Extracellular vesicle (EV) research in Gram-positive bacteria, mycobacteria and fungi was neglected until recently, owing to the presumption that vesicles could not traverse the thick cell walls found in these organisms. EVs are now understood to be produced by all types of microorganism, including those with thick cell walls, and are biologically active. EVs from bacteria, mycobacteria and fungi contain virulence factors, such as toxins, that are involved in pathogenesis and elicit strong host immune responses. For example, Cryptococcus neoformans EVs carry the capsular polysaccharide glucuronoxylomannan, which is an important virulence factor. Interaction of EVs with the host is specific to the microorganism from which the EVs were produced and is based on the lipid content and cargo of the EVs. Research into EVs produced by microorganisms with thick cell walls is a very young field. By learning how these microorganisms use EVs, we hope that researchers will gain insight into pathogenesis, therapeutics and vaccines.

An emerging body of literature demonstrates differences in the gut microbiome (GMB) of patients with major depressive disorder (MDD) compared to healthy controls (HC), as well as the potential benefits of prebiotic, probiotic, and synbiotic treatment. We conducted a systematic review of 24 observational studies (n = 2817), and 19 interventional trials (n = 1119). We assessed alpha diversity, beta diversity, and taxa abundance changes in patients with MDD relative to HC, as well as the effect of prebiotics, probiotics, and synbiotics on depressive symptoms in individuals with clinical or subclinical depression. We observed no significant differences in alpha diversity but a significant difference in beta diversity between patients with MDD and HC. There were fluctuations in the abundance of specific taxa in patients with MDD relative to HC. Probiotic and synbiotic, but not prebiotic, treatment showed a modest benefit in reducing depressive symptoms in patients with MDD over four to nine weeks. The GMB profiles of patients with MDD differ significantly from HC, but further studies are needed to elucidate the benefits of prebiotic, probiotic and synbiotic treatments relative to antidepressants and over longer follow-up before these therapies are implemented into clinical practice.

Summary: Previously, extracellular vesicle production in Gram‐positive bacteria was dismissed due to the absence of an outer membrane, where Gram‐negative vesicles originate, and the difficulty in envisioning how such a process could occur through the cell wall. However, recent work has shown that Gram‐positive bacteria produce extracellular vesicles and that the vesicles are biologically active. In this study, we show that Bacillus subtilis produces extracellular vesicles similar in size and morphology to other bacteria, characterized vesicles using a variety of techniques, provide evidence that these vesicles are actively produced by cells, show differences in vesicle production between strains, and identified a mechanism for such differences based on vesicle disruption. We found that in wild strains of B. subtilis, surfactin disrupted vesicles while in laboratory strains harbouring a mutation in the gene sfp, vesicles accumulated in the culture supernatant. Surfactin not only lysed B. subtilis vesicles, but also vesicles from Bacillus anthracis, indicating a mechanism that crossed species boundaries. To our knowledge, this is the first time a gene and a mechanism has been identified in the active disruption of extracellular vesicles and subsequent release of vesicular cargo in Gram‐positive bacteria. We also identify a new mechanism of action for surfactin.

Pharmacogenomic (PGx) testing has emerged as a compelling strategy that clinicians can use to inform antidepressant medication selection and dosing, but the clinical efficacy of this strategy has been questioned. We systematically reviewed and meta‐analyzed clinical trials for an association between the use of PGx‐guided antidepressant therapy and depressive symptom remission in patients with major depressive disorder (MDD). We included prospective, controlled clinical trials published in English up to July 12, 2022. Data extraction and synthesis adhered to the 2020 Preferred Reporting Items for Systematic Reviews and Meta‐Analyses guidelines. Each trial was assessed for risk of bias and a random‐effects model was used to estimate pooled risk ratios. Thirteen trials comprising 4,767 patients were analyzed, including 10 randomized controlled trials, and three open label trials. Across all included trials, those that received PGx‐guided antidepressant therapy (n = 2,395) were 1.41 (95% confidence interval (CI) = 1.15–1.74, P = 0.001) more likely to achieve remission compared with those that received unguided antidepressant therapy (n = 2,372). Pooled risk ratios for randomized controlled trials and open label trials were 1.46 (95% CI: 1.13–1.88) and 1.26 (95% CI = 0.84–1.88), respectively. These results suggest that PGx‐guided antidepressant therapy is associated with a modest but significant increase in depressive symptom remission in adults with MDD. Efforts to address the heterogeneity in PGx test composition (i.e., genes and alleles tested) and accompanying prescribing recommendations across trials will likely reduce the uncertainty about the efficacy of PGx‐guided antidepressant therapy in the literature.

Many intracellular bacterial pathogens naturally release membrane vesicles (MVs) under a variety of growth environments. For pathogenic bacteria there are strong evidences that released MVs are a delivery mechanism for the release of immunologically active molecules that contribute to virulence. Identification of membrane vesicle-associated proteins that can act as immunological modulators is crucial for opening up new horizons for understanding the pathogenesis of certain bacteria and for developing novel vaccines. In this protocol, we provide all the details for isolating MVs secreted by either mycobacteria or Gram-positive bacteria and for the subsequent identification of the protein content of the MVs by mass spectrometry. The protocol is adapted from Gram-negative bacteria and involves four main steps: (1) isolation of MVs from the culture media; (2) purification of MVs by density gradient ultrucentrifugation; (3) acetone precipitation of the MVs protein content and in-solution trypsin digestion and (4) mass spectrometry analysis of the generated peptides and protein identification. Our modifications are:•Growing Mycobacteria in a chemically defined media to reduce the number of unrelated bacterial components in the supernatant.•The use of an ultrafiltration system, which allows concentrating larger volumes.•In solution digestion of proteins followed by peptides purification by ziptip.

Although clozapine is the most effective pharmacotherapy for treatment-resistant schizophrenia, it is under-utilized, and initiation is often delayed. One reason is the occurrence of a potentially fatal adverse reaction, clozapine-induced agranulocytosis (CIA). Identifying genetic variations contributing to CIA would help predict patient risk of developing CIA and personalize treatment. Here, we (1) review existing pharmacogenomic studies of CIA, and (2) conduct meta-analyses to identify targets for clinical implementation. A systematic literature search identified studies that included individuals receiving clozapine who developed CIA and controls who did not. Results showed that individuals carrying the HLA-DRB1*04:02 allele had nearly sixfold (95% CI 2.20–15.80, p_corrected = 0.03) higher odds of CIA with a negative predictive value of 99.3%. Previously unreplicated alleles, TNFb5, HLA-B*59:01, TNFb4, and TNFd3 showed significant associations with CIA after multiple-testing corrections. Our findings suggest that a predictive HLA-DRB1*04:02-based pharmacogenomic test may be promising for clinical implementation but requires further investigation.