Chimeric Plasmid Research Articles

Development of a novel strategy to reduce diagnostic errors in real-time polymerase chain reaction using probe-based techniques

Real-time PCR assays are valuable tools for the rapid and accurate diagnosis of infectious diseases by identifying the nucleic acid sequences of pathogens. Here, we aimed to develop a recombinant plasmid-based standard for validating the sensitivity of different molecular diagnostic methods adopting the Jonstrup assay (J assay). Chimeric plasmid DNA (cpDNA) harboring various pathogen genes and the target site of the J assay was constructed. Our findings revealed that the J assay could detect a single copy of the target gene similar to digital droplet PCR. The detection sensitivity of each established real-time PCR method for various disease diagnoses was evaluated using the cpDNA. Although most methods showed high sensitivity, similar to that of the J assay, the VHS Garver and SARS-CoV-2 diagnostic methods exhibited detection sensitivities tenfold lower than that of the J assay. To ensure accuracy of results and avoid genetic contamination from positive controls, we introduced an additional probe attachment site emitting distinct fluorescent signals within the cpDNA. Target genes and exogenous sequences within the plasmid DNA were simultaneously detected in a single assay using this unique method. Our approach will help improve the sensitivity of diagnostic methods and develop novel diagnostic methods based on molecular techniques.

Functional features of KPC-109, a novel 270-loop KPC-3 mutant mediating resistance to avibactam-based β-lactamase inhibitor combinations and cefiderocol

To investigate a ceftazidime/avibactam (CZA)-resistant Klebsiella pneumoniae (NE368), isolated from a patient exposed to CZA, expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant (KPC-109). Antimicrobial susceptibility testing was performed by reference broth microdilution. Whole-genome sequencing (WGS) analysis of NE368 was performed combining a short- and long-reads approach (Illumina and Oxford Nanopore Technologies). Functional characterization of KPC-109 was performed to investigate the impact of KPC-109 production on the β-lactam resistance phenotype of various Escherichia coli and Klebsiella pneumoniae strains, including derivatives of K. pneumoniae with OmpK35 and OmpK36 porin alterations. Horizontal transfer of the KPC-109-encoding plasmid was investigated by conjugation and transformation experiments. K. pneumoniae NE368 was isolated from a patient after repeated CZA exposure, and showed resistance to CZA, fluoroquinolones, piperacillin/tazobactam, expanded-spectrum cephalosporins, amikacin, carbapenems and cefiderocol. WGS revealed the presence of a large chimeric plasmid of original structure (pKPN-NE368), encoding a novel 270-loop mutated KPC-3 variant (KPC-109; ins_270_KYNKDD). KPC-109 production mediated resistance/decreased susceptibility to avibactam-based combinations (with ceftazidime, cefepime and aztreonam) and cefiderocol, with a trade-off on carbapenem resistance. However, in the presence of porin alterations commonly encountered in high-risk clonal lineages of K. pneumoniae, KPC-109 was also able to confer clinical-level resistance to carbapenems. Resistance of NE368 to cefiderocol was likely contributed by KPC-109 production acting in concert with a mutated EnvZ sensor kinase. The KPC-109-encoding plasmid did not appear to be conjugative. These findings expand current knowledge about the diversity of emerging KPC enzyme variants with 270-loop alterations that can be encountered in the clinical setting.

OR11-05 High-throughput Screening Identifies TR4 Modulators For Potential Therapeutic Application

