Search Results (155)

Recombinant adeno-associated virus (rAAV) is a preferred vector in gene therapy, although high production costs inhibit widespread adoption. The most common approach for rAAV production involves transfection of HEK293 cells with three plasmids: pTransgene, pRep/Cap and pHelper. Producing sufficient amounts of these plasmids accounts for up to 40% of total batch costs. Initially, this work aimed to increase plasmid yields by replacing the backbones. While this approach increased pHelper yields, pRep/Cap and pTransgene yields were unaffected. A possible reason was identified: pTransgene contains inverted terminal repeat (ITR) sequences that are essential for rAAV production. ITRs have strong secondary structures (including hairpin loops termed B and C arms) that likely interfere with plasmid production. Therefore, targeted deletions were performed within the ITRs. Partial deletions in both the B and C arms of the ITR were most beneficial, as both plasmid yield and transgene expression increased. Importantly, partial deletions did not reduce rAAV yield, as had been previously observed when the B and C arms were fully deleted. In summary, we report a 140% increase in pHelper plasmid production, while the most successful ITR variant increased pTransgene plasmid yields by 57% and transgene expression by 28%, without reducing rAAV yields or transduction efficiency. Full article

Background/Objectives: Acinetobacter baumannii is a major cause of infections in Intensive Care Units (ICUs), driving mortality through high-level antimicrobial resistance. This study utilized whole-genome sequencing (WGS) to evaluate the phenotypic, genotypic and virulence features of carbapenem-resistant A. baumannii (CRAB), which has caused ventilator-associated pneumonia/tracheobronchitis (VAP/VAT) in the ICU of the University Hospital of Split, Croatia. Methods: Over 1 year, lower respiratory tract specimens from 79 VAP/VAT patients were analyzed. CRAB isolates were identified via MALDI-TOF MS and evaluated for antimicrobial susceptibility, and a representative subset underwent WGS and multilocus sequence typing (MLST). Results: Out of 106 specimens, 18 non-duplicate CRAB strains were isolated. Five isolates underwent genomic analysis, identifying two globally distributed, high-risk Pasteur lineages: ST2 and ST492. These lineages displayed distinct resistomes: ST2 carried bla_OXA-23 and bla_ADC-73, while ST492 harbored plasmid-borne bla_OXA-72 (Rep3-T1/AB082 cluster) and bla_ADC-30. All isolates shared aminoglycoside/macrolide-resistance genes, conserved efflux pumps, and virulence determinants (bau, bas, ent, bar) crucial for acinetobactin synthesis and respiratory colonization. Phylogenetic analysis confirmed regional circulation and genetic links to neighboring countries. Conclusions: This study highlights the evolutionary dynamics of endemic CRAB lineages in a Croatian ICU and their global dissemination, which poses a critical threat, demanding strict infection control and novel therapeutics. Full article

A high manufacturing cost of goods (CoG) remains a critical barrier to the broad clinical adoption of gene therapies and is driven in part by limited productivity in adeno-associated virus (AAV) manufacturing. Here, we report an optimized AAV production process developed to markedly increase upstream titers while preserving vector quality and potency. The process combines a redesigned plasmid system, an optimized plasmid ratio, and a novel synthetic transfection reagent and was benchmarked against a conventional triple-plasmid/PEI MAX workflow. Across multiple AAV capsids and independent production runs, the optimized process reproducibly increased crude harvest titers by approximately 10- to 33-fold relative to the standard process, while maintaining key vector quality attributes. Notably, within the detection limits of the assay, rcAAV was undetectable at 1 × 10¹⁰ vg input with the optimized process, whereas the conventional triple-plasmid (with native Rep-Cap sequence)/PEI MAX workflow remained rcAAV-positive under identical conditions. Importantly, the in vivo potency was comparable to that of vectors produced by the conventional process. These results position our optimized AAV production process as a promising strategy to materially reduce AAV manufacturing CoG per patient. Full article

