Search Results (94)

Several strategies can be employed for the identification of novel microbial lipases. Despite the increasing importance of metagenomics in bioprospecting, significant limitations in the expression of recombinant proteins, and lipases in particular, remain. Culture-based bioprospecting approaches are, therefore, still valuable. In this work, a collection of bacterial isolates, mainly of marine origin, was screened for lipase activity through a culture-based approach. Screening for lipolytic bacteria was performed in solid media containing olive oil emulsions and rhodamine B. Positive isolates were subsequently grown in liquid media, to confirm lipase production. Significant hydrolytic activity towards the triglyceride substrates tributyrin and triolein could be observed with the biomass produced, although no lipase activity could be detected in the culture supernatants. Six isolates presenting high activity were characterized as whole-cell biocatalysts, and all were found to be active at temperatures ranging between 25 and 65 °C, and at pH values between 6 and 10.5. Genomic analyses of two of these Gram-negative lipase-producing isolates revealed the presence of several hypothetical genes encoding for lipolytic enzymes, including outer cell-bound enzymes, predicted through the application of machine-learning tools. These natural isolates, containing cell-associated lipases, may therefore be of special interest for application as whole-cell biocatalysts. Full article

Multidrug-resistant Acinetobacter baumannii is a major nosocomial pathogen for which bacteriophages are being explored as alternative antibacterial agents. In this study, we isolated and characterized AUBFM01, a lytic phage active against MDR A. baumannii, and performed an initial assessment of its interaction with PMA-differentiated THP-1 macrophages. AUBFM01 was evaluated by host range testing, adsorption and one-step growth assays, lytic activity, stability testing, biofilm disruption, whole-genome sequencing, and flow cytometry-based macrophage profiling. The phage showed rapid adsorption, a short latent period of approximately 30 min, and a burst size of about 165 phage particles per infected cell. It remained stable under moderate temperature and near-neutral pH conditions and significantly reduced preformed A. baumannii biofilm biomass in vitro. Genomic analysis identified a 41,354-bp double-stranded DNA genome lacking detectable lysogeny-associated genes, antibiotic resistance determinants, and known bacterial virulence factors. In THP-1 macrophages, AUBFM01 exposure was associated with reduced cell viability and with enrichment of a resting/intermediate-like CD86-defined phenotype among the remaining cells, including after endotoxin reduction. These findings identify AUBFM01 as a lytic anti-Acinetobacter phage with antibiofilm activity and notable macrophage-associated effects that warrant further mechanistic and safety investigation. Full article

Members of the genus Brucella are bacterial pathogens of global importance, and their increasing detection in marine mammals has raised concerns for wildlife conservation and public health. In this study, we evaluated the biological and pathogenic characteristics of two Brucella sp. sequence type 27 (ST27) isolates obtained from a dwarf sperm whale (Kogia sima). We compared them with terrestrial and marine Brucella reference strains. We assessed resistance to polymyxin B and human serum complement, intracellular infection dynamics in HeLa epithelial cells, persistence in a murine model, and associated hematological and histopathological changes, and analyzed lipopolysaccharide (LPS) profiles. The Kogia isolates exhibited resistance to polymyxin B and serum complement, comparable to that of B. abortus 2308W and marine mammal Brucella strains. In HeLa cells, the isolates displayed distinct, strain-specific intracellular infection dynamics. In the murine model, both isolates persisted in the spleen and induced granulomatous lesions. However, splenic bacterial loads and histopathological scores were generally lower than those observed with B. abortus 2308W, which exhibited the highest virulence among the strains evaluated. Hematological alterations associated with Kogia isolates were also less pronounced than those induced by B. abortus 2308W, indicating an intermediate and strain-dependent virulence phenotype without evidence of enhanced virulence relative to the terrestrial reference strain. Western blot analyses showed that Brucella sp. ST27 isolates were not recognized by anti-B. abortus or anti-O-antigen monoclonal antibodies, while exhibiting a distinct recognition pattern with anti-B. canis serum, indicating differences in surface antigen composition. Comparative whole-genome analysis identified a limited number of isolate-specific variants affecting coding and intergenic regions. Collectively, these findings highlight phenotypic and genetic features of Brucella sp. ST27 from Kogia sima, which distinguishes it from other marine and terrestrial Brucella strains and supports further investigation into its biological behavior and potential public health relevance. Full article

