Antibiotic resistance represents a critical global health challenge driven by the widespread misuse of antimicrobials and the rapid evolution of multidrug-resistant pathogens. Conventional antibiotics are increasingly limited by poor stability, low bioavailability, toxicity, and reduced efficacy. This study investigates the potential of carbon nanotube-based nanocarriers to enhance antibiotic delivery and efficacy against resistant bacteria. Multi-walled carbon nanotubes were oxidized and functionalized with glucosamine, followed by loading with two antibiotics, ethacridine lactate and sulfamethoxazole, to form nanoconjugates. Characterization studies confirmed successful functionalization and drug loading. Thermogravimetric analysis indicated distinct weight losses corresponding to surface modifications, while Fourier transform infrared spectra verified amide bond formation and drug-nanocarrier interactions. Scanning electron microscopy revealed increased surface roughness and structural defects after functionalization. UV–Vis spectroscopy demonstrated high encapsulation efficiencies (85.37
Brazilian coffee production relies heavily on mineral fertilizers, particularly nitrogen-based inputs. However, the intensive use of these fertilizers is associated with nutrient losses, elevated production costs, and environmental impacts, highlighting the need for more sustainable alternatives. In this context, plant growth-promoting bacteria (PGPB) represent promising resources for improving nutrient use efficiency and reducing dependence on synthetic inputs. Nevertheless, the cultivable bacterial fraction associated with coffee soils remains poorly characterized under contrasting fertilization management systems. The objectives of this study were to isolate, identify, and characterize cultivable bacterial isolates recovered from Coffea arabica soils managed under conventional mineral fertilization (CMF) and organic fertilization, and to screen them for plant growth-promoting traits. Thirty-eight bacterial isolates were recovered and identified by Sanger sequencing of the 16S rRNA gene. Both fertilization systems shared dominant bacterial groups, although each management harbored exclusive bacterial genera. Eleven bacterial isolates from organic fertilization and nine from CMF exhibited growth in nitrogen-free semi-solid NFb medium. All isolates recovered from organic fertilization and several isolates from CMF solubilized tricalcium phosphate. Furthermore, no significant differences in indole-acetic acid production were observed among the bacterial isolates recovered from both fertilization systems. Overall, the cultivable bacterial fraction associated with both management systems exhibited considerable functional diversity and included multifunctional isolates with plant growth-promoting traits. These findings expand current knowledge of cultivable bacterial diversity in coffee soils and highlight the native microbiota as a promising source of candidate microorganisms for future microbial inoculants and microbial consortia adapted to sustainable coffee production.
In endosymbiosis, unicellular organisms, mostly prokaryotes, live inside the cells of a host that may be uni- or multicellular. More than half insect and arthropod species host the obligate endosymbiont Wolbachia spp., an alpha-proteobacterium that, given time, may coevolve with its host and progress from parasitism to strict mutualism, where an obligate endosymbiont avoids rejection by providing advantages to the host such as essential nutrients. It was recently found that in the wild yeasts may host both facultative and obligate endosymbionts. To model host-symbiont mutualist interactions in yeast, we tested two different Saccharomyces cerevisiae riboflavin-synthesis pathway deletion mutants (ΔRIB1 and ΔRIB4), finding that these failed to grow in the absence of exogenous riboflavin (RF), i.e., they were RF-auxotrophic strains. In both mutants Wolbachia infection cured auxotrophy. In addition, Wolbachia infection led to a mild increase in survival to exposure to 1 M NaCl or to 10 mM hydrogen peroxide. The artificial yeast/endosymbiont model proposed here may become a useful tool to explore parasitic, facultative and mutualist associations.
