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Bacterial lysis during treatment of Gram-negative infections can release lipopolysaccharide (LPS) and aggravate inflammation. Here, we engineered two complementary T7 bacteriophages: T7-nluc, a NanoLuc reporter bacteriophage for real-time monitoring of viable bacteria, and T7-phoa, a therapeutic bacteriophage that releases alkaline phosphatase (PhoA) during lysis to reduce LPS bioactivity. Both engineered bacteriophages retained lytic activity similar to that of wild-type T7. In vitro, T7-nluc produced a low-background bioluminescent signal that reflected bacterial burden, whereas T7-phoa released catalytically active PhoA into the extracellular environment. In Galleria mellonella and Danio rerio infection models, T7-nluc enabled dynamic monitoring of infection progression, while T7-phoa improved survival, reduced inflammatory responses, and accelerated inflammatory resolution without compromising bacterial clearance. These findings support a modular bacteriophage engineering strategy that combines bacterial killing, real-time infection monitoring, and local attenuation of LPS-driven inflammation, offering a potential approach for improving bacteriophage-based treatment of Gram-negative infections. Bacteriophage therapy is being reconsidered for treating drug-resistant Gram-negative infections, but there is concern that rapid bacterial lysis may release LPS and worsen inflammation. We used bacteriophage T7 as a platform to test whether bacteriophages can be engineered to both fight bacteria and soften these harmful host responses. First, we created a NanoLuc reporter bacteriophage that produces light only when it grows in live bacteria, confirming that engineered bacteriophages can deliver active proteins directly in infected animals. We then built a therapeutic T7-phoa bacteriophage designed to release enzymatically active alkaline phosphatase upon on-target lysis, thereby providing lysis-coupled local phosphatase activity at the infection site. In both G. mellonella and Danio rerio models, infection-site fluids collected after treatment showed elevated phosphatase activity in the T7-phoa group, and the treatment was associated with lower inflammatory peaks, improved survival, and preserved bacterial clearance. Together, these results support a modular route for bacteriophage-based strategies that couple bacterial killing with real-time reporting and local control of LPS associated inflammation.

The epidemiology of suspected pediatric meningoencephalitis has changed in the era of conjugate vaccines and multiplex PCR. Updated epidemiologic data are needed to adapt diagnostic and therapeutic algorithms to current practice. This retrospective single-center study included children&#x2009;<&#x2009;18 years who underwent lumbar puncture with cerebrospinal fluid multiplex PCR for suspected central nervous system infection at a tertiary pediatric hospital in Germany between 2016 and 2024. Clinical, laboratory, and outcome data were extracted from electronic medical records. Cerebrospinal fluid was analyzed using the BioFire&#xae; FilmArray&#xae; Meningitis/Encephalitis Panel. Statistical analyses included descriptive statistics, nonparametric comparisons, and receiver operating characteristic analyses. Among 1,198 children, definite bacterial meningitis was diagnosed in 13 (1.1%), definite viral meningitis in 80 (6.7%), aseptic meningitis of unknown etiology in 131 (11.0%), confirmed/probable encephalitis in 53 (4.4%), and possible encephalitis in 34 (2.8%). Bacterial meningitis represented 5.8% of meningitis cases. A causative pathogen was identified in all bacterial cases, most commonly Streptococcus pneumoniae (n&#x2009;=&#x2009;7). Enterovirus (n&#x2009;=&#x2009;52) and parechovirus (n&#x2009;=&#x2009;9) predominated in viral meningitis, while an infectious etiology was identified in only 13/53 confirmed/probable encephalitis cases. The Bacterial Meningitis Score showed 80.0% sensitivity and 57.6% specificity. The recently published UK-ChiMES pre- and post-lumbar puncture scores demonstrated sensitivities of 84.6% and 76.9% and specificities of 86.3% and 92.7%, respectively. Bacterial meningitis was rare, whereas viral and etiologically unresolved infections predominated despite routine multiplex PCR. Clinical prediction scores supported risk stratification, with the UK-ChiMES pre-lumbar puncture score showing the most favorable sensitivity-specificity balance.

