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At least 19 recordsLinked to original sources

PubMed2026

Characterisation of Carbapenem-Resistant Raoultella planticola and Structural Analysis of NDM Composite Plasmids.

OBJECTIVE: This study aimed to investigate the molecular characteristics, resistant plasmid structures and phylogeny of a carbapenem-resistant Raoultella planticola (CRRP) strain from a patient with pneumonia to inform antimicrobial resistance control strategies. METHODS: We performed strain identification using MALDI-TOF MS, the BD Phoenix 100 system and whole-genome sequencing (WGS). We assessed antimicrobial susceptibility and resistance gene transfer using PCR, conjugation and stability assays, plasmid structure using a bioinformatics tool and phylogeny using a core-genome phylogenetic tree. RESULTS: WGS confirmed the isolate as R. planticola (average nucleotide identity (ANI) > 98.9% with reference type strains), co-harbouring blaKPC-2 and blaNDM-1. It was resistant to 19 antimicrobial agents and susceptible to only polymyxin, amikacin and chloramphenicol. Resistance genes were present on two conjugative plasmids: pzwx_KPC (IncFIA) and pzwx_NDM (a novel repFIB/repHI5B hybrid assembled via non-homologous end joining). Both plasmids demonstrated efficient transfer and stable inheritance over 12 passages. pzwx_KPC was highly homologous to plasmids from Klebsiella pneumoniae. Phylogenetic analysis revealed the closest relationship with German R. planticola strains. CONCLUSION: CRRP carries highly transmissible and stable resistance plasmids. Strengthened monitoring in immunocompromised patients and improved environmental disinfection are recommended. The risk of misidentification by automated systems underscores the importance of WGS for accurate pathogen identification.

Carbapenem resistance↗

PubMed2025

Global emergence and transmission dynamics of carbapenemase-producing Citrobacter freundii sequence type 22 high-risk international clone: a retrospective, genomic, epidemiological study.

BACKGROUND: Carbapenemase-producing Citrobacter (CPC) species have recently been recognised as emerging pathogens associated with nosocomial infections in humans. The increased rate of Citrobacter freundii infections is a public health concern and there is a paucity of genomic data regarding its global transmission dynamics. We aimed to characterise the genetic features of CPC species, and their associated carbapenemase-encoding plasmids, obtained from hospitalised patients in China and from publicly available global data, with a particular focus on high-risk clones. METHODS: This was a retrospective, genomic epidemiological study of CPC species obtained from a tertiary hospital in Zhejiang Province, China, from March 5, 2013, to March 5, 2023. We used antimicrobial susceptibility testing, short-read and long-read whole-genome sequencing, phylogenomic analysis, and plasmid structure analysis. A global dataset of complete plasmid sequences encoding blaKPC, blaNDM, and blaIMP was constructed from the National Center for Biotechnology Information (NCBI) RefSeq database to provide insights into their diversity and distribution. All carbapenemase-producing Citrobacter freundii genomes from the NCBI GenBank database were incorporated in the comparative genomic analyses. Bayesian phylogeographical analysis and growth rate assays were carried out to characterise the high-risk C freundii sequence type (ST) 22 clone. FINDINGS: 1724 Citrobacter species isolates were collected from diverse clinical specimens, with 48 identified as CPC species. Citrobacter koseri (22 [46%] of 48) and C freundii (20 [42%]) were the predominant CPC species. Comparative analysis found C freundii carried significantly higher median numbers of plasmid replicons (5·0 [IQR 3·3-6·0] vs 2·0 [2·0-3·0]; p<0·0001) and acquired antimicrobial resistance genes (12·0 [7·3-15·8] vs 3·0 [3·0-5·3]; p<0·0001) than did C koseri. Molecular characterisation identified Inc-type plasmids, In823::Kl.pn.I3/In1589-like/In837-like integrons, Tn6296/Tn125/Tn5060 transposons, and insertion sequences (eg, IS26, IS3000, IS5, ISAba125, ISCR1), collectively facilitating the dissemination of carbapenemase genes. Global analysis of 3126 carbapenemase-encoding plasmids found epidemic plasmids with broad host ranges and global diversity. Phylogenetic investigation of predominant carbapenemase-encoding plasmids showed their persistence across geographical regions, temporal spans, and Enterobacterales species, exhibiting high genetic similarity to our clinical plasmids. A phylogenetic tree of 726 global carbapenemase-producing C freundii genomes showed that ST22 (227 [31·3%]) represents the predominant multidrug-resistant clone across community, health-care, and environmental niches. Transmission across continents contributes to the global predominance of the ST22 clone, which carries a high load of resistance genes (median 15·0 [IQR 11·0-17·0] vs 12·0 [3·0-16·0]; p<0·0001) and enhanced plasmid maintenance capacity (median replicons 5·0 [IQR 4·0-7·0] vs 4·0 [3·0-6·0]; p<0·0001) relative to non-ST22 clones. INTERPRETATION: Our study provides evidence to suggest that Citrobacter species are emerging carriers of carbapenem-resistance genes. These findings provide insight into the population structure of CPC species and highlight C freundii ST22 as a prominent high-risk international clone. FUNDING: National Natural Science Foundation of China, National Health Commission Scientific Research Fund-Zhejiang Provincial Major Health Science and Technology Plan Project, Zhejiang Province Natural Science Foundation Project, Outstanding Youth Foundation of Jiangsu Province of China, the Priority Academic Program Development of Jiangsu Higher Education Institutions, and Postgraduate Research and Practice Innovation Program of Jiangsu Province.