Brucella infection is frequently acquired by inhalation, but the pathogen disseminates systemically from the lungs. However, little is known about the interaction of Brucella spp. with the airways. Using a 3D air-exposed organotypic human bronchial tissue model (polarized 16HBE14o- bronchial epithelial cells grown over a collagen matrix containing MRC-5 lung fibroblasts), we analyzed Brucella abortus replication, translocation and cytokine responses over prolonged post-infection times. Apically inoculated B. abortus invaded, replicated and persisted during the whole follow-up (16 days) within the bronchial tissue without inducing cytotoxicity. Viable bacteria were also detected in the conditioned medium (CM) since day five post-infection, indicating release from the basolateral side. In parallel experiments, no invasion or bacterial release was detected for Escherichia coli. The levels of IL-6, IL-8 and MCP-1 were increased in CM from Brucella-infected 3D cultures and in monocultures of polarized bronchial epithelial cells or lung fibroblasts. Collagenase/gelatinase activity was increased in 3D cultures and MRC-5 monocultures. Infection transference from bronchial cells to lung fibroblasts was documented using monocultures. An immune cross-talk was detected, as cytokine levels were increased in fibroblasts stimulated with bronchial CM, and vice versa. These results suggest that the bronchial mucosa can sustain B. abortus persistence, replication and dissemination, and that it induces a proinflammatory response to which both epithelial cells and fibroblasts contribute. Full article

Salmonella is a major bacterial pathogen in low-income countries, where it circulates among humans, animals, and the environment. Children with sickle cell disease (SCD) are particularly vulnerable to severe Salmonella infections. This study aimed to characterize Salmonella isolates causing infections in Senegalese children with SCD. Using antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatic analysis, we investigated antibiotic resistance, serovar diversity, and virulence factors on 23 isolates from SCD patients with diverse clinical infections. The isolates belonged to 12 serovars, with Enteritidis predominating (n = 7). Twenty-two isolates were fully susceptible to antibiotics, while one was multidrug-resistant. Eight isolates (Enteritidis and Typhimurium) carried a virulence plasmid harboring the spvRABCD gene cluster. Core Salmonella pathogenicity islands (SPIs-1 to -5, -11, and -13), as well as SPI-10 and SPI-23, were detected in all isolates, whereas other SPIs were variably present. These results show high serovar diversity and low antimicrobial resistance among Salmonella isolates in children with SCD in Dakar, Senegal. Our findings suggest that strains causing diarrhea in healthy individuals may also cause invasive disease in SCD patients, highlighting the need for dedicated surveillance in this vulnerable population. Full article

Background: Killed Whole Cell Genome-Reduced Bacteria (KWC/GRB), a versatile vaccine platform, can produce very low cost, thermostable, easily manufactured vaccines expressing complex immunogens that include potent immunomodulators. This system supports iterative optimization through a Design–Build–Test–Learn (DBTL) workflow aimed at enhancing immunogenicity. We applied this approach to developing HIV-1 gp41 Membrane-Proximal External Region (MPER) vaccines using the scaffolded MPER antigen, 3AGJ, a recombinant heterologous protein engineered to mimic MPER structures recognized by broadly neutralizing monoclonal antibodies (bNAbs). Methods: Five KWC/GRB vaccines expressing versions of 3AGJ were designed, including versions linked to immunomodulators and multimers of the immunogen. Display on the surface of the bacteria was evaluated by flow cytometry using the broadly neutralizing monoclonal antibody 2F5. Outbred HET3 mice were vaccinated intramuscularly, and MPER-specific antibody responses were assessed by ELISA and by the ability of the vaccines to induce neutralizing antibodies. Neutralization was measured against tier 1 and tier 2 HIV-1 pseudoviruses. Results: All five vaccines were strongly expressed on the bacterial surface and induced clear MPER-specific antibody responses in every mouse. About 33% of the animals showed detectable HIV-1 neutralization. Conclusions: These results demonstrate that a KWC/GRB-based scaffold-MPER (3AGJ) vaccine can elicit HIV-1 neutralizing antibodies in a subset of animals. Although further optimization will be required to improve the consistency and magnitude of neutralizing responses, the findings provide an initial validation of the concept. There are many strategies that can be used to enhance and extend immune responses induced by KWC/GRB vaccines that can be employed to yield improved anti-HIV-1 immune responses. Full article

