines). Spectrometry was used to quantify St. epidermidis depletion as a function of ketamine or PBS intervention (B). Data are means ± SEM. One-way ANOVA was used for statistical analyses, F (2, 6 = 203.2, **P ≤ 0.0001). NS = not significant. Control = absolute control: dH20 condition. These results suggest a potential clinical application for ketamine in microbiology as well as new mechanisms for the therapeutic effects of NMDA receptor antagonist drugs. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Lyme disease is a common tick-borne disease associated with specific clinical features that are typically treated with doxycycline or amoxicillin (Bratton et al.2008). Motivated by our previous findings, we tested the ability of ketamine to limit the growth of Borrelia burgdorferi in vitro. When tested at final concentrations ranging from 1.25% to 10%, ketamine was 100% inhibitory at both 24 and 48 h after establishing cultures using BSK medium. No inhibition of growth occurred at a final dilution (1:320) of ketamine (Table 1). In this context, non-viable B. burgdorferi appeared as thin, non-motile and shortened or contracted structures having blebs relative to the more elongated, highly motile and less tightly coiled B. burgdorferi that were not exposed to ketamine (Fig. 5A and B). Based on these findings and because relatively high levels (74%–88%) of growth inhibition still occurred at a dilution of 1:160, we next wanted to determine at what concentration of ketamine it produced bacterial growth inhibition at or near the 50% mark. In additional experiments, titration of ketamine was adjusted more tightly in 24–48 h cultures in which dilutions of ketamine ranged from 1:60 to 1:240. It was found (data not shown) that, at a dilution of 1:240, inhibition of B. burgdorferi growth ranged from 41% to 57%, which we considered to be the 50% inhibitory dose (ID50). Figure 5. View largeDownload slide Representative photomicrograph of B. burgdorferi cultured with BSK medium and saline diluent, and subsequently visualized using phase-contrast microscopy (A). Representative photomicrograph of B. burgdorferi cultured with BSK medium and ketamine, and subsequently visualized using phase-contrast microscopy (B). The mammalian molecules assumed to mediate the activity of B. burgdorferi include, decorin, fribronectin, glycosaminoglycans and β3-chain integrins (Coburn, Medrano and Cugini 2002). It remains unclear whether native L-glutamate signaling pathways support the function of tick-borne spirochete species. Magnification for (A): ×200. Magnification for (B): ×400. Scale bar = 20 μm. Figure 5. View largeDownload slide Representative photomicrograph of B. burgdorferi cultured with BSK medium and saline diluent, and subsequently visualized using phase-contrast microscopy (A). Representative photomicrograph of B. burgdorferi cultured with BSK medium and ketamine, and subsequently visualized using phase-contrast microscopy (B). The mammalian molecules assumed to mediate the activity of B. burgdorferi include, decorin, fribronectin, glycosaminoglycans and β3-chain integrins (Coburn, Medrano and Cugini 2002). It remains unclear whether native L-glutamate signaling pathways support the function of tick-borne spirochete species. Magnification for (A): ×200. Magnification for (B): ×400. Scale bar = 20 μm. Table 1. Ketamine treatment inhibits the in vitro growth of B. burgdorferi.   % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%    % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%  aResults are reported as mean % inhibition of 3 replicate experiments after culturing strain 297 of B. burgdorferi in BSK medium at designated time intervals. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. View Large Table 1. Ketamine treatment inhibits the in vitro growth of B. burgdorferi.   % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%    % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%  aResults are reported as mean % inhibition of 3 replicate experiments after culturing strain 297 of B. burgdorferi in BSK medium at designated time intervals. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. View Large DISCUSSION Ketamine is both water and lipid soluble allowing convenient dosing by different routes. In humans with major depressive episodes, ketamine is administered intravenously at sub-anesthetic doses (Aaan het Rot et al.2012). In animal models of psychopathology, ketamine at a single intraperitoneal dose (∼10 mg/kg) sustains antidepressant-like phenomena through L-glutamate signaling pathways of the mammalian target of rapamycin [mTOR; (Li et al.2010)]. mTOR is a ubiquitous protein kinase network involved in protein synthesis, including synthesis of certain proteins associated with the formation of dendritic spine structure (Jaworski and Sheng 2006; Bowling and Klann 