The global emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), particularly the ST11-KL64 subclone acquiring pLVPK-like virulence plasmids, represents a critical public health threat. This study investigates the epidemiological dominance and molecular mechanisms underlying ST11-KL64’s fitness advantage over KL47 variants. We performed comparative genomic analysis on 43,722 K. pneumoniae genomes (2011–2022) from 112 countries, focusing on ST11-CRKP strains. Capsular typing (KL64 vs. KL47), virulence gene profiling (aerobactin, RmpADC), and plasmid stability analysis were conducted using Kleborate, RAST, and PlasmidFinder. Plasmid-chromosome interactions were characterized through hybrid assembly approaches. ST11-KL64 demonstrated rapid expansion post-2016, surpassing KL47 as China’s dominant CRKP subtype (40.5
Pseudomonas aeruginosa is a major causative agent of burn wound infections, frequently characterised by multidrug resistance (MDR) and robust biofilm formation, which limit the efficacy of conventional antibiotics. 1,3,4-Oxadiazole derivatives have emerged as potential antimicrobial and antibiofilm agents; however, comprehensive evaluations against clinical MDR isolates from burn patients remain limited. The objective of this study was to investigate the in vitro antibacterial and antibiofilm activities of a synthesised 1,3,4-oxadiazole compound against clinical MDR P. aeruginosa isolates from burn wounds. In addition, the study sought to assess its synergistic potential with gentamicin and imipenem, and to examine its effects on biofilm-associated gene expression. A total of 75 non-duplicate clinical P. aeruginosa isolates were collected from patients admitted to educational hospitals in Hamedan, Iran, including 49 burn-wound isolates and 26 respiratory isolates. From the initial burn-wound collection, nine isolates were selected for detailed antimicrobial and antibiofilm analyses based on their clinical relevance and biofilm phenotype; these comprised seven MDR and two non-MDR isolates, all demonstrating strong or intermediate biofilm formation. Antimicrobial susceptibility and biofilm formation were assessed using the disk diffusion and microtiter crystal violet assays. MIC, MBC, MBIC, and MBEC values were determined for 1,3,4-oxadiazole, gentamicin, and imipenem. Checkerboard assays were performed to evaluate the combined effects of 1,3,4-oxadiazole with gentamicin or imipenem against planktonic and biofilm-associated cells. The effects of sub-inhibitory concentrations of 1,3,4-oxadiazole on lasR and algD expression were evaluated using real-time PCR. Among the 75 clinical isolates, 57 (76
Antibiotic resistance among Gram-negative bacteria, particularly those capable of forming biofilms, has become a serious clinical challenge with limited treatment options. In this study, magnesium oxide nanoparticles (MgO NPs) were synthesized using a green, cost-effective approach based on aqueous leaf extract of Cymbopogon citratus (lemongrass) and evaluated for their antibacterial and antibiofilm potential against multidrug resistant (MDR) clinical isolates of Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Minimum inhibitory concentration (MIC) values determined by broth microdilution ranged from 250 to 500 µg/ml, and minimum bactericidal concentration (MBC) values ranged from 1000 to 2000 µg/ml. Antibacterial activity assessed by the well diffusion method was dose-dependent, with inhibition zones ranging from 18 to 28 mm. The effect of MgO NPs on biofilm formation and eradication of pre-formed biofilms was evaluated using microtiter plate assay with crystal violet staining. Percentage inhibition of biofilm formation ranged from 27 to 97.1
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen frequently associated with multidrug-resistant nosocomial infections. The MexAB-OprM efflux system, a member of the Resistance-Nodulation-Division (RND) family, contributes to intrinsic and acquired resistance by extruding structurally diverse antimicrobial agents, including quinolones, from the bacterial cell. This study aimed to evaluate the association between quinolone resistance phenotypes and the presence of the mexAB-oprM determinant in clinical P. aeruginosa isolates, and to assess the functional contribution of efflux activity using the inhibitor PAβN. This cross-sectional descriptive study was performed on 55 samples of P. aeruginosa isolated from patients visiting the Martyr Zare hospital in the city of Sari, Iran. The genus and types of isolated strains were first characterized by biochemical tests. Antibiotic-resistance patterns of the strains were evaluated based on the disk diffusion method toward 13 common antibiotics and minimum inhibitory concentration (MIC) in the presence of the PAβN inhibitor of levofloxacin antibiotic-2 and ciprofloxacin. Finally, the presence of the mexAB-oprM operon was surveyed using the polymerase chain reaction (PCR) method. In this experiment, the highest bacterial resistance was observed toward nalidixic acid (100
The objectives of this study were to isolate, characterize, and evaluate the biocontrol potential of Staphylococcus aureus-targeting bacteriophages in milk. From wastewater and raw milk samples, a total of 19 lytic phages were isolated. Among them SAKAY1298 and SAKAY1306 phages were selected for detailed phenotypic and genomic analysis based on their lytic profiles, host range and growth kinetics. Both phages exhibited icosahedral capsids with short, non-contractile tails under transmission electron microscopy. The phages exhibited moderate acid tolerance by remaining stable at pH 4 and maintained thermal stability at temperatures up to 50 °C. They also maintained high titers over one year at + 4 °C and -80 °C in tryptic soy broth and Tris-buffered saline (1 ×). Genome analyses revealed that both phages possess complete genomes ( 17.5 kb), encoding 18 phage-related genes and lacking virulence and resistance determinants. Comparative and phylogenetic analyses confirmed assignment to the genus Rosenblumvirus, with 99
Exploring natural products in drug discovery can provide new options for effective treatments for infectious diseases. Biofilms are frequently not considered in most studies of antimicrobial activity, despite their relevance in microbial pathogenicity and resistance to antimicrobials. Here we investigated the antimicrobial and antibiofilm potentials of Ocotea odorifera, a Brazilian endangered species, against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. We assessed the activity of the extract using minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC) and minimal biofilm eradication concentration (MBEC) assays, and a skin infection model. We evaluated the cytotoxicity of the extract in BGM cells and assessed its anti-inflammatory potential. Phytochemical profiling was conducted using classical methods and UHPLC-HRMS/MS. The extract was active against the planktonic cells of all species (MIC = 8 µg/mL, variable MBC values), and eradicated biofilms of S. aureus (MBEC = 256 µg/mL). Phytochemical analysis indicated that erucamide was the most abundant compound in the sample, which is being described for this species for the first time along with other phytomolecules. The extract did not show cytotoxicity and presented anti-inflammatory activity comparable to meloxicam. Our data provides evidence on the antimicrobial activity of O. odorifera, especially its antibiofilm potential, and highlights the need of preservation of endangered species as a valuable source of bioactive metabolites of clinical interest. More studies are necessary to investigate these pharmacological properties in vivo.