Abstract Disclosure: S. Khowal: None. D. Zhang: None. R. Damoiseaux: None. K. Javaherian: None. A.P. Heaney: None. Cushing Disease (CD), due to an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor, leads to excess cortisol and is a life-threatening “orphan disease”. Although surgical removal can be effective, CD frequently recurs. Thereafter, repeat surgery, radiation therapy and/or bilateral adrenalectomy are not always successful and cause significant morbidity. Medical therapy has significantly improved but there is a clear unmet need for efficacious and safe therapies that target the pituitary corticotroph tumor itself to offer biochemical and tumor control. We previously demonstrated that the orphan testicular receptor 4 (TR4) regulates pro-opiomelanocortin gene transcription and ACTH secretion. Herein, we used the Flexi Vector system to identify TR4 modulators by fusing the TR4 LBD (380-615aa) to a 3’ GAL4 DBD to generate a chimeric fusion plasmid, pBIND-TR4-LBD. The pBIND-TR4-LBD construct and an exogenous luciferase reporter construct pGL4.35 (luc2P/9XGAL4UAS), containing 9 copies of a GAL4 responsive upstream activating sequence (UAS), were then transiently transfected into human embryonic kidney (HEK293) cells. Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. In a high throughput screen (HTS) of 27,705 chemicals, we identified several potential “hit” TR4 modulators that induced or inhibited TR4 LBD-directed GAL4UAS-directed luciferase expression by >3 -fold. Compounds from the HTS included ATP-sensitive K+ and calcium channel blockers, steroid hormones and modulators of cell proliferation and inflammation and several agonist and antagonist “hit” compounds induced TR4 >10 fold or inhibited TR4 by 90% respectively with minimal cell toxicity (<10%). In silico analysis of interactions between the TR4 LBD and these potential ligands indicated that the GLU-534 & ARG-565 may be crucial residues involved in TR4 ligand binding. Dose-response curves to calculate EC50 of these potential TR4 hit compounds and mode-of-action characterization are in progress. Our studies have identified several potent TR4 agonistic and antagonistic compounds and lay the framework for the discovery of safe and efficacious lead TR4 ligands that can be further advanced as potential drug therapies for CD and other diseases. Presentation: Friday, June 16, 2023

Genetic background of neomycin resistance in clinical Escherichia coli isolated from Danish pig farms.

Neomycin is the first-choice antibiotic for the treatment of porcine enteritis caused by enterotoxigenic Escherichia coli. Resistance to this aminoglycoside is on the rise after the increased use of neomycin due to the ban on zinc oxide. We identified the neomycin resistance determinants and plasmid contents in a historical collection of 128 neomycin-resistant clinical E. coli isolates from Danish pig farms. All isolates were characterized by whole-genome sequencing and antimicrobial susceptibility testing, followed by conjugation experiments and long-read sequencing of eight selected representative strains. We detected 35 sequence types (STs) with ST100 being the most prevalent lineage (38.3%). Neomycin resistance was associated with two resistance genes, namely aph(3')-Ia and aph(3')-Ib, which were identified in 93% and 7% of the isolates, respectively. The aph(3')-Ia was found on different large conjugative plasmids belonging to IncI1α, which was present in 67.2% of the strains, on IncHI1, IncHI2, and IncN, as well as on a multicopy ColRNAI plasmid. All these plasmids except ColRNAI carried genes encoding resistance to other antimicrobials or heavy metals, highlighting the risk of co-selection. The aph(3')-Ib gene occurred on a 19 kb chimeric, mobilizable plasmid that contained elements tracing back its origin to distantly related genera. While aph(3')-Ia was flanked by either Tn903 or Tn4352 derivatives, no clear association was observed between aph(3')-Ib and mobile genetic elements. In conclusion, the spread of neomycin resistance in porcine clinical E. coli is driven by two resistance determinants located on distinct plasmid scaffolds circulating within a highly diverse population dominated by ST100. IMPORTANCE Neomycin is the first-choice antibiotic for the management of Escherichia coli enteritis in pigs. This work shows that aph(3')-Ia and to a lesser extent aph(3')-Ib are responsible for the spread of neomycin resistance that has been recently observed among pig clinical isolates and elucidates the mechanisms of dissemination of these two resistance determinants. The aph(3')-Ia gene is located on different conjugative plasmid scaffolds and is associated with two distinct transposable elements (Tn903 and Tn4352) that contributed to its spread. The diffusion of aph(3')-Ib is mediated by a small non-conjugative, mobilizable chimeric plasmid that likely derived from distantly related members of the Pseudomonadota phylum and was not associated with any detectable mobile genetic element. Although the spread of neomycin resistance is largely attributable to horizontal transfer, both resistance determinants have been acquired by a predominant lineage (ST100) associated with enterotoxigenic E. coli, which accounted for approximately one-third of the strains.

Co-integrate Col3m bla NDM-1-harboring plasmids in clinical Providencia rettgeri isolates from Argentina.

