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Current Protocols Oct 2023Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can...
Klebsiella pneumoniae is a Gram-negative, rod-shaped bacterium commonly found in the human intestine. Although it typically exists as part of the normal flora, it can also cause healthcare-associated infections with severe consequences. Understanding the specific genes responsible for its virulence through genetic manipulation is crucial for potential therapeutic interventions. However, manipulating K. pneumoniae presents challenges due to its exopolysaccharide capsule. This article presents a comprehensive collection of protocols designed to facilitate the genetic manipulation of K. pneumoniae. By following these protocols, researchers will acquire the necessary skills to prepare electrocompetent cells, utilize electroporation for efficient plasmid DNA introduction, construct isogenic mutants using the λ Red recombinase system, and generate a complementation vector for restoring the phenotypic traits of knockout strains. These protocols provide valuable tools and techniques to navigate the intricacies associated with studying and modifying K. pneumoniae. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparing electrocompetent K. pneumoniae cells Alternate Protocol 1: Preparing electrocompetent K. pneumoniae cells for recombineering Basic Protocol 2: Transforming K. pneumoniae using electroporation Basic Protocol 3: Constructing isogenic mutants in K. pneumoniae using the λ Red recombinase system Support Protocol 1: Confirming a knockout via colony PCR Support Protocol 2: Verifying absence of secondary mutations Basic Protocol 4: Generating unmarked knockout mutants in K. pneumoniae using the pFLP plasmid Basic Protocol 5: Constructing a complementation vector for K. pneumoniae.
Topics: Humans; Klebsiella pneumoniae; Plasmids; Virulence; Mutation; Recombinases
PubMed: 37889096
DOI: 10.1002/cpz1.912 -
Veterinary Microbiology Dec 2023Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA... (Review)
Review
Virulent Rhodococcus equi strains expressing virulence-associated 15-17 kDa protein (VapA) and having a large virulence plasmid (pVAPA) of 85-90 kb containing vapA gene are pathogenic for horses. In the last two decades, following pVAPA, two host-associated virulence plasmid types of R. equi have been discovered: a circular plasmid, pVAPB, associated with porcine isolates in 1995, and a recently detected linear plasmid, pVAPN, related to bovine and caprine isolates. Molecular epidemiological studies of R. equi infection in foals on horse-breeding farms in Japan and many countries around the world have been conducted in the last three decades, and the epidemiological studies using restriction enzyme digestion patterns of plasmid DNAs from virulent isolates have shown 14 distinct pVAPA subtypes and their geographical preference. This short review summarizes previous reports regarding equine-associated pVAPA subtypes in the world and discusses their geographic distribution from the standpoint of horse movements.
Topics: Animals; Horses; Cattle; Swine; Rhodococcus equi; Goats; Virulence Factors; Actinomycetales Infections; Plasmids; Bacterial Proteins; Horse Diseases; Cattle Diseases; Goat Diseases; Swine Diseases
PubMed: 38000208
DOI: 10.1016/j.vetmic.2023.109919 -
Applied Microbiology and Biotechnology Dec 2023The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake,...
The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) bla Escherichia coli and 40 (9.1%) bla Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for bla E. coli, while all bla K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including bla, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that bla E. coli was highly heterogeneous while the bla K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with bla spread, while IncFII-R and IncFII plasmids mediated bla spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including bla. In addition, all of the bla K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried bla- and pLVKP-like virulence plasmids from migratory birds. The bla K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (bla, bla436, bla , qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.
Topics: Humans; Enterobacteriaceae; Escherichia coli; beta-Lactamases; Phylogeny; Lakes; Klebsiella pneumoniae; Plasmids; Anti-Bacterial Agents; Genomics; China; Klebsiella Infections
PubMed: 37861819
DOI: 10.1007/s00253-023-12746-3 -
Science Advances Mar 2024Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented...
Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 μl of NPs containing 19.2 μg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 μl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 μl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.
Topics: Animals; Humans; Swine; Swine, Miniature; Retina; Plasmids; Genetic Therapy; Nanoparticles
PubMed: 38457512
DOI: 10.1126/sciadv.adl3576 -
Nature Microbiology Oct 2023Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera...
Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera patients in 2018. Here, to characterize circulating genotypes, we analysed 260 isolates sampled in Yemen between 2018 and 2019. Eighty-four percent of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sub-lineage T13, whereas 16% were non-toxigenic, from divergent non-7PET lineages. Treatment of severe cholera with macrolides between 2016 and 2019 coincided with the emergence and dominance of T13 subclones carrying an incompatibility type C (IncC) plasmid harbouring an MDR pseudo-compound transposon. MDR plasmid detection also in endemic non-7PET V. cholerae lineages suggested genetic exchange with 7PET epidemic strains. Stable co-occurrence of the IncC plasmid with the SXT family of integrative and conjugative element in the 7PET background has major implications for cholera control, highlighting the importance of genomic epidemiological surveillance to limit MDR spread.