2014). However, inconsistencies still remain in the literature to fully accept and extrapolate this post-synaptic mechanism to the neurobiology of patients treated with ketamine (Aaan het Rot et al.2012). Regardless of the underlying mechanisms for the antidepressant efficacy of ketamine in neurons, we show here that ketamine, at sub-anesthetic doses (50 μg/ml), also functions effectively as an antifungal and antibacterial agent against three invasive microbes. It is unclear, however, whether ketamine acts through mTOR or other L-glutamate-based signaling pathway to arrest Stachybotrys chartarum, Staphylococcus epidermidis or Borrelia burgdorferi growth and overall pathogen expansion. What is known with some certainty is that L-glutamate, GABA (γ-aminobutyric acid) and other amino acid molecules regulate quorum sensing, enhance virulence in bacteria (Waters and Bassler 2005; Dagorn et al.2013), and govern biofilm formation in Bacillus subtilis bacteria (Prindle et al.2015). Although previous studies have established that antifungal agents such as azoles act by eroding ergosterol and β-glucan synthesis from cell membranes (Denning and Bromley 2015), it is not known whether ketamine signaling events might accomplish similar outcomes. Consistent with previous reports (Gocmen, Buyukkocak and Caglayan 2008; Begec et al.2013), our work suggests that ketamine can disrupt the community of bacteria that typically colonize the human skin. In general, key antibiotics inhibit DNA synthesis, RNA synthesis, cell wall synthesis and/or protein synthesis (Kohanski, Dwyer and Collins 2010). Thus, bactericidal and bacteriostatic drugs appear to use different signaling cues to induce cell death or inhibit cell growth, respectively. Again, it is unclear how ketamine or other drugs associated with L-glutamate signal transduction influence bacterial community structure. It will be interesting to determine whether L-glutamate-based mechanisms are used by prokaryotes as a way to regulate metabolism and growth. Along the same lines, it would be of interest to assess whether some of the antidepressant actions of ketamine might be microbially mediated, as a growing body of preclinical literature suggests that the composition and function of the gut microbiome shapes behavior relevant to stress-related disorders (Mayer et al.2014). Indeed, we are learning that psychotropic drugs affect the microbiome in ways that were not clearly anticipated. For example, atypical antipsychotic drugs such as risperidone and olanzapine can alter the composition of microorganisms that live in the gut, and conversely, the bioavailability of drugs used for treating major mood disorders can be modified by the microbiome itself (Davey et al.2013; Dinan, Stanton and Cryan 2013; Mayer et al.2014; Bahr et al.2015). Our findings also show that ketamine is a potent inhibitor of B. burgdorferi growth. To our knowledge, this is the first report showing borreliacidal activity of this species, or any other spirochetal bacterium, by a non-competitive NMDA receptor antagonist drug. Based on our data, the approximate ID50 occurred in cultures having a dilution of 1:240. The physiologic relevance of these findings, in terms of possibly affecting the clinical outcome of certain types of infections, has yet to be determined and should serve as the focal point on future, follow-up studies. In general, there is substantial precedence and experimental support for our current findings and collectively point to the possibility that ketamine's actions might extend to the microbiota to indirectly affect psychological dimensions of anxiety or depression. Although our studies were motivated by the serendipitous findings of ketamine's ability to arrest fungal activity in radish seeds, we also investigated the impact of ketamine on bacterial growth, maintenance and clonal expansion. To this end, we observed that ketamine intervention, at least in vitro, limits the uncontrolled expansion of S. chartarum, St. epidermidis and B. burgdorferi. The resulting depletion of these pathogens is driven through mechanisms that are not yet fully resolved. 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Abstract