The first cases of bla NDM in Argentina were detected in three Providencia rettgeri (Pre) recovered from two hospitals in Buenos Aires city in 2013. The isolates were genetically related, but the plasmid profile was different. Here, we characterized the bla NDM-1-harboring plasmids of the first three cases detected in Argentina. Hybrid assembly obtained from short- and long-read sequencing rendered bla NDM-1 in Col3M plasmids of ca. 320 kb (p15268A_320) in isolate PreM15268, 210 kb (p15758B_210) in PreM15758, and 225 kb (p15973A_225) in PreM15973. In addition, PreM15758 harbored a 98-kb circular plasmid (p15758C_98) flanked by a putative recombination site (hin-TnAs2), with 100% nucleotide ID and coverage with p15628A_320. Analysis of PFGE/S1-nuclease gel, Southern hybridization with bla NDM-1 probe, hybrid assembly of short and long reads suggests that pM15758C_98 can integrate by homologous recombination. The three bla NDM-1-plasmids were non-conjugative in vitro. Moreover, tra genes were incomplete, and oriT was not found in the three bla NDM-1-plasmids. In two isolates, blaNDM-1 was embedded in a partially conserved structure flanked by two ISKox2. In addition, all plasmids harbored aph(3')-Ia, aph(3')-VI, and qnrD1 genes and aac(6´)Ib-cr, bla OXA-1, catB3, and arr3 as part of a class 1 integron. Also, p15268A_320 and p15973A_225 harbored bla PER-2. To the best of our knowledge, this is the first report of clinical P. rettgeri harboring blaNDM-1 in an atypical genetic environment and located in unusual chimeric Col3M plasmids. The study and continuous surveillance of these pathogens are crucial to tracking the evolution of these resistant plasmids and finding solutions to tackle their dissemination. IMPORTANCE Infections caused by carbapenem hydrolyzing enzymes like NDM (New Delhi metallo-beta-lactamase) represent a serious problem worldwide because they restrict available treatment options and increase morbidity and mortality, and treatment failure prolongs hospital stays. The first three cases of NDM in Argentina were caused by genetically related P. rettgeri recovered in two hospitals. In this work, we studied the genetic structure of the plasmids encoding bla NDM in those index cases and revealed the enormous plasticity of these genetic elements. In particular, we found a small plasmid that was also found inserted in the larger plasmids by homologous recombination as a co-integrate element. We also found that the bla NDM plasmids were not able to transfer or move to other hosts, suggesting their role as reservoir elements for the acquisition of resistance genes. It is necessary to unravel the dissemination strategies and the evolution of these resistant plasmids to find solutions to tackle their spread.

Dual gene-activated dermal scaffolds regulate angiogenesis and wound healing by mediating the coexpression of VEGF and angiopoietin-1.

The vascularization of dermal substitutes is a key challenge in efforts to heal deep skin defects. In this study, dual gene-activated dermal scaffolds (DGADSs-1) were fabricated by loading nanocomposite particles of polyethylenimine (PEI)/multiple plasmid DNAs (pDNAs) encoding vascular endothelial growth factor and angiopoietin-1 at a ratio of 1:1. In a similar manner, DGADSs-2 were loaded with a chimeric plasmid encoding both VEGF and Ang-1. In vitro studies showed that both types of DGADSs released PEI/pDNA nanoparticles in a sustained manner; they demonstrated effective transfection ability, leading to upregulated expression of VEGF and Ang-1. Furthermore, both types of DGADSs promoted fibroblast proliferation and blood vessel formation, although DGADSs-1 showed a more obvious promotion effect. A rat full-thickness skin defect model showed that split-thickness skin transplanted using a one-step method could achieve full survival at the 12th day after surgery in both DGADSs-1 and DGADSs-2 groups, and the vascularization time of dermal substitutes was significantly shortened. Compared with the other three groups of scaffolds, the DGADSs-1 group had significantly greater cell infiltration, collagen deposition, neovascularization, and vascular maturation, all of which promoted wound healing. Thus, compared with single-gene-activated dermal scaffolds, DGADSs show greater potential for enhancing angiogenesis. DGADSs with different loading modes also exhibited differences in terms of angiogenesis; the effect of loading two genes (DGADSs-1) was better than the effect of loading a chimeric gene (DGADSs-2). In summary, DGADSs, which continuously upregulate VEGF and Ang-1 expression, offer a new functional tissue-engineered dermal substitute with the ability to activate vascularization.

PMON149 Identification of TR4 Modulators for Treatment of Cushing Disease.