Citrobacter freundii↗

PubMed2025

Emergence of carbapenem-resistant Serratia marcescens co-harboring blaNDM-1, blaKPC-2, and blaSRT-2 in bloodstream infection.

Serratia marcescens is an emerging opportunistic pathogen with high genetic diversity. The emergence and prevalence of carbapenem-resistant S. marcescens poses a major health threat due to its intrinsic resistance to multiple antibiotics, which severely restricts the selection and treatment of antibiotics for S. marcescens infection. This study presents the first documented case in China of a bloodstream infection caused by Staphylococcus epidermidis and S. marcescens strain (designated S96) co-producing blaNDM-1, blaKPC-2, and blaSRT-2. Strain S96 exhibited resistance to nearly all categories of β-lactam antimicrobials, β-lactam/inhibitor combinations, aminoglycosides, quinolones, and other clinical antibacterial agents, with the exception of tigecycline. Our main objective was to characterize the genetic mechanisms underlying its carbapenem resistance and plasmid transfer potential. Whole-genome sequencing revealed blaKPC-2 on a 44,047 bp "IncX6-like" plasmid and blaNDM-1 on a 100,081 bp IncFII(Yp)-type plasmid, alongside chromosomal blaSRT-2 and aac(6')-Ic. "IncX6-like" and IncFII(Yp)-type plasmids are widely distributed among carbapenem-resistant Enterobacteriaceae strains globally. Conjugation experiments demonstrated that the blaNDM-1-carrying plasmid could be successfully transferred to recipient Escherichia coli 600, with no significant fitness cost observed (P > 0.05). The experimental results demonstrate that carbapenem-resistant genes can disseminate among Enterobacteriaceae via plasmid-mediated horizontal transfer between bacterial cells. Comparative genomic analysis revealed plasmid structural homology with global counterparts, demonstrating IS-mediated recombination and horizontal gene transfer. The low adaptive cost of plasmid carriage and multidrug resistance phenotype pose significant challenges for clinical management. This study highlights the need for enhanced clinical surveillance and antibiotic stewardship to curb the spread of such multidrug-resistant pathogens.IMPORTANCECarbapenem resistance in Serratia marcescens is primarily mediated by Klebsiella pneumoniae carbapenemase (KPC), with New Delhi metallo-β-lactamase (NDM) being a relatively uncommon alternative resistance mechanism. KPC-2 and NDM-1 coexisting in S. marcescens is extremely rare clinically. This study reports the first clinical isolate of S. marcescens in China co-harboring blaNDM-1, blaKPC-2, and blaSRT-2. The isolate exhibits multidrug resistance to nearly all β-lactam antibiotics and β-lactam/inhibitor combinations, with low adaptive costs and high dissemination potential. The potential spread of resistance genes through mobile genetic elements poses a serious public health risk. The study underscores the need for enhanced surveillance, rational antibiotic use, and novel strategies to combat resistance. It also provides insights into the evolutionary mechanisms of bacterial resistance, emphasizing the urgent need for interventions to address the growing threat of antimicrobial resistance.