Abstract Ketamine is one of several clinically important drugs whose therapeutic efficacy is due in part to their ability to act upon ion channels prevalent in nearly all biological systems. In studying eukaryotic and prokaryotic organisms in vitro, we show that ketamine short-circuits the growth and spatial expansion of three microorganisms, Stachybotrys chartarum, Staphylococcus epidermidis and Borrelia burgdorferi, at doses efficient at reducing depression-like behaviors in mouse models of clinical depression. Although our findings do not reveal the mechanism(s) by which ketamine mediates its antifungal and antibacterial effects, we hypothesize that a function of L-glutamate signal transduction is associated with the ability of ketamine to limit pathogen expansion. In general, our findings illustrate the functional similarities between fungal, bacterial and human ion channels, and suggest that ketamine or its metabolites not only act in neurons, as previously thought, but also in microbial communities colonizing human body surfaces. antibacterial effects, antidepressant drug, antifungal effects, dissociative anesthetic, glutamate receptors INTRODUCTION Ketamine is a dissociative anesthetic commonly used in pediatric surgery with a relatively wide therapeutic range (Craven 2007). Ketamine also has rapid antidepressant actions in patients with treatment-resistant depression (Berman et al.2000; Liebrenz et al.2007; Aaan het Rot et al.2012; Scheuing et al.2015) and in patients with suicidal ideation (Ballard et al.2014). Animal and human studies suggest that ketamine acts by blocking N-methyl-D-aspartate (NMDA) receptor subunit proteins, which are activated by the excitatory neurotransmitter L-glutamate (Krystal, Sanacora and Duman 2013; Musazzi et al.2013). L-glutamate signaling pathways are found in almost all synapses where they play key roles in both the strengthening and elimination of neural-circuit output (Chen, Tracy and Nam 2007; Noh et al.2010). As such, NMDA receptors are critically involved in brain development and function, including long-term potentiation for sequence learning and prediction (Tajima et al.2016). The actions of ketamine also extend to neural circuits of the zebrafish (Danio rerio) where it acts on L-glutamate signaling pathways to elicit aberrant behaviors that are qualitatively similar to those seen in humans and rodents treated with phencyclidine, another potent NMDA receptor antagonist drug (Riehl et al.2011; Zakhary et al.2011; De Campos, Bruni and De Martinis 2015; Pittman and Hylton 2015). This suggests that L-glutamate and its ionotropic membrane-bound receptors are ancient and widespread molecules with many vertebrate species displaying their excitatory signaling code. A deep homology in basic L-glutamate signaling structure and underlying functional system is also seen in higher plant tissues, especially in the germinating seeds of the radish plant (Raphanus sativus), where homologous full-length cDNA clones encoding a putative L-glutamate receptor are localized to plasma membranes (Kang et al.2006). This would imply that constituent L-glutamate and NMDA gene clusters have expanded throughout eukaryote evolution from an ancestral, equivalent DNA cluster network, long before the origin and speciation of the vertebrate brain. Against this background, we show here that ketamine has antifungal properties, an effect that was serendipitously found while cleaning and disinfecting our cell and tissue culture room of Stachybotrys chartarum, a fungus associated with idiopathic pulmonary hemorrhage (Barnes et al.2002). As this initial finding was derived from a semi-naturalistic setting, we further tested the ability of ketamine to prevent the growth of a potentially invasive bacterium (e.g. Staphylococcus epidermidis), under controlled, experimental conditions. Again, we show here that ketamine can also act as an antibacterial-like agent by inhibiting, at least in our current experiments, microbes that elicit symptoms of inflammation and infection in high-risk patients. Notably, we also find that ketamine prevents the in vitro growth of the tick-borne spirochete Borrelia burgdorferi, a bacterium that causes an expanding erythematous rash, persistent fever, fatigue, headache, myalgias and arthralgias unless treated with antibiotics (Bratton et al.2008; Borchers et al.2015). In general, our comparative experiments illustrate additional dimensions of ketamine's actions on underlying signaling networks of the amino acid L-glutamate. MATERIALS AND METHODS In vitro growth of radish seeds and ketamine treatment Radish seeds were obtained from American Seeds (Plantation Products, MA) and placed in triplicates in closed 24-well cell culture clusters (Corning Incorporated, Corning, NY). The cell culture clusters were placed in an incubator contaminated with Stachybotrys chartarum at 37°C for either 24 h, 48 h or 7 days (radish seeds usually germinate between 5 and 10 days) under a standard 12 h light:dark condition. Seeds were placed onto moist filtered papers, and the cell culture clusters were kept moist for the duration of the experiments or until leaves appeared. Under this experimental protocol, ∼90% of all seeds germinated. Ketamine hydrochloride (100 mg/ml; AmTech Group Inc. distributed by Phoenix Scientific, MO) was applied at different drug concentrations (ranging from 6 μl to 50 μl) per well cell culture cluster. The number of seedlings was adjusted to 3 plants per well. After 24 h of ketamine exposure, seeds were washed (3X) in distilled