Coxiella burnetii, the bacterium responsible for Q fever, is a highly infectious, environmentally resilient, and aerosol-transmissible zoonotic pathogen. While molecular and serological assays are the primary tools for routine diagnosis, isolating viable C. burnetii remains crucial for comprehensive pathogen characterization, including whole-genome sequencing, phenotypic analysis, antimicrobial susceptibility testing, vaccine development, and diagnostic assay validation. This review examines the literature on two key factors for successful C. burnetii recovery: selecting an appropriate specimen source and choosing a compatible cultivation platform. Viable organisms can be recovered from human clinical specimens, such as acute-phase blood and tissues from focal chronic infections (e.g., cardiac valves), provided that bacterial burden and viability are maintained. Animal-derived specimens, especially reproductive tissues, placental material, fetal tissues, milk, urine, feces, and genital secretions, are vital sources for veterinary investigations and zoonotic risk assessment. However, contamination with other microorganisms often complicates recovery from these samples. Tick-derived and environmental samples can broaden epidemiological insights, but their interpretation requires caution, as Coxiella-like endosymbionts and low viable bacterial burdens may hinder culture recovery. Regarding culture platforms, mammalian cell culture, particularly shell vial-based systems, is the most widely applicable method for primary isolation. Embryonated chicken eggs and animal inoculation are still valuable for biological amplification or generating phase variants. Furthermore, host cell-free axenic media have revolutionized C. burnetii propagation, metabolic analysis, and genetic studies. Successful isolation hinges on several factors: specimen type, processing, storage conditions, bacterial viability, strain characteristics, and compatibility with the chosen culture system. Future advancements should incorporate viability-based molecular preselection, high-throughput image-based culture screening, optimized cell-line selection, axenic media with broader strain permissiveness, and standardized reporting of culture attempts. These improvements will expand available isolate collections and diversify studies on C. burnetii biology, epidemiology, and control.

We examined passage-associated phenotypic and transcriptional changes in the clinical Acanthamoeba lugdunensis keratitis isolate KA/E2 using preplanned serial in vivo passage in C57BL/6 mice. Amoebae were recovered from lung but not brain cultures during the first cycle. Brain cultures yielded lineages P2 and P3 during the second and third cycles, respectively, with significantly greater recovery in the third than the second cycle on day 4 after plating. In a separate experiment, P3 infection was associated with greater blood-brain barrier (BBB) permeability and earlier body-weight loss than E2 infection; body weight differed significantly between infected groups only on day 8. Cldn5 and Ocln expression was lower in P3-infected mice than in controls; Tjp1 showed no significant pairwise differences. RNA sequencing identified 794 differentially expressed genes between parental E2 and P3. Upregulated genes were enriched for carbohydrate metabolism and hydrolase annotations and included transcripts annotated as a putative IgA-specific serine endopeptidase-like protein and cysteine proteinase 5. Phosphorylation-related genes were enriched among downregulated genes. These differences were detected after five axenic subcultures of P3, although their stability and heritability remain unknown. Total gelatinolytic activity was higher in P3 excretory-secretory products than in E2. Phenylmethylsulfonyl fluoride reduced activity in both isolates, with no significant difference between isolates after treatment; E-64 produced no significant change in total activity. Serial in vivo passage was associated with brain culture recovery, transcriptional differences, and greater BBB permeability, without establishing causality.

Mosquitoes are important vectors of human pathogens, and their gut microbiome influences traits such as survival, fecundity, pathogen resistance, insecticide resistance, and vector competence. Manipulating this microbiome offers a promising strategy for reducing mosquito-borne disease. While 16S rRNA gene amplicon sequencing has revealed global patterns of gut microbial diversity, the factors driving these patterns remain poorly understood. Here, we used a community-transplantation approach that allows the collection, cryopreservation, and transfer of whole adult microbial communities into axenic (germ-free) mosquitoes to investigate the determinants of microbiome assembly and host-microbe interactions. Using reciprocal transplants between Aedes aegypti Liverpool strains maintained in isolation for nearly 50 years at the University of Wisconsin-Madison (UW) and the Liverpool School of Tropical Medicine (LSTM), we found striking strain-specific responses: UW mosquitoes exhibited altered bacterial composition and developmental defects, whereas LSTM mosquitoes did not. Transplant fidelity depended on donor community composition and recipient genotype, and we observed conserved assembly patterns across the mosquito life cycle. These results reveal how host genetics and microbial community structure interact to shape microbiome assembly and support the development of microbiome-based vector control strategies.