Humans↗

PubMed2026

Gene transfer from NDM-5-producing and OXA-48-producing Enterobacter hormaechei ST79 on contaminated dicloxacillin capsules to other Enterobacterales in Europe, 2020-23: a retrospective, observational, molecular epidemiological study.

BACKGROUND: In February, 2023, an outbreak of Enterobacter hormaechei ST79 carrying blaNDM-5 and blaOXA-48 was linked to contaminated dicloxacillin capsules administered to approximately 79 000 individuals in Denmark. Initial clonal outbreak investigations identified 11 patients with the E hormaechei ST79 outbreak strain, which carried blaNDM-5 on a distinct IncX3 plasmid, and in nine cases, blaOXA-48 was on a distinct IncL plasmid. Interspecies plasmid transfer was observed in one patient, suggesting a potential plasmid-mediated outbreak involving other Enterobacterales. However, no studies have characterised the progression of a clonal outbreak originating from a contaminated medicine into plasmid-mediated dissemination of carbapenemase genes. Hence, we aimed to characterise the clonal and plasmid-mediated spread of carbapenemase genes in this outbreak. METHODS: We conducted a retrospective genomic and epidemiological investigation using existing short-read whole-genome sequencing data from all carbapenemase-producing Enterobacterales (CPE) from the Danish national surveillance, collected between Jan 1, 2014, and Oct 1, 2023. All confirmed CPE isolates were eligible for inclusion. Using in-silico screening for unique fragments of the two outbreak plasmids, we selected 160 isolates for long-read sequencing to obtain complete plasmid sequences for outbreak investigation. Analyses were descriptive and included comparison of sequence identity and coverage to define outbreak-associated plasmids and summary statistics of patient characteristics. FINDINGS: Data from 1829 isolates were obtained. We detected 16 of 53 isolates involved in the outbreak using conventional outbreak detection methods. The remaining 37 isolates were detected using plasmid-specific screening and long-read sequencing. 15 patients carried the outbreak E hormaechei strain, including the 11 patients previously reported. Three of the 15 patients presented with at least one additional bacterial species carrying one or both outbreak plasmids (pDcap_OXA-48 and pDcap_NDM-5). A further 24 patients, sampled between July 1, 2020, and Oct 1, 2023, presented with other Enterobacterales carrying one or both outbreak-associated plasmids but not the original E hormaechei ST79 strain. In four cases, outbreak-associated plasmids differed structurally from the original outbreak plasmid. INTERPRETATION: This study describes how a clonal CPE outbreak caused by a contaminated medicine evolved into a complex plasmid-mediated outbreak involving multiple Enterobacterales species. Most patients related to the outbreak did not present with the original E hormaechei ST79 outbreak strain and were therefore not identified using standard outbreak detection methods. These findings highlight the importance of using plasmid-focused approaches in outbreak investigations. FUNDING: The Danish Ministry of Health, SSI-Seq (cofunded by EU4Health).

Humans↗

PubMed2026

Diverse structures of mcr-10-bearing plasmids and high colistin resistance in Enterobacter cloacae complex clinical isolates from South Korea.

BACKGROUND: The emergence of mcr-mediated colistin resistance in Enterobacter cloacae complex (ECC) poses a significant threat to antimicrobial therapy. Among mcr variants, mcr-10 has been identified in various environments, but its genetic diversity, structural context, and functional role in colistin resistance remain unclear. METHODS: We investigated 183 ECC isolates and identified 60 colistin-resistant strains through minimum inhibitory concentration (MIC) testing. The presence of mcr-10 was screened using reference genomes from NCBI, and whole plasmid sequencing was conducted on mcr-10-positive isolates. The genetic environment of mcr-10 was analyzed via synteny and structural annotation. RESULTS: Whole-plasmid sequencing of eight mcr-10-positive ECC isolates identified three replicon types among the mcr-10-harboring plasmids: IncFIB (n=3), IncFII (n=3), and IncFII/IncFIB (n=2). Although all plasmids shared the xerC-mcr-10 cassette, they lacked a conserved backbone and showed diverse genetic contexts with variable insertion sequences near mcr-10, indicating marked structural heterogeneity. No additional antimicrobial resistance genes were detected on these plasmids. When introduced into E. coli DH5α, the plasmids increased colistin MICs only modestly, whereas representative plasmids transferred into colistin-susceptible E. roggenkampii and E. kobei conferred high-level resistance comparable to that of the parental mcr-10-positive isolates. Consistently, qRT-PCR showed higher mcr-10 expression in ECC transformants than in E. coli under colistin exposure, supporting a hostdependent effect on mcr-10-mediated colistin resistance. CONCLUSION: These findings highlight the diversity of mcr-10-carrying plasmids and suggest that mcr-10-mediated colistin resistance is shaped by the host genetic background. Further studies are needed to clarify the mechanisms underlying mcr-10 expression.