Cronobacter sakazakii is a rare, life-threatening neonatal pathogen. In this study, we profiled antimicrobial resistance genes (ARGs), plasmid replicons, virulence genes, sequence types (STs), and core-genome phylogeny across all publicly available clinical C. sakazakii genomes from the United States (n = 116). All isolates harbored exclusively chromosomally encoded bla_CSA variants. IncFIB and rep cluster 574 co-occurred in 93.1% of isolates as a conserved backbone among 17 replicon types. Among 49 virulence genes, 13 formed a universal core (flagellar motility and type VI secretion); accessory genes such as the yersiniabactin cluster were sporadic. ARG, plasmid replicon, and virulence gene contents were strongly structured by ST, dominated by ST4, ST1, ST8, and ST13. Stratified testing shows most pairwise associations were confounded by clonal lineage, except the IncFIB-rep cluster 574 co-carriage and an IncFII-hcp/tssD association, significant after ST stratification but not multiple testing correction. Core-genome phylogenetics identified 16 clusters, from a large ST4-dominated group to nine singletons, corroborated by ST monophyly; a genomically identical ST1 pair, nine years apart, indicated long-term clonal persistence. Ancestral state reconstruction distinguished stable single acquisitions from features with repeated turnover. Core-genome and gene-content phylogenies demonstrated substantial discordance despite significant overall correlation. These findings provide a genomic framework for clinical C. sakazakii population structure in the United States. Full article

Here we report the draft genome sequences of three Enterococcus faecalis strains, LAB1, LAB10, and LAB11, isolated from the pond water of a tilapia (Oreochromis niloticus) aquaculture farm at the University Nangui Abrogoua, Abidjan, Ivory Coast. These strains were previously characterised for their probiotic, plant growth-promoting (PGP), and nitrifying properties. All three strains were assigned to sequence type ST19 by multilocus sequence typing (MLST). The draft genomes of LAB1, LAB10, and LAB11 consist of 34, 35, and 34 contigs, totalling 2.94 Mb each (GC content 37.40%). Prokka annotation predicted 2872, 2873, and 2875 protein-coding sequences (CDS) for LAB1, LAB10, and LAB11, respectively. Genomic screening revealed no vancomycin resistance genes; however, tet(M) and lsa(A) resistance determinants were identified in all three strains, located on a repUS43-type plasmid replicon. Fourteen virulence factor homologs conserved in the E. faecalis reference strain V583 were detected, including Ebp pili, gelatinase (gelE), Fsr quorum-sensing system, and capsule biosynthesis genes, but no cytolysin operon was identified. Genes associated with stress tolerance (katA, sodA), bile salt hydrolysis (cbh), siderophore transport (fepC, fhuD), and ethanolamine nitrogen metabolism (eutB/eutC) were identified in all three genomes. Pan-genome analysis with the E. faecalis reference strain revealed 551 core gene clusters and 279 gene clusters exclusive to the three aquaculture isolates. Despite their high genomic similarity, we report the three genomes as distinct isolates due to observed differences in their expressed phenotypic properties. These sequences provide a genomic resource supporting the development of multifunctional probiotic consortia for integrated aquaponic systems. Full article

This study characterized six Enterococcus faecium strains isolated from Algerian camel milk to assess their molecular features and antimicrobial potential as food-associated isolates. Identification was performed using phenotypic tests, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16S rRNA gene sequencing. Strain diversity was assessed by repetitive sequence-based polymerase chain reaction (rep-PCR), including (GTG)₅-PCR, BOX-PCR, and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Polymerase chain reaction (PCR) screening for selected virulence-associated and antimicrobial resistance genes showed that the two selected isolates were negative for the targeted markers, indicating a preliminary molecular profile of interest but not allowing definitive safety conclusions without whole-genome sequencing and phenotypic antimicrobial susceptibility testing. Both strains showed antimicrobial activity against Gram-positive and Gram-negative indicators. Strain 9 showed notable inhibition against Klebsiella pneumoniae subsp. pneumoniae ATCC 13883, with an inhibition zone of 23.4 ± 2.3 mm using native supernatant, which increased after concentration. Trypsin treatment abolished activity, supporting the proteinaceous nature of the inhibitory compounds. The enterocin genes entA and entB were detected in strains 9 and 13, indicating genetic potential only. Further genomic, safety, and functional investigations are required. Full article