To study the effect of hyperin on the bacterial community diversity and richness on the leaves surface of Burley tobacco, a variety of Burley tobacco 'Eyan 1' was used as the research material. The day before the harvest, all leaves in the treatment group were sprayed with hyperin, while all leaves in the control group were treated with pure water. Samples were collected on the day of harvesting, as well as two weeks and four weeks after air-curing. The results showed that the application of hyperin could potentially affect the bacterial community diversity and richness on the Burley tobacco leaves. Bacterial community structure, co-occurrence patterns, and predicted metabolic pathways were analyzed. Hyperin treatment was associated with increased relative abundance of several genera, including Agrobacterium, Stenotrophomonas, and Bacillus, which have been previously linked to nitrogen transformation or TSNA reduction. These microorganisms may contribute to TSNA reduction during air-curing, thereby reducing TSNA accumulation in the upper leaves of Burley tobacco. The results of this study can furnish evidence for investigating the impact of hyperin on the microbial community and deepen our understanding of the regulatory mechanisms of hyperin in Burley tobacco.
Hypertension (HTN) is a major global health burden and a leading risk factor for cardiovascular morbidity and mortality. Although numerous studies have explored host genetic factors and molecular mechanisms underlying HTN, increasing evidence indicates that gut microbiota dysbiosis also contributes to disease development. However, the specific microbial genes involved in HTN pathogenesis and their potential therapeutic targeting remain largely unexplored. This study aimed to identify HTN-associated differentially abundant bacterial genes (DAGs), prioritize bacterial key genes (bKGs) from among them, and repurpose potential therapeutic agents targeting these bKGs using an integrated bioinformatics framework. A total of 167 stool (fecal) microbiome samples, comprising 72 samples from HTN patients and 95 samples from HCs, were analyzed using publicly available 16 S rRNA sequencing data. After quality processing and clustering at 97
Antimicrobial resistance and the overuse of antimicrobials have driven the search for innovative therapies, particularly those based on natural compounds and medicinal plant derivatives, aiming to develop new drugs. This study aimed to investigate the antimicrobial and antibiofilm activities of crude ethanolic extract of O. campechianum (CEE-OC), and commercial (AgNPs-C), non-commercial (AgNPs-NC), and Ocimum campechianum-biosynthesized (AgNPs-OC) silver nanoparticles, against 21 Staphylococcus spp. isolates. Antimicrobial activity of rosmarinic acid (RA) against six isolates was also determined. The minimum inhibitory concentration (MIC) of CEE-OC ranged from 6,250 to 781.25 µg/mL, showing the highest antimicrobial activity among the substances evaluated. AgNPs-OC exhibited MIC values approximately fourfold lower than those of CEE-OC. In contrast, AgNPs-C and AgNPs-NC showed no antimicrobial activity at the concentrations tested. CEE-OC and AgNPs-OC inhibited biofilm formation but had no effect on established biofilms. Molecular docking analysis suggested a potential interaction between RA and the NorA efflux pump protein. The results indicated that O. Campechianum extract and biosynthesized silver nanoparticles may represent promising antimicrobial alternatives, although further studies are needed to elucidate the mechanisms underlying their activity.