Colistin↗

PubMed2024

Putative evolution of Myxococcus fulvus 124B02 plasmid pMF1 from a chromosomal segment in another Myxococcus species.

Myxobacteria or order Myxococcales (old nomenclature) or phylum Myxococcota (new terminology) are fascinating organisms well known for their diverse peculiar physiological, taxonomic, and genomic properties. Researchers have long sought to identify plasmids within these organisms, yet thus far, only two organisms from different families have been found to harbor a plasmid. This study delves into the putative evolution of one of these plasmids, i.e., pMF1 present in Myxococcus fulvus 124B02 in the suborder Cystobacterineae and family Myxococcaceae. Here, we first reannotated the pMF1 plasmid genome sequence and identified two additional open reading frames or putative genes which were not annotated until now. We further reported that all pMF1 plasmid genes depict homology with Myxococcus stipitatus CYD1 draft genome (contig 28) and a chromosomal segment of M. stipitatus DSM14675 in a syntenic manner, implying the presence of plasmid-like structure in M. stipitatus CYD1, integrated into its chromosome. To comprehend the relationship among these three species, we conducted phylogenetic analyses using 16S and concatenated housekeeping genes and genome-to-genome distance calculator (GGDC) analysis, which confirmed that M. stipitatus CYD1 is a distinct and novel species within the genus Myxococcus. Overall, this comparative genomic study sheds light on the putative emergence of the pMF1 plasmid from a common ancestor of closely related yet distinct species, M. stipitatus CYD1, possibly through the partition from its chromosome as a segment.IMPORTANCEMyxobacteria are not well known to have plasmids. Until now, only two organisms have been shown to have plasmids, raising a pertinent question about how these plasmids evolved randomly within the phylum Myxococcota. The study presented in this manuscript delves into the emergence of the pMF1 plasmid found in Myxococcus fulvus 124B02, a member of the suborder Cystobacterineae and family Myxococcaceae. Our research addresses this intriguing topic of plasmid identification and evolution within myxobacteria, which are a group of fascinating organisms that have garnered significant interest due to their diverse physiological, taxonomic, and genomic properties.

Plasmids↗

PubMed2026

Genomic characterization of KPC-2 and NDM coproducing carbapenem-resistant Klebsiella pneumoniae in a hospital: discovery of ST1869 clone and a novel hybrid plasmid.