Tenacibaculum maritimum is a major etiological agent of tenacibaculosis in marine fish and represents an increasing concern for Chilean salmon aquaculture; however, updated information on the phenotypic and molecular diversity of circulating isolates is limited. This study characterized 40 isolates recovered from Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and red cusk eel (Genypterus chilensis) obtained from outbreaks between 2020 and 2024. Isolates were analyzed using biochemical and phenotypic assays, multiplex PCR targeting the O-antigen gene cluster (O-AGC), REP-PCR-based genetic fingerprinting, and experimental bath challenges in Atlantic salmon. Phenotypic characterization revealed species-consistent traits but variable gelatin and starch hydrolysis and differences in seawater tolerance. O-AGC typing identified four molecular serotypes (1-0, 3-1, 3-2, and 4-0), with serotypes 1-0 and 3-2 detected for the first time in Chilean salmonids. Genetic fingerprinting distinguished previously described profiles and two novel REP patterns (REP6 and REP7), indicating additional genomic heterogeneity within dominant serotypes. Virulence assays showed cumulative mortality ranging from 0% to 100%, with serotype 3-1 isolates generally associated with higher mortality and serotype 4-0 displaying broad intra-serotype variability. These findings document substantial phenotypic, antigenic, and genetic diversity among Chilean T. maritimum isolates and provide epidemiologically relevant information for disease surveillance and vaccine design in salmon aquaculture. Full article

Transposable elements (TEs) play important roles during development and disease, including through transcriptional activation of TE-associated genes during early human development. Moreover, based on the functional and epigenetic similarities between early development and cancer, TE-associated genes contribute not only to early human development, but frequently contribute to cancer progression. In this study, we hypothesised that recruitment of TE-associated genes during cancer onset occurs through epigenetic regulatory processes, especially involving DNA hypomethylation accompanied by transcriptional upregulation of early developmental pathways, such that, when reactivated inappropriately in later life, they may drive malignancy. It is unknown, however, to what extent DNA methylation changes are critically involved in the transcriptional activation of TE-associated genes. Accordingly, to investigate this we used the RepExpress tool to identify developmentally regulated TE-associated genes in placenta and human embryonic stem cells (hESCs), which we then investigated by targeted deep bisulfite sequencing (TDBS) to determine the methylation status of the identified TE-associated genes in placenta, somatic tissues, and melanoma cell lines. Outcomes suggest that DNA methylation may be one of the regulatory factors underscoring transcriptional activation of TE-associated genes, but that methylation is not necessarily the sole factor involved in regulating the transcriptional activation of TE-associated genes during malignant transformation. Full article

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches. Full article

Despite its clinical importance, Staphylococcus aureus colonization in community populations remains insufficiently understood. This study aimed to determine the prevalence, anatomical distribution (nasal versus throat), and antimicrobial resistance patterns of Staphylococcus aureus colonization in healthy community-dwelling adults and to identify demographic and clinical factors associated with carriage. A cross-sectional study was conducted among 100 community-dwelling adults in Germany, yielding 200 nasal/throat samples. Staphylococcal isolates were identified using MALDI-TOF MS, and antimicrobial susceptibility was determined according to EUCAST guidelines. MRSA and PVL genes were assessed using molecular assays, and genetic relatedness was evaluated by rep-PCR. Associations with demographic and clinical variables were analyzed using multivariable logistic regression in R. Staphylococcus aureus carriage prevalence was 39%, higher in the nose (33%) than the throat (19%), with rare MRSA (3%) and no PVL detection. Significant nasal–throat discordance was observed (p < 0.01), with a fair agreement between sites (κ = 0.34). Resistance patterns among Staphylococcus aureus isolates were dominated by penicillin G resistance (47%), while 35% remained fully susceptible, and multidrug resistance was rare (6%). Multivariable analyses indicated no strong associations between overall, nasal, or throat carriage and age, sex, recent antibiotic use, or other clinical exposures (p > 0.05), with wide confidence intervals, potentially reflecting limited statistical power and only modest model discrimination (AUC 0.65–0.68). These findings indicate that community Staphylococcus aureus colonization is potentially marked by modest prevalence, substantial anatomical discordance, and a low-risk resistance profile, while common demographic and clinical factors contributed little to explaining carriage patterns. Full article

