-
Microbiology Spectrum Dec 2023Carbapenem-resistant (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, can also cause...
Carbapenem-resistant (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.
Topics: Humans; Klebsiella pneumoniae; Anti-Bacterial Agents; Carbapenems; Virulence; Plasmids; Carbapenem-Resistant Enterobacteriaceae; Klebsiella Infections
PubMed: 37982629
DOI: 10.1128/spectrum.03038-23 -
Virulence Dec 2023Emerging mobile colistin resistance () genes pose a significant threat to public health for colistin was used as the last resort to treat multidrug-resistant (MDR)...
Emerging mobile colistin resistance () genes pose a significant threat to public health for colistin was used as the last resort to treat multidrug-resistant (MDR) pathogenic bacterial infections. Hypervirulent (hvKP) is a clinically significant pathogen resulting in highly invasive infections, often complicated by devastating dissemination. Worryingly, the untreatable and severe infections caused by -harbouring hvKP leave the selection of antibiotics for clinical anti-infective treatment in a dilemma. Herein, we screened 3,461 isolates from a tertiary teaching hospital from November 2018 to March 2021, and an -harbouring hvKP FAHZZU2591 with a conjugative plasmid was identified from paediatric sepsis. This is the first report of MCR-8-producing hvKP from paediatric sepsis to our best knowledge. The susceptibility, genetic features, and plasmid profiles of the isolate were investigated. Further, we assessed the virulence potential of FAHZZU2591 and verified its pathogenicity and invasive capacity using a mouse model. The phylogenetic analysis of -bearing revealed that China is the predominant reservoir of the gene, and the clinic is the primary source. Our work highlights the risk for the spread of -positive hvKP in clinical, especially in paediatric sepsis, and the persistent surveillance of colistin-resistance hvKP is urgent.
Topics: Humans; Colistin; Klebsiella pneumoniae; Phylogeny; Anti-Bacterial Agents; Plasmids; Genomics; Sepsis; Klebsiella Infections
PubMed: 36529894
DOI: 10.1080/21505594.2022.2158980 -
Science Advances Apr 2024Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face...
Transfected stem cells and T cells are promising in personalized cell therapy and immunotherapy against various diseases. However, existing transfection techniques face a fundamental trade-off between transfection efficiency and cell viability; achieving both simultaneously remains a substantial challenge. This study presents an acoustothermal transfection method that leverages acoustic and thermal effects on cells to enhance the permeability of both the cell membrane and nuclear envelope to achieve safe, efficient, and high-throughput transfection of primary T cells and stem cells. With this method, two types of plasmids were simultaneously delivered into the nuclei of mesenchymal stem cells (MSCs) with efficiencies of 89.6 ± 1.2%. CXCR4-transfected MSCs could efficiently target cerebral ischemia sites in vivo and reduce the infarct volume in mice. Our acoustothermal transfection method addresses a key bottleneck in balancing the transfection efficiency and cell viability, which can become a powerful tool in the future for cellular and gene therapies.
Topics: Mice; Animals; Transfection; Mesenchymal Stem Cells; Plasmids; Cell Membrane; Cell- and Tissue-Based Therapy
PubMed: 38630814
DOI: 10.1126/sciadv.adk1855 -
Molecular Therapy : the Journal of the... Sep 2023USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene...
USH2A mutations are a common cause of autosomal recessive retinitis pigmentosa (RP) and Usher syndrome, for which there are currently no approved treatments. Gene augmentation is a valuable therapeutic strategy for treating many inherited retinal diseases; however, conventional adeno-associated virus (AAV) gene therapy cannot accommodate cDNAs exceeding 4.7 kb, such as the 15.6-kb-long USH2A coding sequence. In the present study, we adopted an alternative strategy to successfully generate scaffold/matrix attachment region (S/MAR) DNA plasmid vectors containing the full-length human USH2A coding sequence, a GFP reporter gene, and a ubiquitous promoter (CMV or CAG), reaching a size of approximately 23 kb. We assessed the vectors in transfected HEK293 cells and USH2A patient-derived dermal fibroblasts in addition to ush2a zebrafish microinjected with the vector at the one-cell stage. pS/MAR-USH2A vectors drove persistent transgene expression in patient fibroblasts with restoration of usherin. Twelve months of GFP expression was detected in the photoreceptor cells, with rescue of Usher 2 complex localization in the photoreceptors of ush2a zebrafish retinas injected with pS/MAR-USH2A. To our knowledge, this is the first reported vector that can be used to express full-length usherin with functional rescue. S/MAR DNA vectors have shown promise as a novel non-viral retinal gene therapy, warranting further translational development.