UNLABELLED: To characterize the plasmid architecture and molecular background of KPC-NDM coproducing carbapenem-resistant Klebsiella pneumoniae (KN-CRKP) in a South China hospital. Five KN-CRKP isolates were collected, including three from one patient. All underwent Illumina sequencing; two (ST11 and ST1869) additionally had Nanopore sequencing. Antimicrobial susceptibility testing strain sequence types, conjugation assays, resistance gene profiling, plasmid typing, genetic structure comparison, core-genome single nucleotide polymorphisms (SNPs) analysis, and plasmid clustering were performed. All isolates exhibited an imipenem minimum inhibitory concentration (MIC) of ≥128 µg/mL and harbored multiple resistance genes. One isolate (1/5) belonged to ST1869 and co-harbored blaKPC-2 and blaNDM-5. The blaNDM-5-carrying plasmid was a novel IncI1/X3 fusion plasmid that also carried blaCMY-42. Unlike several IncX3 plasmids carrying blaNDM in publicly available KN-CRKP genomes from South China, this IncI1/X3 hybrid lacked a complete conjugative transfer system. ST11 was the predominant clone (4/5), co-harboring blaKPC-2 and blaNDM-1. A rare genetic structure, ΔISKpn6-blaKPC-2-ISKpn28, was identified on IncFII plasmids carrying blaKPC-2. Plasmid clustering analysis of 126 comparative KN-CRKP genomes showed diverse sequence types and plasmid backgrounds associated with the KPC/NDM co-production pattern. The observed plasmid diversity and structural variation in KN-CRKP support continued genomic surveillance, with particular attention to the ST1869 clone, the novel IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare "ΔISKpn6-blaKPC-2-ISKpn28" genetic structure. Expanded genomic data on KN-CRKP are needed to further elucidate its resistance mechanisms and plasmid evolutionary trajectories. IMPORTANCE: The co-production of KPC and NDM carbapenemases in Klebsiella pneumoniae poses a formidable threat to clinical antimicrobial therapy, as these enzymes confer resistance to virtually all β-lactam agents, including carbapenems. Here, we report novel genomic features of KN-CRKP in South China, including the emergence of the ST1869 clone, a unique IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare ΔISKpn6-blaKPC-2-ISKpn28 genetic structure. These findings substantially expand current understanding of plasmid evolution and resistance gene dissemination in this region. The identification of diverse resistance mechanisms and clonal backgrounds supports enhanced genomic surveillance and infection-control awareness for pan-resistant Enterobacterales.

Plasmids↗

PubMed2026

Transferable IncX3-blaKPC-2 plasmid and chromosomal blaCTX-M-27 in a commensal Escherichia coli ST7854 isolated from a healthy companion dog.

OBJECTIVES: Carbapenemase-producing Enterobacterales have disseminated globally, largely via highly transmissible plasmids. Their emergence in companion animals is of particular concern, indicating that clinically important carbapenem-resistance determinants have spread beyond healthcare settings. This study characterized a multidrug-resistant Escherichia coli isolate from a healthy dog in South Korea. METHODS: Antimicrobial susceptibility was determined by standardized reference methods. Hybrid whole-genome assembly resolved chromosomal and plasmid architectures, and plasmid transferability was assessed by conjugation assays. Comparative genomic analysis based on nucleotide identity and alignment coverage evaluated relatedness to publicly available blaKPC-carrying IncX3 plasmids. RESULTS: The E. coli ST7854 isolate was resistant to carbapenems (imipenem MIC = 8 µg/mL), third-generation cephalosporins, fluoroquinolones, tetracycline, gentamicin, phenicols, and trimethoprim-sulfamethoxazole, but susceptible to amikacin and colistin. Assembly resolved a 4.77 Mb circular chromosome and seven plasmids. The ESBL gene blaCTX-M-27 was chromosomally integrated, whereas carbapenem resistance was conferred by blaKPC-2 on a 54,805 bp IncX3 plasmid. This IncX3-blaKPC-2 plasmid was transferred to E. coli J53 at a frequency of 5.91 × 10⁻⁴, whereas the large IncFIB multidrug resistance plasmid was not co-transferred. Comparative analysis revealed extensive structural similarity with Korean clinical IncX3 plasmids, whereas most representative international plasmids shared only the conserved IncX3 backbone. CONCLUSIONS: A commensal E. coli ST7854 isolate from a healthy companion dog carried a conjugative IncX3-blaKPC-2 plasmid and chromosomally integrated blaCTX-M-27. Similarity to Korean clinical plasmids suggests potential human-animal dissemination, and this asymptomatic carriage of mobile carbapenem-resistance determinants supports continued genomic surveillance of antimicrobial resistance in companion animals within a One Health framework.

Bla(CTX-M-27)↗

PubMed2026

Functional analysis of stem-loop structures within the SARS-CoV-2 5' untranslated region using a plasmid-based reporter system.

