Effects of Immunization With the Soil-Derived Bacterium Mycobacterium vaccae on Stress Coping Behaviors and Cognitive Performance in a “Two Hit” Stressor Model

Abstract

Previous studies demonstrate that <i>Mycobacterium vaccae</i> NCTC 11659 (<i>M. vaccae</i>), a soil-derived bacterium with anti-inflammatory and immunoregulatory properties, is a potentially useful countermeasure against negative outcomes to stressors. Here we used male C57BL/6NCrl mice to determine if repeated immunization with <i>M. vaccae</i> is an effective countermeasure in a "two hit" stress exposure model of chronic disruption of rhythms (CDR) followed by acute social defeat (SD). On day -28, mice received implants of biotelemetric recording devices to monitor 24-h rhythms of locomotor activity. Mice were subsequently treated with a heat-killed preparation of <i>M. vaccae</i> (0.1 mg, administered subcutaneously on days -21, -14, -7, and 27) or borate-buffered saline vehicle. Mice were then exposed to 8 consecutive weeks of either stable normal 12:12 h light:dark (LD) conditions or CDR, consisting of 12-h reversals of the LD cycle every 7 days (days 0-56). Finally, mice were exposed to either a 10-min SD or a home cage control condition on day 54. All mice were exposed to object location memory testing 24 h following SD. The gut microbiome and metabolome were assessed in fecal samples collected on days -1, 48, and 62 using 16S rRNA gene sequence and LC-MS/MS spectral data, respectively; the plasma metabolome was additionally measured on day 64. Among mice exposed to normal LD conditions, immunization with <i>M. vaccae</i> induced a shift toward a more proactive behavioral coping response to SD as measured by increases in scouting and avoiding an approaching male CD-1 aggressor, and decreases in submissive upright defensive postures. In the object location memory test, exposure to SD increased cognitive function in CDR mice previously immunized with <i>M. vaccae</i>. Immunization with <i>M. vaccae</i> stabilized the gut microbiome, attenuating CDR-induced reductions in alpha diversity and decreasing within-group measures of beta diversity. Immunization with <i>M. vaccae</i> also increased the relative abundance of 1-heptadecanoyl-sn-glycero-3-phosphocholine, a lysophospholipid, in plasma. Together, these data support the hypothesis that immunization with <i>M. vaccae</i> stabilizes the gut microbiome, induces a shift toward a more proactive response to stress exposure, and promotes stress resilience.