Topics: Animals; Humans; Usher Syndromes; Zebrafish; HEK293 Cells; Mutation; DNA; Plasmids; Extracellular Matrix Proteins
PubMed: 37337429
DOI: 10.1016/j.ymthe.2023.06.012 -
Antimicrobial Agents and Chemotherapy Aug 2023In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high...
In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high levels of resistance to ceftazidime-avibactam (CZA). One of these strains reached high levels of resistance to both CZA and carbapenems and carried two copies of and one copy of located on plasmid pKpQIL. The genomes and plasmids of CZA-resistant ST307 strains were analyzed to identify the molecular mechanisms leading to the evolution of resistance and compared with ST307 genomes at local and global levels. A complex pattern of multiple plasmids in rearranged configurations, coresident within the CZA-carbapenem-resistant K. pneumoniae strain, was observed. Characterization of these plasmids revealed recombination and segregation events explaining why K. pneumoniae isolates from the same patient had different antibiotic resistance profiles. This study illustrates the intense genetic plasticity occurring in ST307, one of the most worldwide-diffused K. pneumoniae high-risk clones.
Topics: Humans; Meropenem; Anti-Bacterial Agents; Klebsiella pneumoniae; Klebsiella Infections; Bacterial Proteins; beta-Lactamases; Ceftazidime; Azabicyclo Compounds; Plasmids; Carbapenems; Microbial Sensitivity Tests
PubMed: 37428086
DOI: 10.1128/aac.00368-23 -
Microbial Biotechnology Jan 2024Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids,... (Review)
Review
Mobile genetic elements (MGEs) are crucial for horizontal gene transfer (HGT) in bacteria and facilitate their rapid evolution and adaptation. MGEs include plasmids, integrative and conjugative elements, transposons, insertion sequences and bacteriophages. Notably, the spread of antimicrobial resistance genes (ARGs), which poses a serious threat to public health, is primarily attributable to HGT through MGEs. This mini-review aims to provide an overview of the mechanisms by which MGEs mediate HGT in microbes. Specifically, the behaviour of conjugative plasmids in different environments and conditions was discussed, and recent methodologies for tracing the dynamics of MGEs were summarised. A comprehensive understanding of the mechanisms underlying HGT and the role of MGEs in bacterial evolution and adaptation is important to develop strategies to combat the spread of ARGs.
Topics: Interspersed Repetitive Sequences; Gene Transfer, Horizontal; Plasmids; Bacteria; Bacteriophages; Anti-Bacterial Agents
PubMed: 38226780
DOI: 10.1111/1751-7915.14408 -
Bioinformatics (Oxford, England) May 2024PlasCAT (Plasmid Cloud Assembly Tool) is an easy-to-use cloud-based bioinformatics tool that enables de novo plasmid sequence assembly from raw sequencing data....
SUMMARY
PlasCAT (Plasmid Cloud Assembly Tool) is an easy-to-use cloud-based bioinformatics tool that enables de novo plasmid sequence assembly from raw sequencing data. Nontechnical users can now assemble sequences from long reads and short reads without ever touching a line of code. PlasCAT uses high-performance computing servers to reduce run times on assemblies and deliver results faster.
AVAILABILITY AND IMPLEMENTATION
PlasCAT is freely available on the web at https://sequencing.genofab.com. The assembly pipeline source code and server code are available for download at https://bitbucket.org/genofabinc/workspace/projects/PLASCAT. Click the Cancel button to access the source code without authenticating. Web servers implemented in React.js and Python, with all major browsers supported.