Female genital mutilation/cutting (FGM/C) is harmful to physical, mental, and reproductive health, though the effect of this practice on a woman's HIV susceptibility is poorly understood. Despite the known associations of FGM/C with short-term vaginal epithelial damage, neither genital inflammation nor the genital microbiome have been explored in women who have undergone FGM/C. In this study we compare the genital immune milieu and microbiome among female sex workers (FSWs) by FGM/C status, hypothesizing that these biological factors are dysregulated in women who have undergone FGM/C, heightening their risk of HIV acquisition. 1003 FSWs in Nairobi, Kenya, were enrolled in the Maisha Fiti study and visited a study clinic up to three times from June 2019 to March 2021. Participants self-reported any previous exposure to FGM/C as well as other relevant sociodemographic factors. Levels of proinflammatory cytokines and soluble E-cadherin (sE-cad), a biomarker of epithelial barrier disruption, were measured by multiplex immunoassay using self-collected cervicovaginal secretion samples provided by HIV-uninfected participants. Genital inflammation was defined using a composite score of inflammatory cytokines previously associated with HIV acquisition. The presence of inflammation was compared longitudinally between groups using mixed models to control for potential confounders including age, bacterial vaginosis (BV) status as defined by Nugent score, and others. Vaginal bacterial abundance, Shannon diversity, and total levels of key vaginal bacteria were measured by qPCR and compared by FGM/C status in an exploratory analysis. 44 of 1003 (4%) participants had undergone Type I or II FGM/C. These participants were older (p < 0.001) and more likely to test positive for herpes simplex virus-2 (HSV-2; p = 0.04), and less likely to have completed primary education (p = 0.03). Among HIV-uninfected participants, there was no evidence that genital inflammation was associated with FGM/C status after controlling for potential confounders (aOR = 0.70; 95% CI: 0.31-1.59; p = 0.40). There was no evidence of a difference in BV prevalence (p > 0.99), total bacterial abundance (p = 0.96), or Shannon diversity (p = 0.15) by FGM/C status. Type I or II FGM/C was not associated with genital inflammation or microbial dysregulation in the long-term among HIV-negative FSWs in this cohort. This may be due to the duration elapsed since FGM/C occurred or the lowered mucosal immune activation previously observed in FSWs.

Outbreaks of cholera pose a major threat to human health. Currently, antibiotics are the most effective treatment against the causative agent, the bacterium Vibrio cholerae. However, the use of antibiotics eventually leads to the emergence of resistant strains, which necessitates the need for alternative approaches. The use of bacteriophages to target the infection by antibiotic-resistant bacteria is one promising alternative. While clearance of Vibrio cholerae with the use of phages has been performed on several animal models, none of these models are naturalistic hosts of V. cholerae. Therefore, we set out to investigate the interaction between V. cholerae and bacteriophage ICP1 both in vitro and in vivo in a naturalistic host, the zebrafish model, Danio rerio. To study the interplay between host, bacteria, and phages, we used a combination of light and ultrastructural imaging techniques, including confocal fluorescence microscopy, serial block face scanning electron microscopy (EM) imaging, and cryogenic EM, which allowed us to investigate both the colonization process by V. cholerae and clearance by the ICP1 bacteriophage. In addition, we determined the effects of the microbiome on this treatment by using germ-free, conventionalized, and monoassociated zebrafish larvae as a host. Independent of the presence and composition of microbiomes used here, V. cholerae efficiently colonized the larval intestine. Finally, we demonstrate significant in vivo clearance of V. cholerae N16961-dsRED by ICP1, underscoring the role of phage-bacteria dynamics in shaping pathogen colonization within the zebrafish larval host. Cholera remains a life-threatening disease that causes recurring outbreaks and significant mortality, particularly in developing and conflict-affected regions. As antimicrobial resistance continues to rise, there is an urgent need to better understand the ecological and microbial dynamics that govern Vibrio cholerae colonization and persistence. This research investigates how V. cholerae interacts with bacteriophages, the host environment, and the resident microbiota within a natural vertebrate host, offering new insights into the factors that influence pathogen clearance and shaping of the gut ecosystem during infection. The powerful combination of serial block-face scanning and cryogenic electron microscopy, fluorescence microscopy, and traditional colony/plaque counting methods revealed previously unobserved aspects of the interplay between host, pathogen, phages, and selected microsymbionts, highlighting phage-driven clearance of V. cholerae during colonization.