Lumpy Skin Disease (LSD) is a transboundary bovine viral disease. It has a significant economic impact and is caused by the Lumpy Skin Disease Virus (LSDV). Effective surveillance tools are essential for the early detection of infection, outbreak control, and assessment of vaccination coverage in endemic regions such as India. In this study, an in-house ELISA based on inactivated whole-virus antigen (WVA) was developed, optimized, and validated for the detection of LSDV antibodies in cattle. Its field applicability was assessed through a cross-sectional seroprevalence survey conducted across five Indian states. A local field isolate of LSDV (strain 5-Chitra) was cultured in MDBK cells, inactivated using binary ethylenimine (BEI), and used as the antigen source. The assay was optimized by checkerboard titration and evaluated against the Serum Neutralization Test (SNT). Diagnostic sensitivity and specificity were evaluated using the receiver operating characteristic (ROC) curve and area under the curve (AUC) analyses, while cross-reactivity was assessed using sera positive for HS, IBR, BQ, MCF, GTP, SPP, CE, FMD, and Brucellosis. Assay reproducibility was confirmed through inter- and intra-laboratory validation. For the seroprevalence study, 3230 cattle serum samples were collected using a stratified random sampling design across five Indian states, and logistic regression analysis of a subset of 1302 samples was performed to assess the influence of age and sex on LSDV seropositivity. Checkerboard titration identified optimal ELISA conditions at 50 ng of antigen per well, a 1:150 serum dilution, and a 1:10,000 dilution of anti-bovine HRP-conjugated secondary antibody. The WVA-ELISA demonstrated excellent diagnostic performance, with 100% sensitivity, 95% specificity, and no cross-reactivity with other ruminant bacterial or viral pathogens, and showed high laboratory reproducibility (κ > 0.96). Seroprevalence ranged from 50.6% to 71.1% across the five states, indicating widespread exposure to LSDV. Risk factor analysis revealed significantly higher odds of seropositivity among calves (≤1 year old) and female cattle, suggesting age- and sex-dependent susceptibility. Full article

Hibernation profoundly alters host–pathogen dynamics by suppressing metabolism and immune function, posing unique challenges for infection control. In this study, we examined how genomic variation modulates infection and physiological traits in temperate bats during hibernation. We combined infection screening, haematology, blood biochemistry, and whole-genome sequencing across five vespertilionid species, identifying over 170,000 single nucleotide variants (SNVs) and assessing their associations with 23 health-related variables. Using the phylogenetically informed treeWAS framework, we detected 515 significant SNVs linked to traits including fungal, protozoan and bacterial infections, acid–base balance, and blood cell indices. These SNVs mapped to 137 unique genes, which were enriched for functional domains related to cytoskeletal dynamics, membrane trafficking, and intracellular signalling (e.g., SH3, C2, BAR, semaphorin). Notably, canonical immune effector genes were underrepresented, and several trait-associated SNVs appeared in blocks across multiple scaffolds, pointing to regulatory loci as key modulators of hibernator health. Our findings support the hypothesis that bats rely on infection tolerance rather than resistance during hibernation, with genomic variation in regulatory and signalling pathways shaping their physiological responses to infection under energy-limited conditions. Full article

Jeju Island, a volcanic island located off the southern coast of the Korean Peninsula, harbors highly specialized microbial communities shaped by its unique geological and climatic diversity. In particular, Baengnokdam Crater Lake at the summit of Mt. Halla represents an extreme, oligotrophic volcanic habitat characterized by intense UV radiation, temperature fluctuations, and limited nutrients. From this environment, a novel bacterial strain, Brevibacillus sp. JNUCC 42, was isolated and subjected to comprehensive taxonomic, genomic, and biochemical analyses. The strain is a Gram-positive, aerobic, rod-shaped bacterium that grows optimally at 30 °C and pH 7.0–9.0 with moderate NaCl tolerance (≤3%). Phylogenetic analysis based on 16S rRNA gene sequencing and genome-scale GBDP confirmed its affiliation to the genus Brevibacillus, forming a distinct lineage closely related to B. laterosporus DSM 25^T. Whole-genome sequencing generated a 4.93 Mb circular chromosome with a GC content of 40.7%. Comparative genomic analyses revealed ANI (87.1%) and dDDH (32.8%) values far below the species threshold, supporting its delineation as a novel species. Chemotaxonomic data further distinguished JNUCC 42 by its predominance of anteiso-C₁₅:₀ (37.24%) and iso-C₁₅:₀ (27.78%) fatty acids and the presence of a unique unidentified aminolipid not detected in the type strain. Genome mining identified 21 biosynthetic gene clusters, including NRPS, PKS, and NRPS–PKS hybrids, suggesting its potential to produce structurally diverse secondary metabolites. One of these metabolites, the cyclic dipeptide maculosin [cyclo(L-Pro-L-Tyr)], was purified from the culture extract and structurally characterized by NMR spectroscopy. Functional assays demonstrated that maculosin significantly inhibited α-MSH-induced melanogenesis and intracellular tyrosinase activity in B16F10 melanoma cells without cytotoxicity up to 100 µM. Collectively, these findings indicate that Brevibacillus sp. JNUCC 42 represents a novel species within the genus Brevibacillus and a promising microbial source of bioactive compounds with potential cosmeceutical applications. Full article

