Among the taxa that were increased in the saliva of cannabis smokers, five belonged to the Streptococcusgenus, and four belonged to the Actinomyces genus. Among the taxa that were enriched in the saliva of non-smoking controls, six belonged to the Neisseria genus . No differences were observed based on quantity of cannabis use or presence of neurological disease history between the heavy users and light users , and between smoked cannabis containing THC and cannabis containing no THC .Nonetheless, the enrichment of A. meyeri was inversely correlated with the age of first cannabis use .To study which taxa may represent the cannabis smoking oralmicrobiome, we reanalyzed and compared the saliva microbiome from tobacco smokers and non-smoker controls from our published data . Consistent with the results from previously published studies, we found increased Streptococcus and decreased Neisseriain the oral microbiome of tobacco smokers compared with those in non-smoking controls , which was similar to cannabis smokers. However, Actinomyces genus was only increased in cannabissmokers but not in tobacco smokers . To further analyze Actinomyces genus bacteria, mobile grow system we have shown four Actinomyces species bacteria that were significantly increased in cannabis smokers .
Only Actinomyces turicensis was increased in tobacco smokers when compared with non-smoker controls . We further analyzed the difference after adjusting for age, sex, and/or alcohol use. All differences between cannabis users and controls identified in the univariate analysis shown in Fig. S1 remained significant after adjusting for sex, age, and alcohol consumption, although P values were attenuated slightly . When comparing tobacco users to controls, only Streptococcus andA. turicensis , but not Neisseria, remained significant after adjusting for sex and age.The smoking and altered microbiome composition may lead to a compromised mucosal epithelial barrier, which results in the translocation of bacteria or microbial products into circulation. Thus, bacterial fragments or whole bacteria can appear in the blood from translocation and thereby influence the immune system . To study oral microbial translocation in cannabis users, we evaluated the plasma levels of IgG antibody against antigens derived from A. meyeri, A. odontolyticus,and N. elongata. Plasma levels of IgGs against A. meyeri antigenstended to increase in the cannabis smokers compared to controls, while similar levels of IgGs against antigens from the other two bacteria were observed .
These results imply that A. meyeri or its antigens may preferentially translocate from the oral mucosa to the circulation in the setting of an altered oral or periodontal environment in cannabis smokers. In a previous study, oral administration of Campylobacter jejuni activated state of neurons in nucleus tractus solitarius and increased c-Fos expression in the hypothalamic paraventricular nucleusas inmice . To determine if cannabis use-associated oral microbiome affects CNS, we inoculated live A. meyeri, A. odontolyticus, and N. elongata into the oral cavity of C57BL/6 mice. A. meyeri and A. odontolyticus are oral commensal bacteria and were enriched in theoral microbiome of cannabis smokers found in this study. N. elongata,which was enriched in non-smoking controls , mobile vertical rack was used as a control. We examined mouse activity through a uniformly cylindricalarena. The behaviors of mice were quantified and shown by global activity, total distance traveled , average speed , and resting time.
The behavior of N. elongata-treated mice in the arenawas comparable with the PBS-treated mice. However, compared with N. elongata-treated mice, A. meyeri-treated mice exhibited decreases in global activity, distance traveled, and mean speed, as well as increases in resting time .Next, we have evaluated amyloid production in mouse brain tissues as it is a marker of neurodegenerative diseases. Although Ab 40tended to be increased in A. meyeri-treated mice, there was no statistical difference between any two groups . Notably, the Ab 42peptide in the brain from A. meyeri-treated mice was increased significantly compared to the control groups . To validate alterations in oral and gut microbiome, we collected the samples from oralswab and stool one week after the final oral administration of live bacteria. Specific bacteria were quantified using qPCR, and the abundance of each bacterium was normalized by total 16S rDNA. We confirm edoral inoculated bacteria by qPCR . Notably, both A.odontolyticus and N. elongata, but not A. meyeri, presented in the orals wab of some mice from the PBS group, suggesting A. odontolyticus and N. elongata may be oral commensal microbiome in mice .