Ketamine intervention limits pathogen expansion in vitro total information

Pat J. Fraley
Ketamine intervention limits pathogen expansion in vitro total information

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.

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; Aan 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).

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.

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. Buy ketamine liquid 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.

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