Three novel polyketides, talaroindiones A-C (1-3), along with two known diphenyl ether derivatives (4 and 5) were isolated from the rice culture medium extract of the marine-derived fungus Talaromyces indigoticus CS-469. The structures of 1-3 were determined by comprehensive spectroscopic analysis and confirmed by single-crystal x-ray diffraction. Their absolute configurations were assigned using TDDFT-ECD calculations, while those of 4 and 5 were established by comparison of their experimental optical rotations with literature values. All compounds were evaluated for their antibacterial activity. Compound 4 exhibited broad-spectrum inhibitory activities against six bacterial strains, showing particularly potent activity against Vibrio harveyi and Vibrio parahaemolyticus with MIC values of 2 &#xb5;g/mL.

Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top priority bacterium in the fight against antimicrobial resistance. Its high propensity to develop resistance, its high virulence, and its ability to form biofilms and persist intracellularly result in difficult-to-treat infections against, which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic, intracellular, and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibited a bactericidal effect on extracellular bacteria with an MIC similar to that of vancomycin; iBPOX mainly inhibited intracellular replication, while HPOX strongly impaired initial biofilm formation. These results prompted us to identify the potential target enzymes of the three OXs via activity-based protein profiling, combined with mass spectrometry. The antibiofilm HPOX compound was indeed found to primarily react with enzymes involved in biofilm formation and associated virulence, while iBPOX and the most active MpPPOX inhibitor targeted multiple (Ser/Cys)-based enzymes. Among these, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multitarget nature of the OXs, which covalently bind to several (Ser/Cys)-based enzymes of interest. Such property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial agents, notably by improving the antibiofilm activity of ineffective or poorly active drugs.

Selenoproteins, a unique class of proteins critical for cellular antioxidant defense, are characterized by the incorporation of selenocysteine (Sec) in their active sites. Sec is co-translationally inserted into proteins via a specialized mechanism that reprograms the UGA codon to encode Sec, involving a specific RNA structure designated the Sec insertion sequence (SECIS) element and several essential enzymes. Although numerous selenoproteins have been identified in prokaryotes (primarily bacteria), the detection of selenoprotein genes in these organisms remains challenging, largely due to difficulties in distinguishing the Sec-encoding UGA codon from standard termination signals. In recent years, computational approaches for predicting selenoprotein genes, along with comparative genomic analyses of Sec-encoding machinery and selenoproteomes, have emerged as a promising and rapidly evolving field, offering new insights into Sec utilization in bacteria and archaea. This review provides a comprehensive overview of the latest advancements in the study of selenoproteins in prokaryotes. We summarize the molecular mechanisms underlying Sec biosynthesis and incorporation, and the structural diversity of SECIS elements in bacteria and archaea. We then describe current computational strategies for the identification of prokaryotic selenoprotein genes and present an updated, extensive catalog of prokaryotic selenoproteins documented to date, emphasizing those with well-established functions. Finally, we discuss recent progress in understanding the evolutionary dynamics of the Sec-encoding system and selenoproteins across prokaryotes, with a focus on the archaea-to-eukaryote transition of Sec machinery and selenoproteins. Overall, this review offers a unified perspective on the identification, functions, and evolution of selenoproteins in prokaryotes.