Bacterial communities play important roles in the plant phyllosphere. Both microbial communities and their hosts exhibit endogenous circadian rhythms while simultaneously responding to environmental changes across the diurnal cycle. However, the interaction between the host and microbiome is still poorly understood. Here, we exploit paired sequencing data of host transcriptome and microbiome derived from diverse maize genotypes in field conditions and under two contrasting diurnal periods. Expression patterns of known maize circadian clock genes were consistent with the expected sampling phases. Groups of co-expressed genes that responded to diurnal periods were associated with nucleic acid-binding, heat stress responses, and photosynthesis. Microbiome analysis revealed only modest differences in alpha diversity between midday and midnight samples. However, beta diversity indicated a significant shift in community composition. Co-occurrence network analysis identified keystone taxa specific to each time point, suggesting time-dependent ecological roles within the phyllosphere microbiome. Cross-correlation analyses between host gene expression and bacterial taxon abundance revealed a greater number of host–microbe associations during the night. Several canonical circadian clock genes significantly correlated with microbial taxa. Our findings provide initial evidence for diurnal associations between host gene expression and leaf-associated bacteriome, suggesting that maize diurnal transcriptional dynamics, including the activity of circadian clock genes, may contribute to shaping the composition and functional potential of the phyllosphere microbiome.
Engineered microorganisms for environmental release (EMERs) hold a considerable potential for bioremediation of sites polluted with urban and industrial emissions. The growing availability of EMERs with phenotypes that can help recovering polluted sites and degraded ecosystems could become a phenomenal tool to tackle some of the most pressing contamination problems at very different scales worldwide. Yet, while their delivery to diverse environmental targets could have an immense benefit, the regulatory roadmap for permitting release-in particular in the European Union-is controlled by strict policies which become actual deterrents for the development of the field. In this Perspective, we have mapped the current state of regulatory affairs and entertain ways of moving from the existing legislation on genetic engineering to an informed discussion about the future of this technology, highlighting several important aspects of the new generation of molecular tools for precision genetic engineering. Furthermore, we advocate advancing towards a fresh mindset in which EMERs are not perceived as artificial intrusions into the natural world, but as our main allies to combat much of the damage that we have inflicted to the planet.
Gastric cancer (GC) remains highly lethal, and although gastric microbiome dysbiosis has been linked to carcinogenesis, the viral component is still poorly explored. Here, we used metatranscriptomics to characterize the gastric virome and evaluate its association with GC and clinicopathological features. We analyzed 238 gastric tissues (214 GC and 24 non-tumors, NT) with clinicopathological data. Viral classification was performed using Kraken2 with the RVDB database. Virome diversity, composition, and clustering were assessed using phyloseq-based analyses, Jensen–Shannon divergence with PAM clustering, and ordination methods. Differential abundance and diversity were evaluated using LEfSe and statistical tests, and viral gene expression was investigated for clinical relevant viruses. We identified 106 viral genera, predominantly bacteriophages and dsDNA viruses, with distinct GC- and NT-associated viral signatures. Clustering revealed three viral community types (GT-1, GT-2, GT-3) that significantly separated GC and NT samples and showed reduced alpha diversity in GC-associated clusters. GT-1 was dominated by Lymphocryptovirus, GT-2 by Gorganvirus, and GT-3 (NT) exhibited the highest diversity. GC tissues were enriched in oncologically relevant viruses, including Lymphocryptovirus (EBV), Cytomegalovirus, and Alphapapillomavirus, whereas several bacteriophages predominated in NT. Virome composition was significantly associated with Lauren histological subtype, but not with clinical stage, tumor location, or neoadjuvant therapy. EBV-high tumors displayed a predominantly latent transcriptional program, with strong expression of ncRNAs (RPMS1, EBERs) and low lytic activity. These findings highlight major virome restructuring in GC and support a potential role of the gastric virome in tumor-associated microbial ecology, warranting further mechanistic and clinical investigation.