water (dH2O) and allowed to sow for at least 5–7 days in a sterile incubator at 37°C. To remove moisture slowly while at the same time maintaining as much of the original shape and texture as possible, germinating radish material exposed to either ketamine or dH20 was dried in an oven at 100°C for 12 h. After this incubation phase, radish plants were allowed to cool (in sealed plastic bags to prevent moisture) at room temperature for 20 min. Then, dried plant material was weighed and the number of leaves counted with the aid of bright-field light microscopy. All experiments were performed during the lights on period. Quantification of mycotoxin molds infecting radish seeds was recorded by an investigator unaware of the experimental conditions with the aid of bright-field light microscopy. In vitro growth of bacterial cultures and ketamine treatment Bacterial cultures and conditions for measuring the effects of ketamine on bacterial growth kinetics were established as previously described by us (Pavia, Pierre and Nowakowski 2000) with slight modifications. In brief, an ATCC-derived isolate of Staphylococcus epidermidis was purchased from Chrisope Technologies (Lake Charles, LA) as a culti-loop. It was initially cultured in BBL trypticase soy broth (Becton Dickinson and Co., Cockeysville, MD), then sub-cultured onto tryptic soy agar and incubated aerobically at 37°C. Cultures were either used immediately in subsequent experiments, or were stored at 4°C or at –70°C until further use. For the St. epidermidis experiments, a known quantity (0.5 μl) of bacteria that had been diluted (4.95 ml) in phosphate-buffered saline (PBS) to a desire concentration was added to sterile small snap-cap tubes containing known concentrations of ketamine (ranging from 6 to 50 μl). Controls contained St. epidermidis mixed with the PBS diluent alone. These mixtures were incubated for 24 h at 37°C. At the end of the incubation period, bacterial numbers were estimated using a colony counter (Redco Science Inc.) and a No. 2, 4 Digits Tally Register (Compass, Taiwan) under blinded outcome conditions (i.e. without knowledge of control or experimental conditions) or based on spectrophotometric measurements (see below). For the static and dynamic (i.e. viability) ketamine–St. epidermidis experiments, we followed a previously published procedure from our group (Kim et al.2004) with slight modifications. In brief, for the static experiments, ketamine (50 μl) or PBS (50 μl) was incubated at 37°C for 4 h on standard 2 ml LB agar plates (n = 3/group) to allow the incorporation and diffusion of the above solutions into specific sites of the LB agar plates. After 4 h of incubation, the LB agar plates were removed and allowed to dry for 2 h at 37°C. A 50 μl aliquot of St. epidermidis was spread directly onto the LB agar plate and then incubated overnight at 37°C. Bacterial growth was visualized directly on the LB agar plate and photographs were then taken as representative of the biomass results. For the dynamic (i.e. viability) experiments, bacteria solutions were standardized (∼ 1.0 × 108 cells/ml) to an absorbance value between 0.1 and 0.2 at 625 nm. To each aliquot, ketamine (50 μl) or PBS (50 μl) was added. All solutions were then incubated at 37°C with constant shaking (200 rpm) for 24 h and the absorbance at 625 nm read for each aliquot (n = 3/group). For the ketamine–Borrelia burgdorferi experiments, mixed cultures of late log-phase B. burgdorferi (strain 297) and ketamine hydrochloride were established in sterile 1 cc screw-cap tubes (Nunc vials, Thermo Fisher Scientific, Suzhou, Jiangsu, P. R. China). Each tube contained 30 μl of B. burgdorferi suspension (final concentration, 5 × 107B. burgdorferi/ml) and 290 μl of Barbour Stoenner Kelly (BSK) medium. Ketamine or vehicle (PBS) was added to designated tubes in 10 μl volumes (n = 3 per condition). This yielded a final concentration of 1:10. From these suspensions, 2-fold dilutions were made using BSK medium until an endpoint dilution was reached in which there was no inhibition of bacterial growth. Therefore, each tube contained approximately 1.5 × 106B. burgdorferi in a final volume of 330 μl. Cultures were kept airtight by screwing the caps tightly followed by incubation at 35°C. After incubating the cultures for 24 and 48 h, the number of B. burgdorferi in each separate tube was counted microscopically as previously described (Pavia, Wormser and Norman 1997). The percentage inhibition of growth was calculated as follows: [1 – (number of motile B. burgdorferi + ketamine/number of motile B. burgdorferi + saline] × 100. Statistical analysis Data are reported as means ± SEM. One-way ANOVA followed by Mann–Whitney rank sum test and Student's t-tests were performed using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Statistically significant differences were defined