The potential occurrence of zinc oxide nanoparticles (ZnONPs) within healthcare and sanitary products labelled as containing bulk ZnO has become an emerging issue, due to their controversial biotoxicity derived from their recently reported enzyme-like behaviour. To monitor and shed light on it, a multidisciplinary approach has been devised in the present work. Firstly, biotoxicity tests of bulk ZnO and ZnONPs were performed on nine different microorganism strains (yeasts and bacteria), characteristic pathogen and commensal human microbiota inhabiting affected human tissues. Secondly, assorted commercial healthcare and sanitary products containing bulk ZnO (with undeclared ZnONPs) were analyzed by single particle inductively coupled plasma mass spectrometry (spICP-MS). With this aim, an ultrasonic probe-assisted extraction using Triton X-100 1% was applied, and commercial ZnONP powder was used as an internal quality control material. The evaluation of analytical performance features in terms of trueness (particle number concentration and equivalent size), precision, and limits of detection in size and concentration proves the suitability of the methodology regarding preservation of native size, size distribution, and number of NPs. Biotoxicity assays evidenced higher inhibition halos for ZnONPs than for bulk ZnO with a clear size-dependent NPs behaviour and substantial variability in bacterial susceptibility. Minimum inhibitory concentrations (MIC) needed to act on pathogens resulted clearly able to compromise commensal microbiota. Hence, the obtained findings reveal potential toxicity risks and the need to control them using appropriate tools, as some commercial samples presented ZnONP percentages outside of a safety scenario, as well as the advisability of reassessing the legal framework.

Pseudomonas aeruginosa is a prevalent multidrug-resistant&#xa0;pathogen responsible for severe infections. Rapid, ultrasensitive, and selective detection of this bacterium is essential for effective clinical diagnostics and infection control. Here, we report a novel dual-mode biosensing platform based on two-dimensional metal-organic framework-stabilized iron nanoclusters (MOF-Fe NCs) for the simultaneous electrochemical and colorimetric detection of P. aeruginosa. The MOF-Fe NCs display synergistic properties, including exceptional electrocatalytic activity and robust peroxidase-like (POD-like) behavior. Specific capture of the target bacteria by a surface-immobilized F23 aptamer modulates electrochemical signaling and suppresses the POD-like activity for colorimetric readout. The dual-mode bioassay achieves a broad detection range from 101 to 108 CFU/mL, with detection limits of 1.7 CFU/mL (electrochemical) and 1.0 CFU/mL (colorimetric), and demonstrates high selectivity against other bacterial species. This work provides a robust, self-validating sensing strategy with significant potential for point-of-care diagnostics and real-time infection monitoring.

Candida albicans is a common opportunistic fungal pathogen that asymptomatically colonizes most humans. Although typically a benign commensal, dysbiosis caused by antibiotic use, immune dysfunction, or epithelial barrier disruption can trigger fungal overgrowth and infection, ranging from superficial mucosal disease to life-threatening systemic candidiasis. New preclinical infection models are needed to dissect C. albicans pathogenesis in vivo across distinct infection stages and with different measurable host outcomes. The planarian Schmidtea mediterranea was previously established as an invertebrate host for studying host-pathogen interactions during C. albicans infection. S. mediterranea relies entirely on conserved innate immune mechanisms capable of overcoming infection with pathogenic microorganisms, including bacteria and fungi. Planarians' remarkable regenerative capacity and accessible stem cell populations make this organism a tractable model to analyze early immune responses, tissue repair, and pathogen clearance in vivo. This model supports simultaneous analysis of fungal virulence and host transcriptional responses, providing valuable insights into infection dynamics. Here, an updated protocol with detailed modifications, standardized procedures, and optimized steps for infecting S. mediterranea with C. albicans has been presented, designed to enhance reproducibility and enable systematic studies of fungal pathogenesis and host defense.