Francisella tularensis is a zoonotic pathogen transmitted to humans through contact with infected animals, arthropod vectors, or contaminated environmental sources. Although replication occurs primarily within eukaryotic host cells, persistence outside a host is thought to be critical for long-term maintenance and transmission in natural ecosystems. To better understand persistence in aquatic habitats outside of mammalian hosts, we studied virulent strains of F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B) incubated in a planktonic state (free living in water) under various axenic aquatic conditions. After 1 year in cold, nutrient-poor treatments, the type B strain (FSC200) caused disease in mice and remained culturable on laboratory media, whereas the type A strain (Schu S4) caused disease but with delayed onset and became non-responsive to laboratory growth conditions. In 1.5-year persistence experiments, type B still caused disease in mice, but with delayed onset, whereas type A no longer caused disease. The rate of bacterial decline depended on subspecies, temperature, and the initial inoculum size. We further explored the persistence of type A and type B populations in natural pond water, which corroborated findings from axenic conditions and revealed that both strains perpetuated disease from a non-replicative inactive persistent state. This study provides timeframes for the persistence of virulent planktonic F. tularensis subspecies tularensis and holarctica populations in various aquatic conditions, contributing to increased understanding of host-independent survival as a component of the perpetuation of tularemia.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives in host cells by resisting hostile antimicrobial defenses. However, the molecules and mechanisms that contribute to Mtb's intracellular survival are not fully understood. We have previously reported that Rv0148, a putative short-chain dehydrogenase/reductase, plays a significant role in Mtb stress response and virulence in in vitro and in vivo models. To further understand the role of Rv0148 in regulating global functions of Mtb, we performed comparative pilot proteomic analysis between Δrv0148 mutant, and wild-type (WT) strains grown in axenic cultures. Mass spectrometry-based proteomics analysis identified 586 and 626 statistically significant differentially abundant proteins (SSDAP) in the WT and Δrv0148 mutant, respectively. Ontology analysis revealed that proteins involved in biological processes, including bacterial adaptation to host responses and protein homeostasis, were significantly more abundant, while peptidoglycan biosynthesis was less abundant in the mutant than the WT. Further network analysis revealed dysregulation of proteins involved in bacterial stress response, cell wall components, ribosomal and secretory proteins, suggesting impaired cell wall and translation machinery in Δrv0148. Functional categorization of differentially regulated proteins in Δrv0148 showed broad reprogramming in intermediary metabolism and stress adaptation. These findings suggest that Rv0148 may influence remodeling of cell wall components and bacterial physiology, potentially balancing Mtb survival and stress adaptation.

Corynebacterium bovis is an important opportunistic pathogen of immunodeficient mice and the etiologic agent of Corynebacterium -associated hyperkeratosis (CAH). Although disease severity varies, specific vendor-derived microbiomes have been shown to protect against CAH. Preinoculation with nonpathogenic Corynebacterium amycolatum prior to C. bovis infection has been demonstrated to limit disease severity. To define community features associated with protection, axenic outbred athymic nude mice stocks (A, B, and C) were reassociated with donor microbiomes from four vendor sources (A1, A2, B, and C) and topically challenged with C. bovis . C. amycolatum was added to the A1 microbiome in a separate group. Skin (Stock A) and fecal (Stocks A, B, and C) microbiomes were evaluated at 21 days post-inoculation via shotgun metagenomic sequencing. The A2 microbiome, previously associated with resistance to clinical disease and minimal skin pathology, exhibited greater cutaneous microbial evenness after challenge and significantly lower relative abundance of C. bovis than all other microbiome groups. Addition of C. amycolatum to the A1 microbiome did not fully confer disease protection and relative abundance was low, suggesting that sustained colonization by this organism was insufficient to explain the protective phenotype seen with the A2 microbiome. C. kroppenstedtii was detected in all A2 skin samples and was absent from all other groups. Several anaerobic taxa, including Duncaniella dubosii, D. muris, Bacteroides caecimuris , and Muribaculum gordoncarteri , were uniquely detected or enriched in the A2-associated microbiome, whereas Mammaliicoccus lentus and Staphylococcus nepalensis were absent from A2 but present in all nonprotective groups. Gut alpha diversity did not differ significantly among microbiomes, although several taxa, including segmented filamentous bacteria, were enriched in A2 feces. These findings associate resistance to CAH with preservation of cutaneous community structure, suppression of C. bovis prominence, and distinct microbial taxa. The identified organisms represent candidates for future mechanistic studies of microbiome-mediated colonization resistance.