The 5' untranslated region (5'UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains highly conserved stem-loop structures that regulate viral gene expression. This study investigated the functional contributions of selected 5'UTR stem-loop elements to reporter gene expression using a plasmid-based mammalian expression system. Five constructs were tested using a non-integrating plasmid: the wild-type (WT) 5'UTR fused to GFP under the CMV promoter, and four deletion variants (ΔB, ΔC, ΔD, and ΔE) corresponding to deletions of stem-loop 4 (SL4), SL4.5, SL5, and SL5a, respectively. Following transfection into HEK293 cells, GFP fluorescence was quantified using a fluorescence microplate reader, and relative GFP transcript abundance was assessed by RT-qPCR. Deletion of SL4 (ΔB) resulted in marked reduction in both fluorescence and relative transcript abundance compared to WT construct, indicating substantially reduced reporter gene expression. In contrast, deletion of SL4.5, SL5, or SL5a did not produce the pronounced reduction observed for ΔB, although descriptive RT-qPCR analysis indicated differences in relative transcript abundance among these variants. Statistical analysis of fluorescence data demonstrated significant differences among constructs (one-way ANOVA, p < 0.05). Because the reporter assay was based on plasmid expression, the observed differences likely reflect combined contributions from transcription, transcript abundance, RNA stability, and translation rather than translation alone. These findings demonstrate that the SL4 region contributes substantially to reporter gene expression in this experimental system, whereas the remaining stem-loop regions examined exert comparatively modest effects. This study provides additional insight into the functional organization of the SARS-CoV-2 5'UTR and establishes a framework for future investigations aimed at distinguished the transcriptional, post-transcriptional, and translational contributions of individual RNA structural elements.

5' Untranslated Regions↗

PubMed2027

Molecular Cloning and Reverse Genetics.

This chapter describes a detailed molecular biology protocol for introducing specific point mutations into the chikungunya virus (CHIKV) genome using a reverse genetics strategy. The method utilizes an overlapping PCR-based approach to generate a mutated DNA fragment, which is then cloned into a pre-engineered CHIKV infectious clone plasmid. The protocol covers all major steps, from the initial PCRs to create the mutated insert to its digestion and ligation into the vector. It also includes procedures for bacterial transformation, colony screening via PCR and Sanger sequencing to confirm the mutation, and plasmid purification via miniprep. The document is structured with a clear introduction, a list of all required reagents and equipment, and a step-by-step methods section.

Cloning, Molecular↗

PubMed2026

Genomic diversity, antimicrobial resistance, and virulence-associated characteristics of Escherichia coli recovered from poultry hatchery-box material.

Hatcheries are early points of exposure of newly hatched chicks to Escherichia coli from eggshell debris, dust, meconium, and the hatchery environment. This study used descriptive genomic surveillance to characterize population structure, antimicrobial resistance, virulence-associated genes, plasmid replicons, and genetic relatedness in a purposively selected collection of E. coli recovered from hatchery-box material in a Czech commercial poultry hatchery. From 83 samples collected between 2019 and 2023, 409 isolates were recovered. Following within-sample reduction based on antimicrobial susceptibility profiles and PCR screening for selected APEC-associated virulence genes, 91 isolates enriched for cefotaxime-medium recovery or APEC-associated gene profiles were selected for Illumina whole-genome sequencing. The sequenced collection comprised 35 sequence types across nine phylogenetic groups or clades; phylogroup B1 (59/91) and ST162 (28/91) predominated. ColV-associated markers were detected in 88 isolates, acquired resistance genes in 61, and a multidrug-resistant phenotype in 31. Seventeen isolates were resistant to ceftazidime; eight carried ESBL or plasmid-mediated AmpC determinants, while eight additional isolates had the chromosomal ampC promoter substitution C-42T as the only identified determinant associated with this phenotype. Closely related isolates, including ST131, ST162, and ST675, were recovered from different samples or parent-farm groups within restricted sampling periods. These findings document genomically diverse, resistance- and virulence-associated E. coli in hatchery-box material and identify genetically related isolates that warrant investigation through structured longitudinal sampling. Because sampling was unequal and isolates were purposively selected, the findings do not provide prevalence estimates or demonstrate source, transmission, persistence, or pathogenicity.

APEC-associated virulence genes↗

PubMed2026

Emergence of a Tn7-associated blaVIM-1 within IncC plasmids in ST46 Providencia stuartii from Northern Italy.