Abstract Cushing Disease (CD) is caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor that causes excess adrenal-derived cortisol. It is a life-threatening "orphan disease" with an annual health care cost >7-fold higher than average patients. Surgical removal is the current first-line therapy but the disease frequently recurs. Repeat surgery, radiation therapy and bilateral adrenalectomy are not always successful and associated with significant morbidity. Currently available drugs or those in clinical trials for CD do not target the pituitary corticotroph tumor itself and escape from control is common with long-term use. A clear unmet need for efficacious and safe therapies that offer biochemical and tumor control exists. We hypothesize that direct targeting of corticotroph tumors to modulate ACTH and in turn, glucocorticoid secretion is the optimal way to treat CD. We recently demonstrated that the orphan testicular receptor 4 (TR4, also known as NR2C2) is a potent regulator of hypothalamic-pituitary-adrenal (HPA) axis function and directly regulates pro-opiomelanocortin (POMC) gene transcription and ACTH secretion. To identify and characterize TR4 small molecule modulators, we used the Flexi Vector system to fuse the TR4 224-615aa LBD to the 3' GAL4 DBD to generate a chimeric fusion plasmid (pBIND-TR4-LBD) that constitutively expresses a Renilla luciferase serving as an internal reporter control. We then transiently transfected our chimeric pBIND-TR4-LBD construct into human embryonic kidney (HEK293) cells which express an exogenous reporter construct pGL4.35 (GloResponse, luc2P/9XGAL4UAS-HEK293 Cell Line, Promega) that is driven by multiple copies of a GAL4 responsive upstream activating sequence (UAS). Following treatment with TR4 ligands or modulators, our TR4-LBD undergoes a conformational change and recruits additional cofactors and RNA polymerase II to the complex to drive GAL4-directed firefly luciferase expression. This dual-luciferase format allows us to detect compound cytotoxicity or off-target change in expression during chemical screening, greatly improving data quality. In a pilot drug screen of a LOPAC library and using robust z-score statistics (z-score>3), representing a luciferase to renilla induction > 3 standard deviations of baseline variability, we have identified several G-protein coupled ion channel regulators as potential "hit" TR4 modulators. These identified GPCRs regulate phosphatidylinositol and cyclic AMP effector systems, and activate several signaling pathway kinases. Mode-of-action and dose-response evaluation to calculate EC50 of these potential TR4 hit compounds is under investigation and lead compounds will be further validated using a POMC transactivation assay in murine and human corticotroph tumor cells. Our studies have begun to identify and rigorously validate compounds that efficiently and specifically abrogate TR4 actions to inhibit ACTH secretion and may lead to the discovery of safe and efficacious lead TR4 inhibitory compounds that can be further advanced as potential drug therapies for CD. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

The diversity in antimicrobial resistance of MDR Enterobacteriaceae among Chinese broiler and laying farms and two mcr-1 positive plasmids revealed their resistance-transmission risk.

This research aimed to investigate the microbial composition and diversity of antimicrobial resistance genes (ARGs) found in Chinese broiler and layer family poultry farms. We focused on the differences in resistance phenotypes and genotypes of multidrug-resistant Enterobacteriaceae (MDRE) isolated from the two farming environments and the existence and transmissibility of colistin resistance gene mcr-1. Metagenomic analysis showed that Firmicutes and Bacteroides were the dominant bacteria in broiler and layer farms. Many aminoglycoside and tetracycline resistance genes were accumulated in these environments, and their absolute abundance was higher in broiler than in layer farms. A total of 526 MDRE were isolated with a similar distribution in both farms. The results of the K-B test showed that the resistance rate to seven antimicrobials including polymyxin B and meropenem in broiler poultry farms was significantly higher than that in layer poultry farms (P ≤ 0.05). PCR screening results revealed that the detection rates of mcr-1, aph(3’)Ia, aadA2, blaoxa–1, blaCTX–M, fosB, qnrD, sul1, tetA, and catA1 in broiler source MDRE were significantly higher than those in layers (P ≤0.05). A chimeric plasmid p20432-mcr which carried the novel integron In1866 was isolated from broiler source MDRE. The high frequency of conjugation (10–1 to 10–3) and a wide range of hosts made p20432-mcr likely to play an essential role in the high detection rate of mcr-1, aph(3’)-Ia, and aadA2 in broiler farms. These findings will help optimize disinfection and improve antimicrobial-resistant bacteria surveillance programs in poultry farms, especially broilers.