A quartz crystal microbalance-based biosensor for the specific detection of the first transgenic common bean (L.) cultivar (BRS FC401 RMD) with resistance to Bean golden mosaic virus (BGMV) was developed. The immobilization chemistry relies on the strong bond between the thiolated probe and the gold electrode surface. The probe sequence is internal to a region of the BGMV rep gene that was introduced into the common bean genome. The sensor’s analytical performance was determined using synthetic oligonucleotides. Real samples of transgenic and wild-type bean seeds were also tested. Sample pretreatment consisted only of enzymatic fragmentation, followed by a thermal denaturation step combined with blocking oligonucleotides. Different biosensor regeneration approaches were studied. Immobilization showed good reproducibility (CV% of 5.8%). The biosensor proved specific for both synthetic oligonucleotides and non-amplified genomic DNA. A linear detection range of 0–1.4 ng/µL was observed, with a detection limit of 0.18 ng/µL. Three sequential detections were performed without loss of surface activity. The results demonstrate the biosensor’s potential for direct, real-time, label-free detection of DNA samples for field screening of transgenic common bean cultivars. Full article

Background: Hospital wastewater (HWW) is a critical reservoir for carbapenem-resistant Gram-negative bacteria. Methods: Between November 2024 and August 2025, sixty 24 h composite wastewater samples were collected from five tertiary hospitals. Of the 244 carbapenem-resistant isolates recovered, 34 bla_NDM-1-positive Acinetobacter isolates were subjected to phenotypic, genotypic, and plasmid analyses. Results: Eleven species were identified among the 34 carbapenem-resistant Acinetobacter isolates, predominantly non-baumannii Acinetobacter (NBA). All isolates were carbapenem-resistant (34/34, 100%) with high-level MICs (meropenem MIC_50/90, 32/64 mg/L; imipenem MIC_50/90, >128/>128 mg/L); 21% (7/34) of isolates were resistant to colistin, and resistance to ceftazidime, cefepime, and trimethoprim-sulfamethoxazole was 100%, 94%, and 76%, respectively. Core-genome SNP analysis revealed highly similar isolates across hospitals within the same season (1-2 SNPs) or within the same hospital across seasons (19 SNPs). Genomic analysis showed that bla_NDM-1 was present in all isolates (34/34, 100%), with plasmid carriage in 85.3% (29/34); bla_OXA-58 co-occurred in 62.1% (18/29), mainly on Rep_3 plasmids (19/29), especially R3-T28 (15/29) that frequently carried bla_OXA-58 (10/15). Two unclassified plasmids co-harboring bla_NDM-1 and bla_OXA-23 were detected in Acinetobacter tandoii isolates. The bla_NDM-1 gene was embedded in a conserved Tn125-like structures with variable flanks. Conclusions: Overall, carbapenem-resistant Acinetobacter from hospital wastewater frequently carried Rep_3 plasmid-borne bla_NDM-1, especially R3-T28 and often co-occurring with bla_OXA-58, within a conserved Tn125-like core structures. These findings highlight HWW as a potential hotspot for dissemination of carbapenem resistance and support routine genomic surveillance under a One Health framework. Full article