Long-term biodegradation of soil microplastics such as polyethylene terephthalate (PET) in situ remains inadequately addressed due to the limited expression of efficient PET degrading enzymes in engineered bacteria. Here, we developed a quorum-sensing (QS)-based protein expression system (XylS-LuxI/LuxR) that enhanced reporter green fluorescent protein (GFP) expression by 44-fold in Escherichia coli (E. coli). Using this system, we constructed whole-cell PET biodegraders expressing PET hydrolases (FASTPETase-MHETase) and leaf-branch compost cutinase (LCCICCG) in E. coli and Pseudomonas putida (P. putida). Soil-based assays using crude enzymes and E. coli XylS-QS-LCCICCG cells showed >80% degradation of bis(2-hydroxyethyl) terephthalate (BHET) within 30 min. Furthermore, Agde-LCCICCG was identified as the most effective signal peptide (SP) for protein secretion in E. coli, whereas LCCICCG without a SP performed best in P. putida. Engineered E. coli achieved up to 63% PET nanoparticle degradation over 30 days, while P. putida reached 42.3% within 20 days in nonsterilized soil, substantially outperforming wild-type controls and indicating synergistic interactions with native microbiota. These results demonstrate that XylS-QS-based systems enable efficient, self-regulated whole-cell PET biodegradation in soil environments, providing new insights for the development of efficient biodegradation strategies of environmental plastic waste.

A histological safety profile is essential for non-antibiotic microbial interventions in aquaculture. This study assessed the multi-organ histological biocompatibility of a novel actinobacterial strain, Agrococcus sp. RKDAS_1, in Nile tilapia (Oreochromis niloticus) under controlled conditions. Juvenile tilapia were fed diets with varying concentrations of RKDAS_1 (105, 107, and 109 CFU g- 1 feed) for 60 days. Tissues from the gill, intestine, liver, and heart were analysed through standard histological methods, employing semiquantitative scoring and intestinal morphometry. Across all examined organs, RKDAS_1 supplementation did not induce inflammatory responses, degenerative lesions, or structural disruptions, which are indicative of tissue-level toxicity. Gill architecture was intact, with normal hepatocyte arrangements and no necrosis or fibrosis, while cardiac tissues showed a normal structure. Intestinal morphology maintained epithelial integrity, displaying dose-related variations in villus height and goblet cell density. Intermediate-dose live hepatic sections showed reduced cytoplasmic vacuolation compared to controls, though the difference was not statistically significant. The semi-quantitative histopathological evaluation showed that the tissue structure remained intact across different treatments. The results collectively suggest that Agrococcus sp. RKDAS_1 did not cause any noticeable histopathological damage, indicating enhanced biocompatibility. The findings suggest that Agrococcus sp. RKDAS_1 is compatible with biological tissues without causing significant damage. However, these conclusions are restricted to structural analysis and do not ensure functional improvements or overall safety beyond tissue examination. The findings provide a critical starting point for future research, which will delve into molecular, immunological, and long-term exposure studies. These investigations aim to evaluate the biotherapeutic potential of RKDAS_1 thoroughly.

Traditional assessments of poultry litter microbiology and physicochemical composition often rely on static tables or two-dimensional visualizations that may not capture the spatial heterogeneity within the pen environment. This protocol describes interactive three-dimensional (3D) visualizations that integrate microbial enumeration data and environmental parameters across a poultry pen. A grid-based layout was used to approximate pen sampling, and simulated datasets were first generated to demonstrate the workflow. The experimental dataset was used to evaluate spatial gradients relative to environmental features, including the water line, feeders, and pen entrance. Data were processed in RStudio using plotly to generate interactive 3D plots. Aerobic bacterial loads ranged from 5.9 to 8.6 log&#x2081;&#x2080; CFU/g, with significantly higher abundance near the water line (P = 0.023) and lower counts at increasing distance from the feature. The resulting surface plots visually highlighted clustering of microbial populations around moisture-rich areas and demonstrated the utility of the framework for interpreting spatial patterns of microbial communities in poultry litter. Although further evaluation under commercial poultry conditions is needed, the current protocol provides a reproducible method for visualizing and analyzing spatial heterogeneity in poultry litter datasets.