Under global climate change, temperature stress constrains the stable large-scale cultivation of microalgae. Here, we evaluated the role of algal-associated bacteria in the temperature tolerance of Isochrysis zhanjiangensis and identified a functional bacterium with application potential. The presence of the associated bacterial community increased the final cell density of I. zhanjiangensis, reaching 1.28-fold that of the axenic control under heat stress. Analysis of 16S rRNA gene amplicon sequencing revealed that heat stress reshaped the bacterial community and significantly enriched Labrenzia. At an optimal algae-to-bacteria ratio of 1:50, Labrenzia algicola 520 promoted algal growth under both heat and cold stress, with elevated chlorophyll content, osmolyte accumulation, and antioxidant capacity. Genomic and phenotypic analyses further revealed multiple growth-promoting traits, including indole-3-acetic acid (IAA) biosynthesis, siderophore production, antioxidant activity, and biofilm formation. Moreover, L. algicola 520 maintained its beneficial effects under xenic conditions and increased the biomass of Isochrysis galbana by 29.6% in scaled-up winter cultivation. Collectively, these findings establish L. algicola 520 as a promising probiotic candidate for improving the production stability of commercial microalgae under fluctuating temperature conditions and provide a basis for developing bacteria-based bioaugmentation strategies in microalgal cultivation.

Visceral leishmaniasis (VL), caused by Leishmania donovani, is one of the most lethal parasitic diseases, causing 30000 deaths annually. Pentavalent antimonials, with limited effectiveness, cytotoxicity, drug resistance, and therapeutic limitations, underscore the urgent need for the development of novel, selective, and cost-effective antileishmanial agents. The Indian pharmaceutical industry, a major global economic sector, is showing growing interest in mushrooms in developing alternative drugs against several diseases. Thus, this study focuses on the antileishmanial potential of the hydroalcoholic extract of Amanita princeps, a wild edible mushroom, with detailed mechanistic analysis. In our research, the cell-inhibitory activity of the extract was evaluated by the MTT assay against promastigotes, axenic amastigotes, and intracellular amastigotes, yielding IC50 values of 21.20, 30.26, and 38.93 μg/mL, respectively, with high selectivity indices (>10). A detailed mechanistic study revealed that the extract can disrupt the NO-arginase axis, suppress antioxidant enzymes (SOD and catalase), and elevate lipid peroxidation, ultimately causing intracellular oxidative stress. Further, flow cytometric and fluorescence microscopic study confirmed elevated reactive oxygen species, mitochondrial membrane depolarization, nuclear fragmentation, sub-G0/G1 cell cycle arrest, leading towards apoptosis as evidenced by AO/EtBr dual staining. Furthermore, the extract was able to restore host immunological balance by upregulating pro-inflammatory cytokines (IL-12β, TNF-α) and suppressing anti-inflammatory mediators (IL-10, TGF-β). Finally, GC-MS, network pharmacological and molecular docking study identified Urs-12-en-28-ol, a ursane triterpenoid with the highest affinity against DHFR (-9.7 kcal/mol) and demonstrated significant in vitro antileishmanial activity. Conclusively, our findings establish Amanita princeps as a promising candidate for next-generation antileishmanial drug development.

Leishmaniasis poses a significant public health challenge, highlighting the urgent need for safer and more effective therapies. Here, we investigated the antileishmanial activity and mechanism of action of gold(I)-triphenylphosphine complexes against Leishmania amazonensis. AuPPh3Cl and AuPPh3MTZ showed potent activity against promastigote and axenic amastigotes, with low micromolar EC50 and favorable selectivity toward mammalian cells. Both compounds reduced infection rate and parasite burden in infected macrophages. Mechanistically, the complexes targeted mitochondrial function, resulting in increased oxygen consumption associated with dissipation of the mitochondrial membrane potential, without affecting respiratory coupling. These findings were accompanied by increased mitochondrial superoxide production, indicating a pro-oxidant effect. AuPPh3Cl induced higher hydrogen peroxide levels than AuPPh3MTZ, suggesting ligand-dependent differences in redox modulation. In addition, treated parasites displayed elevated NADPH levels, consistent with the activation of compensatory antioxidant responses. Both compounds inhibited trypanothione reductase activity in parasite extracts and with the purified enzyme. Notably, AuPPh3Cl showed greater selectivity toward the parasite enzyme over human glutathione reductase. Overall, these findings support a multitarget mechanism involving mitochondrial dysfunction and disruption of the trypanothione-dependent redox system, leading to oxidative imbalance and parasite death. Gold(I)-triphenylphosphine complexes represent promising candidates for the development of novel antileishmanial agents.