OBJECTIVES: To characterize the genomic features and resistance determinants of carbapenem-resistant Providencia stuartii isolates circulating in Northern Italy, with a focus on the genetic context of blaVIM-1. METHODS: Five P. stuartii isolates collected between 2022 and 2024 from interconnected healthcare facilities underwent molecular characterization. Antimicrobial susceptibility testing was performed according to EUCAST 2025 criteria. Whole-genome sequencing was conducted using Illumina technology, followed by resistome, plasmidome, and phylogenetic analyses. Comparative genomics was used to investigate the genetic environment of blaVIM-1. RESULTS: All isolates belonged to the emerging ST46 lineage and exhibited an extensively drug-resistant phenotype, remaining susceptible only to amikacin. The blaVIM-1 gene was located on a ∼100 kb mobilizable IncC plasmid shared across all isolates. Notably, blaVIM-1 was embedded within a 13 kb Tn7 transposon carrying a complete set of transposition genes and inserted into a class 1 integron structure. Comparative analysis revealed no full homology with previously described IncC plasmids, suggesting a novel genetic arrangement. Phylogenetic analysis demonstrated close relatedness among Italian isolates (<33 SNPs), supporting local clonal circulation, while showing clear separation from previously described NDM-producing ST46 strains. CONCLUSIONS: This study describes the rare association of blaVIM-1 with a Tn7 transposon in P. stuartii, highlighting the genomic plasticity of IncC plasmids and their role in the emergence of new resistance platforms. The identification of this structure in a high-risk lineage underscores the potential for further dissemination of carbapenem resistance in healthcare settings.

IncC↗

PubMed2026

Host-aware Identification of Intrinsic Gene Expression Biopart Parameters using Combinatorial Libraries.

Model-based design in synthetic biology is limited because bioparts are typically characterised by relative metrics that vary across genetic and physiological contexts. To address this, we introduce a host-aware framework for quantitatively characterising bioparts in combinatorial libraries of plasmid-based constitutive expression constructs. The approach integrates a digital twin of Escherichia coli, conditioned on measured growth rate, with model-in-the-loop parameter identification to separate biopart-associated properties from host-dependent effects. Using structured combinatorial libraries, we identify mechanistically interpretable, transferable parameters for plasmid origins, promoters and ribosome binding sites. In particular, we define an intrinsic translation initiation capacity that captures the dominant RBS-associated contribution to translation while context-dependent expression emerges from host physiology and local sequence context. The resulting parameterisation accurately predicts protein synthesis across physiological conditions, supports incremental library expansion, and reveals localised failures of modularity, providing a scalable foundation for predictive host-aware design in synthetic biology.

Escherichia coli↗

PubMed2026

Adaptation of lentiviral vectors for viral gene therapy and their impact on host cell biology.

BACKGROUND: Lentiviral vectors (LVVs) are used as a viral gene therapeutic and were derived from human immunodeficiency virus subtype 1 (HIV-1). LVVs are used to deliver and induce the stable expression of transgenes through genome integration. Current clinical LVV delivery systems do not include HIV-1 major accessory genes; however, critical structural and non-structural HIV-1 proteins are encoded by the 4-plasmid combination that composes the 3rd generation LVV transduction systems. LVVs use HIV-1-like mechanisms for viral genome integration and both transgene delivery and expression. LVVs rely on host cell machinery to transcribe and translate transgenes for either knocking down disease-causing genes and/or supplying functional genes in a targeted disease. LVVs integrate into host intronic and intergenic regions due to genomic accessibility, but there are no known biases toward specific target integration motifs. MAIN BODY: Investigation of LVV integration has uncovered the generation of chimeric LVV-host transcripts and altered host transcript splicing patterns. Several Food and Drug Administration (FDA)-approved LVV-derived therapies are used for treating diseases ranging from beta thalassemia to sickle cell anemia. An increasingly popular application of LVV is in the generation of chimeric antigen receptor (CAR) T cell therapies, which change and enhance T cell antigen specificity and effector function in liquid cancers. In November 2023, all CAR T cell therapies were placed under FDA investigation due to higher-than-expected rates of malignant transformation, hospitalization, and death in treated individuals. LVV integrations driving oncogene expression could be a cause for malignancy development. Current methods for resolving LVV integration patterns are technically limited by the sequencing approach applied allowing for only limited characterization of LVV integration profiles and altered host gene regulation. CONCLUSIONS: A comprehensive understanding of LVV integration and its consequences is necessary for understanding how these events influence host cell gene regulation and splicing, possibly identifying tunable variables for enhanced positive clinical outcomes. Here, we review the development of LVV systems, what is known about LVV integration patterns, technologies used to characterize patterns of LVV integration, and what is understood about the subsequent impact on host cell gene regulation and its potential linkage to patient malignancies.

