Microbiology-Sgm最新文献

R-pyocins are phage tail-like protein complexes produced by Pseudomonas aeruginosa that deliver a single, lethal hit by depolarizing the target cell membrane. Unlike phages, R-pyocins lack capsids and DNA, and their killing is highly specific, being determined by tail fibre proteins that recognize subtype-specific LPS receptors on susceptible strains. Five known subtypes (R1-R5) vary in host range, with R5 displaying the broadest activity. R-pyocin expression is tightly regulated by the SOS response, linking their release to environmental stress. Their non-replicative mechanism and metabolic independence make them especially promising for targeting multidrug-resistant and biofilm-associated P. aeruginosa infections, such as those seen in cystic fibrosis and chronic wounds. Preclinical studies support their therapeutic potential, and bioengineering approaches have extended their target range. With their high specificity, rapid action and adaptability, R-pyocins are strong candidates for next-generation precision antimicrobials.

Klebsiella pneumoniae is a prominent opportunistic pathogen increasingly associated with multidrug resistance and virulence. One of the main mechanisms of antimicrobial resistance in K. pneumoniae is active efflux, primarily mediated by the resistance-nodulation-division (RND) family of pumps. AcrAB-TolC is the key RND efflux pump in K. pneumoniae, regulated by the transcriptional activator RamA and its repressor RamR. Although overexpression of AcrAB-TolC has been linked to drug resistance in various clinical strains, its physiological roles in K. pneumoniae remain insufficiently studied. In this study, we generated isogenic deletions of acrB and ramR in both the genetically tractable K. pneumoniae Ecl8 and the virulent ATCC 43816 strains. We examined the phenotype of the ΔacrB and ΔramR mutants by assessing antimicrobial susceptibility, biofilm formation, growth under infection-related conditions and both in vitro and in vivo infection models. Loss of acrB increased susceptibility to drugs, decreased biofilm formation and reduced in vitro virulence in both Ecl8 and ATCC 43816. However, only in Ecl8 was the loss of AcrB found to diminish growth under infection-like conditions and decrease in vivo virulence in the Galleria mellonella infection model. In contrast, in ATCC 43816, it had no effect. Our findings suggest that AcrAB-TolC exhibits strain-specific physiological functions, highlighting its dual role in antimicrobial resistance and pathogenicity, and thereby broadening our understanding of efflux-mediated adaptations in K. pneumoniae. Exploring the broader functions of RND efflux pumps in K. pneumoniae can provide insights into the potential effects of targeting them with inhibitor molecules.

Deep-sea ecosystems remain poorly understood due to exploration challenges. Despite the advancements metagenomics have brought to the understanding of the ocean microbiome, the diversity of marine viruses, particularly in the deep sea, is still not well characterized. In this study, we analysed the impact of depth on the composition and diversity of marine viruses in deep-sea waters at a global scale. Raw reads from deep-sea shotgun DNA sequences were retrieved from the Tara and Malaspina expeditions, encompassing depths from 270 to 4,005 m. A total of 80 samples containing viral reads were identified and analysed through a comprehensive bioinformatics pipeline, including quality assessment, taxonomic classification and metabolic annotation. The analysis reveals that microbial viral diversity significantly decreases with depth, with shallower waters exhibiting higher species richness. We determined that a substantial proportion of deep-sea viral sequences remains unclassified - up to 31.9% at depths of 270-1,000 m and 9.6% at 2,400-4,005 m. Additionally, a higher abundance of auxiliary metabolic genes was observed at shallower depths, indicating potential roles in host metabolism and adaptation. Our findings reveal the deep ocean as a vast, largely unexplored source of microbial viral diversity. This research emphasizes how depth influences viral diversity and community makeup in deep-sea environments, underscoring the need for further exploration to fully grasp their complexity and ecological roles.

Tularemia is a zoonotic disease caused by Francisella tularensis. Most human cases are caused by F. tularensis ssp. tularensis (type A) or F. tularensis ssp. holarctica (type B), with the former considered more virulent. For this reason, type A isolates are often the benchmark for the testing of new vaccines or antibiotics. However, both subspecies cause considerable disease and can differ in their responsiveness to medical countermeasures. Accordingly, there is a need to identify and characterize representative type B isolates that are available to qualified research institutions to ensure the development of future vaccines or antibiotics is efficacious against both subspecies. The type B isolate OR96-0246 was identified as a strain that can address this need and was subsequently characterized. For in vitro characterization, the OR96-0246 strain was examined for growth in media and for its ability to form biofilm. As the LPS is an essential virulence factor, the O-antigen was characterized through western analysis. For future medical countermeasure testing for biodefence concerns, pneumonic challenges with animal modelling would be required. Therefore, using the OR96-0246 strain, we implemented animal models that encompassed BALB/c mice, Fischer 344 rats and cynomolgus macaques. Mice were challenged via intranasal instillation with varying doses of OR96-0246, and the LD₅₀ was determined to be 1 c.f.u. We progressed to Fischer 344 rats, which are a better-suited rodent model to gauge vaccine efficacy. When challenging the rats by whole body aerosolization with various doses of OR96-0246, the LD₅₀ was determined to be 138 c.f.u. Finally, a staircase challenge design was applied to three cynomolgus macaques, each receiving a different aerosolized dose of OR96-0246 to determine an estimated LD₅₀ for non-human primates (NHPs). Two out of the three NHPs succumbed to the challenge. The animal that received the lowest dose (2.1×10⁴ c.f.u.) survived but did demonstrate clinical signs of infection. Samples from the challenged rats and NHPs were collected for histopathology characterization. Generally, the pathological changes observed in both models were similar, consisting primarily of multifocal bronchopneumonia in the lung and necrotic lesions in the spleen. This animal model development with type B strains of F. tularensis will be essential to properly evaluate new antimicrobials and vaccines to protect against tularemia.