Background/Objectives: Recombinant adeno-associated virus (rAAV) has become a leading vector in gene therapy. However, manufacturing limitations may result in replication-competent AAV (rcAAV) contamination of clinical rAAV products, posing safety risks. Rigorous testing is therefore essential, and the use of accurately calibrated rcAAV reference standard materials is critical for ensuring assay stability and reliability. A disadvantage of the widely used Tissue Culture Infectious Dose 50 (TCID₅₀) assay is its high variability. This study introduces an optimized TCID₅₀ assay for the precise quantification of infectious rcAAV particles. Methods: We developed a TCID₅₀ assay tailored to rep2-based rcAAV, optimizing key aspects such as viral infection conditions, qPCR reaction systems, and standard curve preparation. We employed an innovative strategy to prepare the standard curve using serial dilutions of rcAAV in cell lysate, ensuring alignment with the test sample matrices. Results: The rcAAV-derived standard curve demonstrated exceptional linearity (R² > 0.99), sensitivity (LOQ ≈ 38 copies), and reproducibility, enabling robust endpoint qPCR analysis. The optimized assay significantly improved the precision of the TCID₅₀ assay, as an inter-assay coefficient of variation (CV) of 11.4% was achieved. Conclusions: This refined TCID₅₀ assay is a reliable method for calibrating infectious titers of rcAAV reference standard materials, thereby enabling the standardization of rcAAV testing. Full article

The coexistence of antibiotic resistance genes (ARGs), particularly those conferring resistance to last-resort antibiotics, is increasingly present in lesser-studied bacterial species. Tigecycline is currently one of the last important barriers in the treatment of carbapenem-resistant bacterial infections, whose resistance gene, tet(X), is prevalent across multiple bacterial genera, but the coexistence of tet(X3) and tet(X4) in Acinetobacter sp. is rarely observed. Here, we report a strain co-harboring the chromosomal tet(X3) and plasmid-borne tet(X4) isolated from a commercial beef cattle farm in Henan province, China. The strain exhibited resistance to ampicillin, gentamicin, chloramphenicol, sulfamethoxazole, tetracycline, doxycycline, tigecycline, and omadacycline. Based on whole-genome sequencing (WGS), the strain was identified as A. indicus using Average Nucleotide Identity (ANI) and digital DNA–DNA hybridization (dDDH). Chromosomal tet(X3) was identified in the genetic context, ISVsa3-XerD-tet(X3)-res-ISVsa3. The plasmid-located tet(X4) with the genetic context, ISVsa3-abh-tet(X4)-ISVsa3, and 14 additional resistance genes were located in multiple pdif modules. Two different typing methods, the Rep-based strategy (designed for A. baumanii) and MOB-typer, identified the tet(X4)-positive plasmid as GR31 and rep_cluster_1656, respectively. Conjugation assay failed to observe the transfer of the tet(X4)-positive plasmid into recipients, E. coli J53 and Salmonella LGJ2. The co-occurrence of tet(X3) and tet(X4) in Acinetobacter may suggest a risk of rapid dissemination of tigecycline resistance and the hidden presence of numerous undetected bacteria co-carrying high-risk ARGs in the agroecological system, both of which should cause particular concern. Full article

This study evaluated the viability, microbiological composition, functional traits, and safety of probiotic bacteria isolated from commercial products marketed as containing Limosilactobacillus reuteri. Viable cell counts, biochemical characterization, strain-level identification, functional properties, gastrointestinal tolerance, and safety attributes were assessed. Among the evaluated products, only four presented colony-forming units (CFU) counts consistent with label claims (products E, F, G, and H), while two showed no detectable viable microorganisms (products B and L). All isolates were Gram-positive, catalase-negative, and predominantly rod-shaped. rep-PCR analysis revealed strain homogeneity in most products, whereas others (products A and K) exhibited heterogeneous microbial compositions. Molecular identification based on 16S rRNA sequencing showed a predominance of Lmb. reuteri and Lacticaseibacillus rhamnosus, with some products containing additional species such as Lactiplantibacillus plantarum and Lactobacillus acidophilus. Functional assays demonstrated strain-dependent proteolytic and diacetyl-producing capacities, as well as variable tolerance to simulated gastrointestinal conditions. Most strains preferentially produced L-lactate, although some generated substantial amounts of D-lactate. All isolates were susceptible to antibiotics recommended by EFSA, except for intrinsic vancomycin resistance, and no transferable virulence markers, biogenic amine production, or Salmonella contamination were detected. Furthermore, virulence-related genes such as hdc, tdc, odc, hyl, cylA, and ace were not identified. Overall, the results highlight pronounced discrepancies between label claims and microbiological quality among commercial probiotic products and reinforce the importance of strain-level characterization to ensure safety, functional performance, and regulatory compliance. Full article