Topics: Software; Plasmids; Cloud Computing; Computational Biology; Sequence Analysis, DNA; Internet
PubMed: 38696761
DOI: 10.1093/bioinformatics/btae299 -
Antimicrobial Agents and Chemotherapy Dec 2023Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant , but carbapenem resistance due to acquisition of...
Carbapenems are considered last-resort antibiotics for the treatment of infections caused by multidrug-resistant , but carbapenem resistance due to acquisition of carbapenemase genes is a growing threat that has been reported worldwide. carbapenemase () is the most common type of carbapenemase in Canada and elsewhere; it can hydrolyze penicillins, cephalosporins, aztreonam, and carbapenems and is frequently found on mobile plasmids in the Tn transposon. This means that alongside clonal expansion, can disseminate through plasmid- and transposon-mediated horizontal gene transfer. We applied whole genome sequencing to characterize the molecular epidemiology of 829 carbapenemase-producing isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2010 to 2021. Using a combination of short-read and long-read sequencing, we obtained 202 complete and circular -encoding plasmids. Using MOB-suite, 10 major plasmid clusters were identified from this data set which represented 87% (175/202) of the Canadian -encoding plasmids. We further estimated the genomic location of incomplete -encoding contigs and predicted a plasmid cluster for 95% (603/635) of these. We identified different patterns of carbapenemase mobilization across Canada related to different plasmid clusters, including clonal transmission of IncF-type plasmids (108/829, 13%) in clonal complex 258 and novel repE(pEh60-7) plasmids (44/829, 5%) in ST316, and horizontal transmission of IncL/M (142/829, 17%) and IncN-type plasmids (149/829, 18%) across multiple genera. Our findings highlight the diversity of genomic loci and indicate that multiple, distinct plasmid clusters have contributed to spread and persistence in Canada.
Topics: Humans; Canada; beta-Lactamases; Plasmids; Bacterial Proteins; Klebsiella pneumoniae; Anti-Bacterial Agents; Carbapenems; Genomics; Klebsiella Infections; Microbial Sensitivity Tests
PubMed: 37971242
DOI: 10.1128/aac.00860-23 -
Microbiology (Reading, England) Jul 2023This short primer is intended to give an overview of bacterial plasmids for those not yet familiar with these fascinating genetic elements. It covers their basic... (Review)
Review
This short primer is intended to give an overview of bacterial plasmids for those not yet familiar with these fascinating genetic elements. It covers their basic properties but does not attempt to cover the diversity of phenotypic properties that can be encoded by plasmids, and includes suggestions for further reading.
Topics: Bacteria; Conjugation, Genetic; DNA, Bacterial; Gene Transfer, Horizontal; Logic; Plasmids
PubMed: 37395112
DOI: 10.1099/mic.0.001336 -
Genes Feb 2024Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production... (Review)
Review
Until very recently, the major use, for gene therapy, specifically of linear or circular DNA, such as plasmids, was as ancillary products for viral vectors' production or as a genetic template for mRNA production. Thanks to targeted and more efficient physical or chemical delivery techniques and to the refinement of their structure, non-viral plasmid DNA are now under intensive consideration as pharmaceutical drugs. Plasmids traditionally carry an antibiotic resistance gene for providing the selection pressure necessary for maintenance in a bacterial host. Nearly a dozen different antibiotic-free gene vectors have now been developed and are currently assessed in preclinical assays and phase I/II clinical trials. Their reduced size leads to increased transfection efficiency and prolonged transgene expression. In addition, associating non-viral gene vectors and DNA transposons, which mediate transgene integration into the host genome, circumvents plasmid dilution in dividing eukaryotic cells which generate a loss of the therapeutic gene. Combining these novel molecular tools allowed a significantly higher yield of genetically engineered T and Natural Killer cells for adoptive immunotherapies due to a reduced cytotoxicity and increased transposition rate. This review describes the main progresses accomplished for safer, more efficient and cost-effective gene and cell therapies using non-viral approaches and antibiotic-free gene vectors.
Topics: Anti-Bacterial Agents; Genetic Vectors; Plasmids; Transfection; Transgenes
PubMed: 38540320
DOI: 10.3390/genes15030261