Humans↗

PubMed2026

Modified Plasmids and Inverted Terminal Repeats Enhance Adeno-Associated Virus Production and Performance.

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.

Humans↗

PubMed2025

The functional study of novel KLHL3 missense mutations associated with pseudohypoaldosteronism type II.

BACKGROUND: Pseudohypoaldosteronism type II (PHA II) is an inherited tubulopathy, clinically defined by three hallmark features, including secondary hypertension, hyperchloremic metabolic acidosis, and persistent hyperkalemia occurring despite maintained glomerular filtration function. Herein, we aim to investigate the association of kelch like family member 3 (KLHL3) gene mutations with PHA II. METHODS: Compound heterozygous KLHL3 mutations were identified through whole-exome sequencing and Sanger validation. AlphaFold-based structural modeling, site-directed mutagenesis of Flag-tagged plasmids, and co-immunoprecipitation (Co-IP)/immunoblotting in vivo were combined to analyze mutant protein interactions and ubiquitination effects. RESULTS: A Chinese patient was identified with two previously unreported KLHL3 variants (c.131G > A [p.R44Q] and c.744 C > G [p.Y248*]), exhibiting a biochemical triad of asymptomatic hyperkalemia, mild metabolic acidosis, and borderline hypertension. Administration of thiazide diuretics effectively normalized the patient’s hyperkalemia and hypertension. A p.R44Q missense mutation predicted as variants of uncertain significance (VOUS) by American College of Medical Genetics and Genomics (ACMG) guidelines, and a p.Y248* nonsense mutation predicted as variants of likely pathogenic. Functional study revealed that the two KLHL3 mutations impair its ubiquitination of with-no-lysine kinase 1 (WNK1) and with-no-lysine kinase 4 (WNK4), and further increase phosphorylation of both SPAK (sterile20/sporulation-specific protein-1 related proline/alanine-rich kinase)/OSR1 (oxidative stress response kinase-1) and Na-Cl-cotransporter (NCC). CONCLUSIONS: Our study characterized two previously unreported KLHL3 mutations, followed by comprehensive in vitro functional analyses to elucidate their pathophysiological contributions at the molecular level.

Humans↗

PubMed2004

Ternary complex factor-serum response factor complex-regulated gene activity is required for cellular proliferation and inhibition of apoptotic cell death.

Members of the ternary complex factor (TCF) subfamily of the ETS-domain transcription factors are activated through phosphorylation by mitogen-activated protein kinases (MAPKs) in response to a variety of mitogenic and stress stimuli. The TCFs bind and activate serum response elements (SREs) in the promoters of target genes in a ternary complex with a second transcription factor, serum response factor (SRF). The association of TCFs with SREs within immediate-early gene promoters is suggestive of a role for the ternary TCF-SRF complex in promoting cell cycle entry and proliferation in response to mitogenic signaling. Here we have investigated the downstream gene regulatory and phenotypic effects of inhibiting the activity of genes regulated by TCFs by expressing a dominantly acting repressive form of the TCF, Elk-1. Inhibition of ternary complex activity leads to the downregulation of several immediate-early genes. Furthermore, blocking TCF-mediated gene expression leads to growth arrest and triggers apoptosis. By using mutant Elk-1 alleles, we demonstrated that these effects are via an SRF-dependent mechanism. The antiapoptotic gene Mcl-1 is identified as a key target for the TCF-SRF complex in this system. Thus, our data confirm a role for TCF-SRF-regulated gene activity in regulating proliferation and provide further evidence to indicate a role in protecting cells from apoptotic cell death.

Alleles↗

PubMed2026

Whole-genome sequence of the type strain and two field strains of Arsenicicoccus dermatophilus causing pododermatitis in greater flamingos (Phoenicopterus roseus).

The complete genome sequence of the type strain KM894/11T and two field strains of Arsenicicoccus dermatophilus (KM18/12; KM9/12) isolated from foot skin lesions of captive greater flamingos was determined using Oxford Nanopore and Illumina sequencing technologies. The genomes differ structurally by a large ~650 kb inverted chromosomal fragment and plasmid content.

dermatitis↗