Structural variants and modifications of hammerhead ribozymes targeting influenza A virus conserved structural motifs

The naturally occurring structure and biological functions of RNA are correlated, which includes hammerhead ribozymes. We proposed new variants of hammerhead ribozymes targeting conserved structural motifs of segment 5 of influenza A virus (IAV) (+)RNA. The variants carry structural and chemical modifications aiming to improve the RNA cleavage activity of ribozymes. We introduced an additional hairpin motif and attempted to select ribozyme-target pairs with sequence features that enable the potential formation of the trans-Hoogsteen interactions that are present in full-length, highly active hammerhead ribozymes. We placed structurally defined guanosine analogs into the ribozyme catalytic core. Herein, the significantly improved synthesis of 2′-deoxy-2′-fluoroarabinoguanosine derivatives is described. The most potent hammerhead ribozymes were applied to chimeric short hairpin RNA (shRNA)-ribozyme plasmid constructs to improve the antiviral activity of the two components. The modified hammerhead ribozymes showed moderate cleavage activity. Treatment of IAV-infected Madin-Darby canine kidney (MDCK) cells with the plasmid constructs resulted in significant inhibition of virus replication. Real-time PCR analysis revealed a significant (80%–88%) reduction in viral RNA when plasmids carriers were used. A focus formation assay (FFA) for chimeric plasmids showed inhibition of virus replication by 1.6–1.7 log10 units, whereas the use of plasmids carrying ribozymes or shRNAs alone resulted in lower inhibition.

Resistome and virulome accretion in an NDM-1-producing ST147 sublineage of Klebsiella pneumoniae associated with an outbreak in Tuscany, Italy: a genotypic and phenotypic characterisation

Carbapenemase-producing Enterobacterales (CPE), particularly those producing metallo-β-lactamases, are among the most challenging antibiotic-resistant pathogens, causing outbreaks of difficult-to-treat nosocomial infections worldwide. Since November 2018, an outbreak of New Delhi metallo-β-lactamases-positive CPE (NDM-CPE) has emerged in Tuscany, Italy. In this study, we aimed to investigate the NDM-CPE associated with the outbreak and characterise the responsible Klebsiella pneumoniae clone. We used whole-genome sequencing and bioinformatic analysis to characterise NDM-CPE isolates that caused bloodstream infections in 53 patients at 11 hospitals in Tuscany and that were collected between Jan 1, 2018, and July 5, 2019 (ie, the early phase of the outbreak and preceding months). The CPE isolates characterised in this study were isolated and identified at the species level and as NDM producers by six diagnostic microbiology laboratories that serve the 11 hospitals. We used comparative genomic analysis, antimicrobial susceptibility testing, plasmid conjugal transfer assays, evaluation of virulence potential in the Galleria mellonella infection model, and serum bactericidal assays to further characterise the clone causing the outbreak. The outbreak was sustained by an ST147 K pneumoniae producing NDM-1, which had a complex resistome that mediated resistance to most antimicrobials (except cefiderocol, the aztreonam-avibactam combination, colistin, and fosfomycin). The clone belonged to a sublineage of probably recent evolution, occurred by the sequential acquisition of an integrative and conjugative element encoding the yersiniabactin siderophore, an FIB(pQil)-type multiresistance plasmid carrying blaNDM-1, and a transferable chimeric plasmid, derived from virulence elements of hypervirulent K pneumoniae, carrying several resistance and virulence determinants. Infection of G mellonella larvae revealed a variable virulence potential. The behaviour in serum bactericidal assays was different from typical hypervirulent K pneumoniae strains, with variable grades of serum resistance apparently associated with mutations in specific chromosomal loci (csrD, pal, and ramR). This description of a sublineage of ST147 K pneumoniae with a complex resistome and virulome that is capable of sustaining a large regional outbreak adds to existing research on the evolutionary trajectories within high-risk clones of K pneumoniae. Global surveillance programmes are warranted to track the dissemination of these lineages, and to prevent and control their spread. Italian Ministry of Health and Department of Experimental and Clinical Medicine, University of Florence.

