Virginia–Maryland College of Veterinary Medicine

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories.Significance StatementThe prefrontal cortex of porcine display unique, ramified microglia which are sensitive to germ-free conditions whereby they display alterations in morphology with a more transcriptionally reactive signature. These findings indicate that microglia are regionally sensitive to stimuli in the periphery, and studies in lissencephalic mammalian models may not be directly correlative to other higher-order species. The neuroanatomical heterogeneity of microglia across species is informative and understudied, but necessary, to draw conclusions on the array of perturbations spanning neurodevelopmental trajectories in health and disease.

Usutu virus (USUV, Flaviviridae) is a mosquito-borne, neuroinvasive flavivirus that is maintained in a transmission cycle between Culex spp. mosquitoes and passerine birds. USUV shares close ecological and antigenic similarity to West Nile virus (WNV). Our study focused on North American Cx. tarsalis and Cx. pipiens, both important in WNV transmission. At day 10, we found that Cx. pipiens were competent for USUV while Cx. tarsalis had limited evidence for transmission. Additionally, Cx. pipiens transmission significantly increased by day 21 to 33%. We found the minimum threshold for infection of Cx. pipiens to be 5 log₁₀ PFU/mL when using an artificial bloodmeal and 6 log₁₀ PFU/mL when using a live avian host. These results provide support that North American Cx. pipiens are competent vectors for USUV but require a high USUV dose for infection. Together, these data provide insight into the potential efficiency of North American Cx. pipiens for USUV transmission.

The emergence of vancomycin-resistant Enterococcus (VRE) has posed a significant global health threat, especially in healthcare settings where treatment options are increasingly limited due to rising antibiotic resistance. Maslinic acid, a naturally occurring pentacyclic triterpene found in olives, is known for its diverse biological activities, including anti-inflammatory, anticancer, antioxidant, and antimicrobial effects. In this study, we investigated the antibacterial and antibiofilm potential of maslinic acid against VRE. Initial screening identified maslinic acid as a potent hit, with minimum inhibitory concentrations (MICs) ranging from 4 to 8 µg/mL across 13 clinical enterococcal isolates, including multidrug-resistant strains. Time-kill assays demonstrated bacteriostatic activity comparable to linezolid, while cytotoxicity and hemolysis assays confirmed its safety in mammalian cells. Furthermore, maslinic acid disrupted established Enterococcus faecalis biofilms by approximately 50%, whereas linezolid was not effective against biofilms. Notably, maslinic acid significantly reduced bacterial burden in a Caenorhabditis elegans infection model by 80%, outperforming linezolid. These findings highlight maslinic acid as a promising candidate for the development of new therapies targeting VRE, with the added advantage of antibiofilm activity and a favorable safety profile.

Due to the ever-increasing threat of methicillin-resistant Staphylococcus aureus (MRSA), we have embarked on a campaign to discover novel antibacterial agents which are effective at eradicating both MRSA, as well as acting on pre-formed MRSA biofilms. Using the known scaffold of N-(1,3,4-oxadiazol-2-yl)benzamides, we performed a halogenation study which has led to the identification of HSGN-2241. This novel compound was found to effectively kill many multi-drug resistant Gram-positive clinical isolates in a bactericidal manner. The growth inhibition mechanism of HSGN-2241 was found to be via potassium ion release and subsequent depolarization of the cell membrane. As an initial safety test, HSGN-2241 was found to not lyse human red blood cells, so it is a promising lead compound for the treatment of bacterial infections caused by Staphylococcus aureus.

Vancomycin-resistant enterococci (VRE) represent a growing threat to public health due to their increasing prevalence and limited treatment options. The urgent need for alternative therapeutics highlights the value of novel approaches such as drug repurposing to identify effective antimicrobial agents. In this study, we screened a library of 1,135 antibacterial compounds to identify candidates active against VRE. Fifty-eight compounds demonstrated measurable activity, with ridinilazole and CRS3123 emerging as the most promising. Both exhibited potent antibacterial activity in the nanomolar range against multiple Enterococcus species, including vancomycin-resistant and vancomycin-sensitive E. faecium, E. faecalis, E. hirae, and E. durans. CRS3123 displayed exceptional potency, with MIC values of < 0.007 µg/mL for most strains. Ridinilazole exhibited a broader MIC spectrum with E. faecium highly sensitive (< 0.007–0.25 µg/mL) and E. faecalis less susceptible (0.5–>64 µg/mL). Both compounds also displayed low cytotoxicity in Vero cells and no hemolytic activity, suggesting a favorable safety profile. In vivo studies using the C. elegans model confirmed potent efficacy, with CRS3123 reducing both E. faecium and E. faecalis burden similar to linezolid while ridinilazole were effective against E. faecium only. These findings support ridinilazole and CRS3123 as promising candidates for further VRE therapeutic development.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-29583-1.

Cryptococcosis is an opportunistic fungal infection affecting individuals with compromised immunity, particularly those with HIV. The limited accessibility to effective treatments and treatment-related toxicities underline the need for more effective therapeutic options. In this study, we conducted a whole-cell screening of ~ 3,700 FDA-approved drugs and clinical molecules against the Cryptococcus neoformans H99 strain. The anti-mycobacterial agent SQ109 was identified as one of the most potent hits, with broad antifungal activity. SQ109 exhibited potent activity against Cryptococcus spp., with an MIC90 of 4 μg/mL. In the time-kill assay, SQ109 demonstrated a fungicidal activity on proliferating cryptococcal cells in a concentration-dependent manner. Unlike fluconazole (FLC) and flucytosine (5-FC), C. neoformans showed a negligible tendency to develop resistance to SQ109 during frequent passaging. Furthermore, SQ109 exhibited a potent efficiency in the murine model of cryptococcal infection, resulting in a 50% survival rate among animals treated with 25 mg/kg for 10 consecutive days. The transcriptomic analysis revealed that SQ109 disrupts ergosterol biosynthesis, affecting membrane integrity and oxidative homeostasis. Additionally, molecular docking and structural analysis indicated that squalene synthase protein ERG9 is the most likely target of SQ109 within the ergosterol biosynthesis machinery of cryptococcal cells. Notably, SQ109 potentiates the activity of the standard antifungal FLC, as well as other ergosterol inhibitors, with a fractional inhibitory concentration Index (ΣFICI) ranging from 0.38 to 1. These findings highlight the therapeutic potential of SQ109 in combating cryptococcal infections, both as a standalone therapy and as an adjuvant to FLC monotherapy.