Antimicrobial resistance has intensified the search for novel antimicrobial and wound-healing agents. Ludwigia octovalvis (Jacq.) P.H. Raven is traditionally used to treat infections, inflammation, and skin disorders, although its in vitro biotechnological potential remains largely unexplored. This study reports the first successful establishment of L. octovalvis callus cultures, their characterization by gas chromatography-mass spectrometry (GC-MS), and the evaluation of their antimicrobial and wound-healing activities. Friable calluses were induced from leaf and node explants of axenic seedlings using combinations of 6-benzylaminopurine (BAP), kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D), and α-naphthaleneacetic acid (NAA). Optimal callus induction was achieved in leaf explants cultured on full-strength Murashige and Skoog (MS) medium supplemented with BAP (4.44 µM) and 2,4-D (0.45 µM). After six months of subculture, the calluses showed morphological uniformity and stable biomass production. Growth kinetics followed a specific growth rate of 0.047 d-1 and a doubling time of 14.54 days. GC-MS analysis identified fatty acids and phytosterols as the main components. The ethyl acetate extract significantly improved wound healing in vivo, while the methanolic extract exhibited antibacterial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). These findings demonstrate that L. octovalvis callus cultures constitute a sustainable source of bioactive compounds with promising therapeutic potential.

Swine wastewater is a nutrient-rich waste stream with considerable potential for cyanobacterial cultivation and resource recovery. However, its impacts on microbial community dynamics and antibiotic resistance genes (ARGs) remain poorly understood. This study evaluated the cultivation of Arthrospira platensis in a medium containing 25% swine wastewater and 75% Zarrouk medium (SWW25) using a flat-panel photobioreactor. A. platensis achieved a biomass concentration of 1.58 ± 0.02 g/L, a biomass productivity of 0.11 ± 0.01 g/L/d, and a specific growth rate of 0.26 ± 0.01 d-1. Simultaneously, the system removed 98.5% of ammonium nitrogen and 80% of orthophosphate. Metagenomic analysis indicated changes in bacterial community composition during A. platensis cultivation. Microbial community analysis revealed shifts in relative abundance after cultivation, likely associated with changes in community composition following Arthrospira biomass enrichment, while Proteobacteria, Firmicutes, and Synergistota remained among the dominant phyla. In addition, ARG profiling indicated a decline in ARG diversity and read counts during cultivation, whereas tet(X) remained the only detectable ARG after A. platensis cultivation. Collectively, these findings suggest that A. platensis cultivation under non-axenic conditions was associated with biomass production, nutrient removal, and changes in microbial community composition and ARG profiles in swine wastewater.

Genome-resolved phylogenomics reveals widespread misclassification of metal-reducing bacteria historically assigned to Geobacter based on 16S rRNA gene phylogeny, and highlights species that persist only as 16S rRNA entries without genomes for robust taxonomic resolution. Here, we resolve two such lineages by integrating whole-genome phylogeny with average amino acid identity (AAI) and percentage of conserved proteins (POCP) across 418 dereplicated genomes of Desulfuromonadia. We report a draft genome of the psychrophilic iron-reducing bacterium Geobacter psychrophilus (100% completeness). Phylogenomic analyses place both Geobacter psychrophilus and the GTDB placeholder genus g__JACRCG01 within the family 'Pseudopelobacteraceae', outside Geobacteraceae sensu stricto. Within this framework, G. psychrophilus forms a distinct, well-supported lineage separated from neighbouring genera by discontinuities in AAI and POCP, supporting its reclassification as Irobacter psychrophilus comb. nov. Additionally, we show that Geosyntrophus acetoxidans, a non-axenic syntrophic bacterium, forms a coherent genus with 51 other environmental genomes (placeholder genus g__JACRCG01), for which we propose the replacement name Geosyntrophus gen. nov. Comparative genome analysis revealed conserved family-level metabolic traits together with genus-specific differences in respiratory metabolism, while ANI-based clustering identified substantial species-level diversity within both proposed genera. Metagenome and 16S rRNA-gene survey data further show that Geosyntrophus and Irobacter occur in broadly similar aquatic and subsurface habitats spanning from the Arctic to the Antarctic. Together, these results resolve the taxonomy of two previously ambiguous Desulfuromonadales lineages and shed light on their environmental distribution.