CTX-M-15-producing Klebsiella pneumoniae ST273 associated with nasal infection in a domestic cat

The aim of this study was to investigate the genetic context of expanded-spectrum β-lactam resistance in a Klebsiella pneumoniae strain causing a hard-to-treat nasal infection in a domestic cat. A K. pneumoniae isolate was recovered from a 4-year-old male cat hospitalised in a veterinary hospital in Paraíba, Northeastern Brazil. Following phenotypic confirmation of multidrug resistance by the disk diffusion method, the genome was sequenced using an Illumina MiSeq system. Multilocus sequence typing (MLST) and structural features related to antimicrobial resistance were determined by downstream bioinformatics analyses. The strain was confirmed as sequence type 273 (ST273) K. pneumoniae harbouring a variety of genes conferring antimicrobial resistance to phenicols tetracyclines, aminoglycosides, β-lactams, fosfomycin, sulfonamides and quinolones. Two plasmids were identified. Plasmid p114PB_I co-harboured a set of plasmid-borne resistance genes [blaCTX-M-15, blaTEM-1, qnrS1, tetD, tetR, sul2, aph(6)-Id, aph(3'') and cat2]. Notably, the multiresistance region was characterised as a chimeric plasmid structure sharing high sequence homology with several plasmids from Enterobacteriaceae. The second plasmid (p114PB_II) was characterised as a plasmid present in many genomes belonging to K. pneumoniae. The genetic context of the plasmid sequences harboured by a veterinary pathogenic K. pneumoniae isolate reveals the high complexity of horizontal gene transfer mechanisms in the acquisition of antimicrobial resistance genes. The emergence, dissemination and evolution of antimicrobial resistance must be investigated from a One Health perspective.

Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicumNRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5’ coding region and the 5’ upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Construction of a Chimeric Plasmid Vector and Its Investigation for Usage as an Internal Control for Detection of Shiga Toxin-Producing Escherichia coli by Polymerase Chain Reaction and Real-Time PCR

Background: Shiga-toxigenic Escherichia coli (STEC) is an important foodborne pathogen and considered a worldwide problem. The infection can cause severe illnesses, including hemolytic uremic syndrome. Traditional methods for STEC detection are currently not done in clinical laboratories due to the cost and related long turnaround time, which necessitates the development of quick and reliable alternative detection methods. Objectives: In this study, the application of PCR and real-time PCR methods for rapid detection of STEC in DNA extracts of stool samples was investigated. Methods: A synthetic control DNA vector, containing target genes of stx1, stx2, EhxA, and eae, was designed to evaluate the validity of these methods. Moreover, efficiency of primers targeting the genes, limit of detection, repeatability, sensitivity, and specificity of real-time PCR assay was compared with PCR results. Results: Repeatability of real-time PCR assay showed percent coefficient of variation of 3.3% - 16.1% and sensitivity of 66% to 100%. Efficiency of all the primers was 100% with a detection limit of 103 copies of DNA per reaction for both of the two assays. Comparison of the results showed that real-time PCR was more sensitive than conventional PCR. The chimeric vector interfered with template DNA when it was used as an internal control in PCR and real-time PCR assays; however, its application as external control was approved for detection of all targeted genes in STEC. Conclusions: These results confirmed application of the chimeric vector as an external control in detection of STEC by both PCR and real-time PCR assays, but its usage as an internal control was not approved in this study.

Molecular and genetic characterization of the pOV plasmid from Pasteurella multocida and construction of an integration vector for Gallibacterium anatis

BackgroundThe pOV plasmid isolated from the Pasteurella multocida strain PMOV is a new plasmid, and its molecular characterization is important for determining its gene content and its replicative properties in Pasteurellaceae family bacteria. MethodsAntimicrobial resistance mediated by the pOV plasmid was tested in bacteria. Purified pOV plasmid DNA was used to transform E. coli DH5α and Gallibacterium anatis 12656-12, including the pBluescript II KS(−) plasmid DNA as a control for genetic transformation. The pOV plasmid was digested with EcoRI for cloning fragments into the pBluescript II KS(−) vector to obtain constructs and to determine the full DNA sequence of pOV. ResultsThe pOV plasmid is 13.5 kb in size; confers sulfonamide, streptomycin and ampicillin resistance to P. multocida PMOV; and can transform E. coli DH5α and G. anatis 12656-12. The pOV plasmid was digested for the preparation of chimeric constructs and used to transform E. coli DH5α, conferring resistance to streptomycin (plasmid pSEP3), ampicillin (pSEP4) and sulfonamide (pSEP5) on the bacteria; however, similar to pBluescript II KS(−), the chimeric plasmids did not transform G. anatis 12656-12. A 1.4 kb fragment of the streptomycin cassette from pSEP3 was amplified by PCR and used to construct pSEP7, which in turn was used to interrupt a chromosomal DNA locus of G. anatis by double homologous recombination, introducing strA-strB into the G. anatis chromosome. ConclusionThe pOV plasmid is a wide-range, low-copy-number plasmid that is able to replicate in some gamma-proteobacteria. Part of this plasmid was integrated into the G. anatis 12656-12 chromosome. This construct may prove to be a useful tool for genetic studies of G. anatis.