Topics: Humans; Cholera; Vibrio cholerae O1; Yemen; Plasmids; Genomics
PubMed: 37770747
DOI: 10.1038/s41564-023-01472-1 -
The Journal of Antimicrobial... Sep 2023Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the...
OBJECTIVES
Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the environment, or human and animal guts (One Health concept). The aim of our study was to evaluate whether subdosages of antibiotics administered in veterinary medicine could enhance plasmid transfer and, consequently, resistance gene exchange in gut microbiota.
METHODS
Conjugation frequencies were determined with Escherichia coli strains carrying IncL- (blaOXA-48) or IncI1-type (blaCTX-M-1) plasmids subjected to a series of subinhibitory concentrations of antibiotics used in veterinary medicine, namely amoxicillin, ceftiofur, apramycin, neomycin, enrofloxacin, colistin, erythromycin, florfenicol, lincomycin, oxytetracycline, sulfamethazine, tiamulin and the ionophore narasin. Treatments with subinhibitory dosages were performed with and without supplementation with the antioxidant edaravone, known as a mitigator of the inducibility effect of several antibiotics on plasmid conjugation frequency (PCF). Expression of SOS-response associated genes and fluorescence-based reactive oxygen species (ROS) detection assays were performed to evaluate the stress oxidative response.
RESULTS
Increased PCFs were observed for both strains when treating with florfenicol and oxytetracycline. Increased expression of the SOS-associated recA gene also occurred concomitantly, as well as increased ROS production. Addition of edaravone to the treatments reduced their PCF and also showed a decreasing effect on SOS and ROS responses for both plasmid scaffolds.
CONCLUSIONS
We showed here that some antibiotics used in veterinary medicine may induce transfer of plasmid-encoded resistance and therefore may contribute to the worldwide spread of antibiotic resistance genes.
Topics: Animals; Humans; Anti-Bacterial Agents; Oxytetracycline; Edaravone; Reactive Oxygen Species; Escherichia coli; Plasmids; Drug Resistance, Microbial; Gene Transfer, Horizontal
PubMed: 37486104
DOI: 10.1093/jac/dkad226 -
BMC Microbiology Dec 2023The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.
OBJECTIVE
The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.
METHODS
Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.
RESULTS
In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.
CONCLUSION
In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.
Topics: Vancomycin; RNA, Guide, CRISPR-Cas Systems; Vancomycin Resistance; CRISPR-Cas Systems; Anti-Bacterial Agents; Plasmids; Bacterial Proteins
PubMed: 38049763
DOI: 10.1186/s12866-023-03136-w -
Cell Reports Methods Dec 2023We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection...
We created a generalizable pipeline for antibiotic-resistance-gene-free plasmid (ARGFP)-based cloning using a dual auxotrophic- and essential-gene-based selection strategy. We use auxotrophic selection to construct plasmids in engineered E. coli DH10B cloning strains and both auxotrophic- and essential-gene-based selection to (1) select for recombinant strains and (2) maintain a plasmid in E. coli Nissle 1917, a common chassis for engineered probiotic applications, and E. coli MG1655, the laboratory "wild-type" E. coli strain. We show that our approach has comparable efficiency to that of antibiotic-resistance-gene-based cloning. We also show that the double-knockout Nissle and MG1655 strains are simple to transform with plasmids of interest. Notably, we show that the engineered Nissle strains are amenable to long-term plasmid maintenance in repeated culturing as well as in the mouse gut, demonstrating the potential for broad applications while minimizing the risk of antibiotic resistance spread via horizontal gene transfer.
Topics: Animals; Mice; Anti-Bacterial Agents; Escherichia coli; Plasmids; Drug Resistance, Microbial; Cloning, Molecular
PubMed: 38086386
DOI: 10.1016/j.crmeth.2023.100669 -
Current Genetics Dec 2023pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible...
pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3'-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments. Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation. Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.
Topics: Plasmids; Escherichia coli; Recombinant Proteins; Anti-Bacterial Agents; Aminoglycosides
PubMed: 37938343
DOI: 10.1007/s00294-023-01276-0 -
Journal of Visualized Experiments : JoVE Jun 2024Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of...
Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope. Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.
Topics: Animals; Microinjections; Gene Transfer Techniques; Drosophila; Plasmids; Embryo, Nonmammalian; Animals, Genetically Modified; Integrases
PubMed: 38912770
DOI: 10.3791/66679