Post-transcriptional regulation can be mediated by small regulatory RNAs (sRNAs) in bacteria, which can act by base-pairing to a target mRNA. The discovery and mechanistic validation of base-pairing sRNAs in multidrug-resistant Acinetobacter baumannii has been hampered by the lack of genetic tools to assess RNA-RNA interactions. Here, we created two compatible plasmids for A. baumannii, which addresses this need. The newly designed plasmids validated the known Aar sRNA-carO mRNA and a new interaction of sRNA44 and the mRNA of the biofilm-associated protein Bap. The new plasmid system should accelerate the mechanistic characterization of sRNAs in A. baumannii.

To develop a low-cost, environmentally friendly taurine fermentation method for sustainable marine fish culture using feed derived from photosynthetically produced agricultural products, it is crucial to study cysteine sulfinic acid decarboxylase (CSAD), a key enzyme in the taurine biosynthetic pathway in applicable microorganisms. In this study, a method was devised to screen for CSAD genes using Escherichia coli growth as an indicator, based on sulfur assimilation following the decarboxylation of l-cysteic acid, a taurine precursor compound. The E. coli used has a double deletion mutation of cysA (sulfate/thiosulfate ABC transporter) and ssuD (FMNH₂-dependent alkanesulfonate monooxygenase) genes. If needed, an additional defect in enzyme genes, such as cysC (adenylyl-sulfate kinase), which participates in the pathway reducing sulfate to sulfite, is also introduced. Using this method, it was demonstrated that the glutamic acid decarboxylase gene from Yarrowia lipolytica possesses CSAD activity. The identified decarboxylase was further confirmed to act on l-cysteine sulfinic acid. Additionally, two observations made during method refinement to reduce background growth in screening are discussed: that SsuD is involved in sulfur assimilation from an unknown sulfur compound and that certain mscK (mechanosensitive channel) missense mutations enable external sulfate above a specific concentration to enter the cell.

Cellular proliferation relies on the successful coordination and completion of genome replication and segregation. To help achieve this, many bacteria utilize regulatory pathways that ensure DNA replication initiation only occurs once per cell cycle. When dysregulated, loss of DNA replication control can have severe consequences. In Escherichia coli, it has been established that hyper-initiation of DNA synthesis leads to pleiotropic genome instability and cell death. Therefore, targeting DNA replication initiation proteins to promote hyper-initiation may be an approach to generate novel antimicrobials. However, the pathways and potential consequences of replication hyper-initiation in Gram-positive species remain enigmatic. To address this question, we devised genetic systems to artificially induce hyper-initiation in the model organism Bacillus subtilis and the pathogen Staphylococcus aureus. In both species, hyper-initiation elicited cellular degeneration culminating in growth inhibition by cell death. During this process in B. subtilis, temporal analyses revealed the early onset of the DNA damage response, followed by membrane depolarization and cell lysis. This phenotype could be suppressed by removing pathways that repair damaged DNA, suggesting that cell death is a consequence of conflicts between DNA replication and repair. In S. aureus, cells quickly accumulated striking morphological changes associated with rapid loss of chromosomal DNA and death via a lysis-independent pathway. Moreover, inducing hyper-initiation in S. aureus was observed to decrease bacterial survival during infection of murine macrophages. Taken together, the data suggest that stimulating initiation of bacterial DNA synthesis could be an alternative approach to inhibiting microbial growth, particularly in combination with compounds that inhibit or poison DNA repair, akin to cancer therapies.

We determined the mechanism of resistance to seven chemical series with potent activity against Mycobacterium tuberculosis. Resistant mutants were isolated against the aminothiazoles, phenylhydrazones, 8-hydroxyquinolines, nitazoxanides, phenyl alkylimidazoles, morpholino thiophenes and trifluoromethyl pyrimidinones. We demonstrated that mutations in several components of the Esx-3 type VII secretion system (EccA3, EccB3, EccC3 and EccD3) conferred resistance to these disparate scaffolds. We conclude that mutations in Esx-3 are a common mechanism of resistance to anti-tubercular agents, which may have clinical relevance for new drugs.

Artificial intelligence (AI) and machine learning (ML) are reshaping microbiology, enabling rapid antibiotic discovery, resistance prediction and clinical diagnostics. For microbiologists, the goal is not to build new algorithms but to recognize when ML is appropriate, how to prepare data and how to interpret outputs responsibly. This primer takes that practical stance - driving the ML car rather than rebuilding the engine. At a high level, ML learns from complex patterns, often noisy data. In antibiotic discovery, ML models help identify compounds in biological data and design new ones from scratch using generative AI. In microbiome studies, where measurements are compositional, sparse and often confounded, ML helps uncover community structure and link taxa or functions to phenotypes. In pathogen genomics, supervised models map sequence-derived features (e.g. k‑mers, SNPs and gene presence/absence) to outcomes such as species identity, antimicrobial susceptibility or MIC. Unsupervised learning supports exploration, including clustering, latent gradients and dimensionality reduction for visualization. Across these settings, success hinges less on exotic architectures than on sound problem framing, careful preprocessing and experimental validation.