as P ≤ 0.05. RESULTS Ketamine intervention prevents the growth of fungi Indoor building materials, including air-conditioners, are often heavily colonized by fungi and bacteria with the mold Stachybotrys chartarum being a prominent resident of ongoing dampness or water damage. Our cell and tissue culture room, unfortunately, was colonized by the aforementioned fungus, which was seen on seeds of the radish plant (Raphanus sativus; Fig. 1A and C). Interestingly, radish seeds treated with 24 μl ketamine for 24 h showed little or no signs of colonization as observed by bright-field light microscopy (Fig. 1B and D). This finding was further demonstrated by quantifying the number of mycotoxin molds on seeds colonized by S. chartarum. The majority of untreated seeds showed a significant number of mycotoxin molds (≥200) on their surface shell as compared with seeds treated with ketamine (≤25; unpaired Student's t test, P ≤ 0.05). Remarkably, nearly 80% of all seeds treated with ketamine were completely devoid of mycotoxin molds. Figure 1. View largeDownload slide Ketamine intervention limits fungal growth and expansion in radish seeds. Ketamine at a concentration of 24 μl dissolved in 12 ml dH20 was applied to radish seeds (n = 3 seeds/well) for 24 h. Untreated (A) and treated (B) radish seeds were then visualized by bright- eld light microscopy and the number of myocotoxin molds (arrows) counted for each well. Ketamine exposure (D) also limits S. chartarum growth in germinating radish plants relative to untreated germinating plants (C; arrow). Similar results were obtained with ketamine concentrations of 12 μl dissolved in 12 ml dH20 and 6 μl dissolved in 12 ml dH20 (data not shown). Scale bar = 0.5 cm for A and B; 1 cm for C and D. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 1. View largeDownload slide Ketamine intervention limits fungal growth and expansion in radish seeds. Ketamine at a concentration of 24 μl dissolved in 12 ml dH20 was applied to radish seeds (n = 3 seeds/well) for 24 h. Untreated (A) and treated (B) radish seeds were then visualized by bright- eld light microscopy and the number of myocotoxin molds (arrows) counted for each well. Ketamine exposure (D) also limits S. chartarum growth in germinating radish plants relative to untreated germinating plants (C; arrow). Similar results were obtained with ketamine concentrations of 12 μl dissolved in 12 ml dH20 and 6 μl dissolved in 12 ml dH20 (data not shown). Scale bar = 0.5 cm for A and B; 1 cm for C and D. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Radish seeds were evaluated for the development of morphological untoward signs potentially associated with ketamine treatment as this drug is thought to cause neuronal death and weakening of neural circuits in children and infant mice undergoing surgery (Wang et al.2014; Disma et al.2016). Treatment of ketamine beginning on the day radish seeds (n = 27) were placed to sow did not prevent normal plant development as germination was not significantly different from seeds (n = 27) not treated with the aforementioned NMDA receptor antagonist drug (data not shown). More specifically, the shapes and sizes of plant organs such as leaves and stems as well as the coloring of the leaves did not vary as a function of ketamine treatment when compared to untreated radish seeds. In addition, dry weight of 1-week germinating radish plants did not differ markedly between untreated (mean ± SEM = 0.0784 g ± 0.02) vs. ketamine-treated groups (mean ± SEM = 0.1477 g ± 0.22; Mann–Whitney rank sum test, P ≥ 0.05), thus suggesting that ketamine treatment preserves normal seed development without inducing apparent cell toxicity. Although these data do not identify a set of mechanistic events that are specifically associated with the putative antifungal properties of ketamine, they nevertheless provide fundamental insight into additional dimensions of psychotropic drugs that act at evolutionarily conserved gated ion channels. Ketamine intervention prevents the growth of bacteria Bacteria possess several classes of gated ion channels, such as calcium-gated potassium channels and ionotropic glutamate receptor channels that are structurally similar to those found in vertebrate neurons (Prindle et al.2015). Therefore, we wondered whether the actions of ketamine also extended to Staphylococcus epidermidis, a gram-positive bacterium that is part of the normal human flora, primarily on the skin surface. In this context, this particular bacterium is commonly found in catheter-associated infections and in blood cultures from neutropenic patients (Adam, Baillie and Douglas 2002). Treatment with 50 μl ketamine for 24 h resulted in a significant diminution in the number of bacterial colonies growing on standard LB agar plates (Fig. 2A and B). Indeed, both visual inspection and St. epidermidis