With the aim of developing a new tool to meet the increasing demand for food safety, a whole-cell-based system able to detect the presence of cobalt contamination along the pasta production chain was constructed. This system is based on bacterial cells engineered with a plasmid containing the eGFP gene under the control of a promoter sequence, and is able to elicit a fluorescence signal when activated. The promoters of four stress-responsive genes (DnaK, GroE, UspA, and ZntA) were used to test their responsiveness to cobalt; the promoter of the UspA gene, coding for a universal stress protein, was chosen. The UspA promoter was activated by cobalt, and the system described was highly sensitive, successfully detecting low concentrations of cobalt within complex food matrices derived from durum wheat seeds when exogenous cobalt was added. In food matrices tested alone, a fluorescence signal was present only in bran and fine bran, confirming that these parts of the wheat seed are the ones in which contaminants accumulate. Conversely, in the other matrices derived from the inner part of grains, no signal was detected. The findings reported contribute to the development a new, effective and sensitive tool for monitoring cobalt contamination, offering a valuable approach to enhance food safety control. Full article

Foodborne illnesses associated with Bacillus cereus represent a persistent public health concern. In this study, we described the isolation and characterization of a novel bacteriophage, ΦBc24, from mud samples, which showed lytic activity against foodborne pathogen B. cereus. Transmission electron microscopy revealed that ΦBc24 exhibited a myovirus morphotype. Biological assays demonstrated that its narrow host range was restricted to B. cereus strains and efficient lytic activity, characterized by a latent period of 10 min and a burst size of 40 PFU per infected cell. The phage exhibited high physicochemical stability, tolerating pH values of 2–12, temperatures of 4–50 °C, salinity up to 1 M NaCl, and ultraviolet exposure, while effectively suppressing host bacterial growth for up to six hours. Whole-genome sequencing showed that phage ΦBc24 possessed a double-stranded DNA genome of 160,311 bp, with 39.48% GC content, and 269 predicted open reading frames (ORFs). Remarkably, 11 tRNA genes were identified, whereas no genes associated with lysogeny, virulence, or antimicrobial resistance were detected. Phylogenetic analysis suggested that ΦBc24 belongs to the genus Caeruleovirus, subfamily Bastillevirinae, family Herelleviridae. Taken together, these results highlight the biological robustness and genomic safety of ΦBc24, supporting its potential as a biocontrol candidate against the foodborne pathogen B. cereus. Full article

Periodontitis (PD) is a multifactorial, progressive inflammatory disease affecting the teeth-supporting tissues, characterized by an imbalance of the oral microbiota and the presence of bacterial biofilms leading to host response. Nowadays, reliable biochemical markers for early and objective diagnosis, and for predicting disease progression, are still lacking. Our previous proteomic investigations revealed the significant overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in periodontal pocket tissue, gingival crevicular fluid (GCF), and tooth-surface-collected material (TSCM) from PD patients in comparison to periodontally healthy controls, proposing it as a possible biomarker of PD. This study aimed to evaluate the expression of GAPDH in saliva, a more accessible, non-invasive, and clinically relevant oral sample. The whole saliva was analyzed by a preliminary mass spectrometry-based proteomic approach, identifying significantly increased levels of GAPDH also in salivary samples from periodontal-affected subjects. These data were further validated by enzyme-linked-immunosorbent assay (ELISA). Additionally, protein–protein interaction networks were generated through the Human Protein Atlas database, using different datasets (OpenCell, IntAct, and BioGRID). Bioinformatic analysis provided noteworthy GAPDH-associated networks potentially relevant to periodontal pathology. The scientific significance of this study lies in the detection of salivary GAPDH as a novel strategy to advance periodontal clinical diagnostics from the perspective of a non-invasive screening test. In correlation with other protein markers, salivary GAPDH could constitute a promising set of distinctive and predictive targets to enhance early diagnosis of PD, disease monitoring, and treatment planning in periodontology. Full article