Bacterial lysates, which contain cell wall components and intracellular metabolites, have emerged as important modulators of macrophage immune function through interactions with pattern recognition receptors (PRRs). Ultrasonication is an effective technique for extracting these bioactive constituents. The goal of this work was to examine the immunomodulatory and cytoprotective effects of ultrasonically lysed Bifidobacterium breve (BB-UL) in J774A.1 macrophages activated by Pam3CSK4. J774A.1 macrophages were pre-exposed to BB-UL (equivalent to 10⁸ CFU/mL) for 12 h before stimulation with Pam3CSK4 (500 ng/mL). Cell viability was determined via the MTT assay, intracellular reactive oxygen species (ROS) were quantified using the DCFDA assay, and apoptosis was assessed with Annexin V-FITC/PI staining. Gene expression levels of Sirt1, Nfe2l2, Hmox1, Rela, Il1b, Il6, Tnf, Bax, and Bcl2 were analyzed by RT-qPCR, while secreted cytokines were measured using ELISA. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test (n = 3). Pam3CSK4 exposure significantly decreased cell viability to approximately 50
Carbapenemase-producing Klebsiella pneumoniae remains a major driver of multidrug resistance, traditionally linked to healthcare-associated infections but its detection in community-acquired infections represents a worrying shift. We aimed to characterize community-origin KPC- or NDM-producing K. pneumoniae isolates from two cities in São Paulo State, Brazil. A laboratory-based surveillance was conducted, including 111 non-duplicate K. pneumoniae isolates from urine of non-hospitalized patients. Polymyxin B MICs were determined by broth microdilution. All isolates underwent Illumina WGS. In silico analyses used Kleborate and Roary were used for bioinformatics analyses. 76
Klebsiella aerogenes is an opportunistic pathogen increasingly associated with healthcare-associated infections and severe respiratory complications. However, it’s in vivo adaptation during host colonization remains poorly understood. We characterized three clonal K. aerogenes ST93 isolates sequentially recovered on days 4, 14, and 19 of hospitalization from a patient with fatal viral pneumonia and concurrent abdominal sepsis. While initially identified generically as Klebsiella spp. by automated clinical systems, Whole-Genome Sequencing and Average Nucleotide Identity confirmed their identity at the species level. Genomic analysis revealed an in vivo plasmid curing event characterized by the loss of a 6.1 kb blaOXA−232-carrying plasmid in the final isolate (18281). Despite this plasmid loss, isolate 18281 sustained persistent carbapenem resistance due to a conserved ompK36 chromosomal mutation. Phenotypically, 18281 exhibited significantly higher lethality in Galleria mellonella larvae and induced progressive body weight loss in dexamethasone-treated mice compared to the other isolates. This enhanced virulence strongly correlated with a tissue-specific, compartmentalized transcriptional reprogramming, characterized by a marked 15.66-fold up-regulation of the yersiniabactin gene irp1 within hepatic tissues, whereas the colibactin gene clbA was consistently downregulated in vivo across both isolates and organs. Our findings demonstrate that intra-host microevolution of K. aerogenes involves structural plasmid instability and targeted transcriptional remodeling during host adaptation. This localized adaptive behavior underscores the clinical threat posed by transitional clonal variants in critically ill patients undergoing viral-mediated immune dysregulation.
Streptococcus suis (S. suis) is an important zoonotic pathogen. Biofilm formation contributes to persistent and chronic infections, increases the difficulty of bacterial eradication, and may pose a threat to public health. Clinically, S. suis infection is mainly treated with antibacterial drugs. However, biofilm-forming S. suis exhibits enhanced drug tolerance, and conventional drugs are often unable to eradicate established biofilms effectively. At present, screening traditional Chinese medicine monomer drugs to interfere with the formation of biofilms has become a promising strategy for controlling S. suis biofilms. In this study, the antibiofilm effect of tubuloside A (TA) on S. suis ATCC700794 and the associated metabolic changes were investigated using untargeted metabolomics. The minimum inhibitory concentration (MIC) of TA against S. suis ATCC700794 was determined by the broth microdilution method. The effects of TA were studied using crystal violet staining. The morphology of TA treated ATCC700794 cells was observed by scanning electron microscopy. Differentially abundant metabolites were screened using metabolomics and bioinformatics analyses. The MIC of TA was 64 µg/mL, whereas 1/2 MIC (32 µg/mL) of TA significantly inhibited biofilm formation without markedly affecting bacterial growth under the tested conditions and reduced biofilm structural formation. After treatment with 1/2 MIC of TA, 65 annotated metabolites met the screening criteria, including 23 upregulated and 42 downregulated metabolites. Bioinformatic analysis showed that the metabolic changes in S. suis ATCC700794 after TA treatment were mainly associated with glycine, serine and threonine metabolism, purine metabolism, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, the citrate cycle, arginine and proline metabolism, and pyruvate metabolism. This study provides preliminary metabolomic evidence that TA-mediated inhibition of S. suis biofilm formation is associated with alterations in amino acid metabolism and central carbon metabolism, offering candidate metabolic clues for future mechanistic studies. This study is the first to show that tubuloside A inhibits biofilm formation in Streptococcus suis at sub-inhibitory concentrations. This study reveals the metabolic changes associated with the antibiofilm effect of tubuloside A using untargeted metabolomics. This study suggests that amino acid metabolism and central carbon metabolism are associated with the metabolic response of Streptococcus suis to tubuloside A treatment.