quantification confirmed this observation. That is, our experimental protocol generated a population of untreated colonies that grew exponentially (mean ± SEM = ≥4666.7 ± 210.8), and a ketamine-treated population that was limited in colony-formation, maintenance and spatial expansion (mean ± SEM = 32 ± 26.04; Mann–Whitney rank sum test, P ≤ 0.004). Figure 2. View largeDownload slide Ketamine intervention limits bacterial growth and clone expansion in vitro. Photomicrographs depict untreated bacterial colonies (A) and ketamine-treated bacterial colonies (B). Note the significant bacterial inhibition produced by 50 μl ketamine following 24 h of drug exposure. Scale bar for both photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 2. View largeDownload slide Ketamine intervention limits bacterial growth and clone expansion in vitro. Photomicrographs depict untreated bacterial colonies (A) and ketamine-treated bacterial colonies (B). Note the significant bacterial inhibition produced by 50 μl ketamine following 24 h of drug exposure. Scale bar for both photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Static experiments carried out along with dynamic (i.e. viability) experiments further indicated that ketamine arrested microbial growth after 24 h of drug treatment. To assess the ability of different concentrations of ketamine to inhibit colony-formation and clonal expansion, we treated the aforementioned bacterium with decreasing concentrations of ketamine. As expected, untreated bacterial colonies expanded exponentially with some colonies exceeding ≥5000 clones. In sharp contrast, bacterial colonies treated with 50 μl, 25 μl but not 12.5 μl ketamine resulted in a profound colony growth inhibition (≤4 clones; Fig. 3A–D). Additional confirmatory experiments further recapitulated the above findings, with the 50 μl ketamine concentration associated with the greatest inhibition of colony-formation (Fig. 4A and B). Collectively, these data suggest that ketamine has broad-spectrum activity against gram-positive pathogens such as St. epidermidis and a eukaryotic fungus (e.g. S. chartarum) implicated in building-related illness. Figure 3. View largeDownload slide Application of ketamine at various doses limits bacterial growth and clone expansion in vitro. Staphylococcus epidermidis cultures were grown on standard LB agar plates and quantification was performed on the resultant cellular biomass. Representative images of bacterial colonies: (A) control conditions (i.e. PBS-treated bacteria; ≥5000 colonies). (B) Staphylococcus epidermidis treated with 12.5 μl ketamine (≥2000 colonies). (C) Staphylococcus epidermidis treated with 25 μl ketamine (≤800 colonies). (D) Staphylococcus epidermidis treated with 50 μl ketamine (≤30 colonies). Note the significant inhibition and limited expansion of clones (in C and D) after incubation with ketamine for 24 h. Scale bar for all photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 3. View largeDownload slide Application of ketamine at various doses limits bacterial growth and clone expansion in vitro. Staphylococcus epidermidis cultures were grown on standard LB agar plates and quantification was performed on the resultant cellular biomass. Representative images of bacterial colonies: (A) control conditions (i.e. PBS-treated bacteria; ≥5000 colonies). (B) Staphylococcus epidermidis treated with 12.5 μl ketamine (≥2000 colonies). (C) Staphylococcus epidermidis treated with 25 μl ketamine (≤800 colonies). (D) Staphylococcus epidermidis treated with 50 μl ketamine (≤30 colonies). Note the significant inhibition and limited expansion of clones (in C and D) after incubation with ketamine for 24 h. Scale bar for all photomicrographs = 100 μm. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 4. View largeDownload slide Static (A) and dynamic (i.e. viability; B) experiments further demonstrate the ability of ketamine in limiting the uncontrolled expansion of St. epidermidis in vitro. A representative photomicrograph depicts bacterial colonies with and without ketamine during 24 h of exponential growth on standard LB agar plates. Note that application of 50 μl ketamine (K) to a given site of the LB agar plate successfully repels St. epidermidis invasion and colonization (A; red dotted lines). Spectrometry was used to quantify St. epidermidis depletion as a function of ketamine or PBS intervention (B). Data are means ± SEM. One-way ANOVA was used for statistical analyses, F (2, 6 = 203.2, **P ≤ 0.0001). NS = not significant. Control = absolute control: dH20 condition. These results suggest a potential clinical application for ketamine in microbiology as well as new mechanisms for the therapeutic effects of NMDA receptor antagonist drugs. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Figure 4. View largeDownload slide Static (A) and dynamic (i.e. viability; B) experiments further demonstrate the ability of ketamine in limiting the uncontrolled expansion of St. epidermidis in vitro. A representative photomicrograph depicts bacterial colonies with and without ketamine during 24 h of exponential growth on standard LB agar plates. Note that application of 50 μl ketamine (K) to a given site of the LB agar plate successfully repels St. epidermidis invasion and colonization (A; red dotted lines). Spectrometry was used to quantify St. epidermidis depletion as a function of ketamine or PBS intervention (B). Data are means ± SEM. One-way ANOVA was used for statistical analyses, F (2, 6 = 203.2, **P ≤ 0.0001). NS = not significant. Control = absolute control: dH20 condition. These results suggest a potential clinical application for ketamine in microbiology as well as new mechanisms for the therapeutic effects of NMDA receptor antagonist drugs. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. Lyme disease is a common tick-borne disease associated with specific clinical features that are typically treated with doxycycline or amoxicillin (Bratton et al.2008). Motivated by our previous findings, we tested the ability of ketamine to limit the growth of Borrelia burgdorferi in vitro. When tested at final concentrations ranging from 1.25% to 10%, ketamine was 100% inhibitory at both 24 and 48 h after establishing cultures using BSK medium. No inhibition of growth occurred at a final dilution (1:320) of ketamine (Table 1). In this context, non-viable B. burgdorferi appeared as thin, non-motile and shortened or contracted structures having blebs relative to the more elongated, highly motile and less tightly coiled B. burgdorferi that were not exposed to ketamine (Fig. 5A and B). Based on these findings and because relatively high levels (74%–88%) of growth inhibition still occurred at a dilution of 1:160, we next wanted to determine at what concentration of ketamine it produced bacterial growth inhibition at or near the 50% mark. In additional experiments, titration of ketamine was adjusted more tightly in 24–48 h cultures in which dilutions of ketamine ranged from 1:60 to 1:240. It was found (data not shown) that, at a dilution of 1:240, inhibition of B. burgdorferi growth ranged from 41% to 57%, which we considered to be the 50% inhibitory dose (ID50). Figure 5. View largeDownload slide Representative photomicrograph of B. burgdorferi cultured with BSK medium and saline diluent, and subsequently visualized using phase-contrast microscopy (A). Representative photomicrograph of B. burgdorferi cultured with BSK medium and ketamine, and subsequently visualized using phase-contrast microscopy (B). The mammalian molecules assumed to mediate the activity of B. burgdorferi include, decorin, fribronectin, glycosaminoglycans and β3-chain integrins (Coburn, Medrano and Cugini 2002). It remains unclear whether native L-glutamate signaling pathways support the function of tick-borne spirochete species. Magnification for (A): ×200. Magnification for (B): ×400. Scale bar = 20 μm. Figure 5. View largeDownload slide Representative photomicrograph of B. burgdorferi cultured with BSK medium and saline diluent, and subsequently visualized using phase-contrast microscopy (A). Representative photomicrograph of B. burgdorferi cultured with BSK medium and ketamine, and subsequently visualized using phase-contrast microscopy (B). The mammalian molecules assumed to mediate the activity of B. burgdorferi include, decorin, fribronectin, glycosaminoglycans and β3-chain integrins (Coburn, Medrano and Cugini 2002). It remains unclear whether native L-glutamate signaling pathways support the function of tick-borne spirochete species. Magnification for (A): ×200. Magnification for (B): ×400. Scale bar = 20 μm. Table 1. Ketamine treatment inhibits the in vitro growth of B. burgdorferi.   % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%    % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%  aResults are reported as mean % inhibition of 3 replicate experiments after culturing strain 297 of B. burgdorferi in BSK medium at designated time intervals. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. View Large Table 1. Ketamine treatment inhibits the in vitro growth of B. burgdorferi.   % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%    % Inhibitiona  Dilution of ketamine  24 h of culture  48 h of culture  1:10  100%  100%  1:20  100%  100%  1:40  100%  100%  1:80  100%  100%  1:160  86%–88%  74%–82%  1:320  0%  0%  aResults are reported as mean % inhibition of 3 replicate experiments after culturing strain 297 of B. burgdorferi in BSK medium at designated time intervals. Ketamine hydrochloride, USP: equivalent to 100 mg/ml ketamine. View Large DISCUSSION Ketamine is both water and lipid soluble allowing convenient dosing by different routes. In humans with major depressive episodes, ketamine is administered intravenously at sub-anesthetic doses (Aaan het Rot et al.2012). In animal models of psychopathology, ketamine at a single intraperitoneal dose (∼10 mg/kg) sustains antidepressant-like phenomena through L-glutamate signaling pathways of the mammalian target of rapamycin [mTOR; (Li et al.2010)]. mTOR is a ubiquitous protein kinase network involved in protein synthesis, including synthesis of certain proteins associated with the formation of dendritic spine structure (Jaworski and Sheng 2006; Bowling and Klann 2014). However, inconsistencies still remain in the literature to fully accept and extrapolate this post-synaptic mechanism to the neurobiology of patients treated with ketamine (Aaan het Rot et al.2012). Regardless of the underlying mechanisms for the antidepressant efficacy of ketamine in neurons, we show here that ketamine, at sub-anesthetic doses (50 μg/ml), also functions effectively as an antifungal and antibacterial agent against three invasive microbes. It is unclear, however, whether ketamine acts through mTOR or other L-glutamate-based signaling pathway to arrest Stachybotrys chartarum, Staphylococcus epidermidis or Borrelia burgdorferi growth and overall pathogen expansion. What is known with some certainty is that L-glutamate, GABA (γ-aminobutyric acid) and other amino acid molecules regulate quorum sensing, enhance virulence in bacteria (Waters and Bassler 2005; Dagorn et al.2013), and govern biofilm formation in Bacillus subtilis bacteria (Prindle et al.2015). Although previous studies have established that antifungal agents such as azoles act by eroding ergosterol and β-glucan synthesis from cell membranes (Denning and Bromley 2015), it is not known whether ketamine signaling events might accomplish similar outcomes. Consistent with previous reports (Gocmen, Buyukkocak and Caglayan 2008; Begec et al.2013), our work suggests that ketamine can disrupt the community of bacteria that typically colonize the human skin. In general, key antibiotics inhibit DNA synthesis, RNA synthesis, cell wall synthesis and/or protein synthesis (Kohanski, Dwyer and Collins 2010). Thus, bactericidal and bacteriostatic drugs appear to use different signaling cues to induce cell death or inhibit cell growth, respectively. Again, it is unclear how ketamine or other drugs associated with L-glutamate signal transduction influence bacterial community structure. It will be interesting to determine whether L-glutamate-based mechanisms are used by prokaryotes as a way to regulate metabolism and growth. Along the same lines, it would be of interest to assess whether some of the antidepressant actions of ketamine might be microbially mediated, as a growing body of preclinical literature suggests that the composition and function of the gut microbiome shapes behavior relevant to stress-related disorders (Mayer et al.2014). Indeed, we are learning that psychotropic drugs affect the microbiome in ways that were not clearly anticipated. For example, atypical antipsychotic drugs such as risperidone and olanzapine can alter the composition of microorganisms that live in the gut, and conversely, the bioavailability of drugs used for treating major mood disorders can be modified by the microbiome itself (Davey et al.2013; Dinan, Stanton and Cryan 2013; Mayer et al.2014; Bahr et al.2015). Our findings also show that ketamine is a potent inhibitor of B. burgdorferi growth. To our knowledge, this is the first report showing borreliacidal activity of this species, or any other spirochetal bacterium, by a non-competitive NMDA receptor antagonist drug. Based on our data, the approximate ID50 occurred in cultures having a dilution of 1:240. The physiologic relevance of these findings, in terms of possibly affecting the clinical outcome of certain types of infections, has yet to be determined and should serve as the focal point on future, follow-up studies. In general, there is substantial precedence and experimental support for our current findings and collectively point to the possibility that ketamine's actions might extend to the microbiota to indirectly affect psychological dimensions of anxiety or depression. Although our studies were motivated by the serendipitous findings of ketamine's ability to arrest fungal activity in radish seeds, we also investigated the impact of ketamine on bacterial growth, maintenance and clonal expansion. To this end, we observed that ketamine intervention, at least in vitro, limits the uncontrolled expansion of S. chartarum, St. epidermidis and B. burgdorferi. The resulting depletion of these pathogens is driven through mechanisms that are not yet fully resolved. 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Pathogens and DiseaseOxford University Press

Published: Mar 1, 2018

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