Background/Objectives: Nocardia seriolae and Aeromonas veronii are two important pathogens that can affect a wide range of fish species and cause substantial economic losses. However, a vaccine that simultaneously protects fish from these two bacterial infections is not yet available. Methods: Three formalin-inactivated whole-cell vaccines prepared from N. seriolae and A. veronii (Monovalent Av, Monovalent Ns and Bivalent Av-Ns) were generated, and their efficacy was evaluated through a range of tests. The immune-related gene expression in the spleen and head kidney, enzyme activity, and specific antibody levels in serum were also detected. Results: All groups of vaccinated fish exhibited increased serum enzymatic activity compared with control fish, which peaked at week 3 after vaccination; in particular, that of the Bivalent Av-Ns group increased remarkably. The expression of immune-related genes in the spleen, head, and kidneys increased after immunization and were significantly enhanced (p < 0.05) in the bivalent vaccine group. Specific antibodies were produced at the 1st wpv, peaked at the 4th to the 5th wpv, and then decreased at the 6th wpv in all vaccinated groups. The Monovalent Av and Monovalent Ns against A. veronii and N. seriolae showed 56.67% and 22.22% RPS, respectively. Moreover, Bivalent Av-Ns offered 33.33% and 76.67% RPS for single infection with N. seriolae or A. veronii, as well as providing 44.44% RPS for dual infection with combined N. seriolae and A. veronii. Conclusions: Our findings indicate that the administration of the A. veronii and N. seriolae bivalent vaccine can protect largemouth bass from both bacterial infections. Full article

Pseudomonas aeruginosa is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we developed a whole-cell aptamer selection method for P. aeruginosa using an Eppendorf-tube-based SELEX system, where bacterial cells were directly incubated with an ssDNA library. This configuration enhanced the recovery of bound aptamers and overcame the cell quantity limitations often encountered in microtiter-plate-based SELEX. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. Molecular docking analysis revealed that aptamer T1 possessed the highest target selectivity. To demonstrate diagnostic applicability, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the conventional detection antibody. In this format, the AuNP–aptamer complex bound to the target bacteria and was captured by a specific antibody immobilized on the test line. The LFIA achieved a visual detection limit of 2.34 × 10² CFU/mL within 15 min, showing high specificity and suitability for point-of-care applications. This study presents the first demonstration of an aptamer–antibody hybrid LFIA for bacterial detection and highlights the potential of aptamers as low-cost, rapidly synthesized recognition elements adaptable for the detection of other infectious agents. Full article

Introduction: X-linked agammaglobulinemia (XLA) is a prototypical inborn error of immunity (IEI) caused by mutations in the BTK gene, leading to a profound deficiency of mature B cells and severe pan-hypogammaglobulinemia. The Epstein-Barr virus (EBV), which primarily infects B lymphocytes, is believed to be unable to establish persistence in these patients due to the lack of its natural reservoir. Indeed, current evidence supports that EBV infection is typically refractory in individuals with XLA. Methods: We describe the clinical and molecular characterization of a 10-year-old male patient with genetically confirmed XLA who developed EBV viremia, hemophagocytic lymphohistiocytosis (HLH), and EBV-positive cutaneous T cell lymphoma. Diagnosis was supported by flow cytometry, serology, quantitative PCR, EBER in situ hybridization, histopathology, and whole-exome sequencing. Results: Despite the complete absence of peripheral B cells, EBV was detected in leukocytes and multiple tissues, indicating active infection. The patient developed HLH and a T cell lymphoma with EBER-positive infiltrates. Genetic analysis revealed a nonsense mutation in BTK (1558C>T, R520*), confirming XLA. The clinical course included multiple episodes of neutropenia, viral and bacterial infections, and severe systemic inflammation. Conclusions: This is the first documented case of an XLA patient with confirmed BTK mutation presenting with clinical features more consistent with chronic active EBV infection. These findings challenge the prevailing paradigm that XLA confers protection against EBV-related diseases and further support the possibility of EBV noncanonical reservoirs leading to immune dysregulation. EBV should also be considered in the differential diagnosis of XLA patients presenting with systemic inflammation or lymphoproliferative disease. Full article