Bacterial Expression and Characterization of Recombinant β-Xylosidase from the Thermophilic Xylanolytic Bacterium Bacillus sp.

With the passage of time, energy sources are decreasing day by day. In order to meet the world's demand, much attention is being paid to the study of enzymes with xylanolytic activity as a potential means of generating energy. A thermophilic xylanolytic bacterium, Bacillus sp., was isolated from naturally decaying material by enrichment culture and serial dilution methods. The bacterium was grown in MH medium at 50°C and pH 7 for 10 h. The xylanolytic Bacillus sp. produced clear yellow haloes around the colonies in the presence of p-nitrophenyl beta-D-xylopyranoside (pNPX) as a substrate. After condition optimization, it was found that the organism produced the higher level of xylosidase activity after 14 h in the presence of arabinose as a carbon source and ammonium sulfate as a nitrogen source in the pH 7 medium of at 55°C. The maximum β-xylosidase activity after optimizing the culture condition was 5.0 U/mL. Later this thermophilic Bacillus sp. was used as a donor in cloning of the β-xylosidase gene. A genomic library of Bacillus sp. was prepared by digesting the genomic DNA of the Bacillus with the restriction endonuclease BamHI, ligating the fragments in the pUC18 cloning vector and then transforming the competent E. coli DH5α cells with the resultant chimeric plasmid. The β-xylosidase gene was identified by screening the transformants in duplicates on LB agar plates overlaid with pNPX as a substrate. Commercial production of β-xylosidase to be used as a methanol-producing enzyme can help to overcome fuel shortages.

The Use of a Chimeric Rhodopsin Vector for the Detection of New Proteorhodopsins Based on Color.

Student microbial ecology laboratory courses are often conducted as condensed courses in which theory and wet lab work are combined in a very intensive short time period. In last decades, the study of marine microbial ecology is increasingly reliant on molecular-based methods, and as a result many of the research projects conducted in such courses require sequencing that is often not available on site and may take more time than a typical course allows. In this work, we describe a protocol combining molecular and functional methods for analyzing proteorhodopsins (PRs), with visible results in only 4–5 days that do not rely on sequencing. PRs were discovered in oceanic surface waters two decades ago, and have since been observed in different marine environments and diverse taxa, including the abundant alphaproteobacterial SAR11 group. PR subgroups are currently known to absorb green and blue light, and their distribution was previously explained by prevailing light conditions – green pigments at the surface and blue pigments in deeper waters, as blue light travels deeper in the water column. To detect PR in environmental samples, we created a chimeric plasmid suitable for direct expression of PRs using PCR amplification and functional analysis in Escherichia coli cells. Using this assay, we discovered several exceptional cases of PRs whose phenotypes differed from those predicted based on sequence only, including a previously undescribed yellow-light absorbing PRs. We applied this assay in two 10-days marine microbiology courses and found it to greatly enhance students’ laboratory experience, enabling them to gain rapid visual feedback and colorful reward for their work. Furthermore we expect this assay to promote the use of functional assays for the discovery of new rhodopsin variants.

Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effectors among X. citri strains

BackgroundXanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains.ResultsWe utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains.ConclusionsWe demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

A novel phosphoester-based cationic co-polymer nanocarrier delivers chimeric antigen receptor plasmid and exhibits anti-tumor effect.

Chimeric antigen receptor T cells (CAR-T cells) targeting of CD19 antigen has been proven to be effective and successful in B cell acute lymphoblastic leukemia. The traditional CAR delivery systems have several problems such as poor biosafety, low loading capacity, and low transfection efficiency. Utilization of nanocarriers for CAR delivery offers new possibilities for CAR-T treatment. In the present study, an anti-CD19 CAR expression lentivirus plasmid was constructed for CAR delivery and immunotherapy. In addition, a three-segment amphiphilic co-polymer, methoxy polyethylene glycol-branched polyethyleneimine-poly(2-ethylbutyl phospholane) (mPEG-bPEI-PEBP) was synthesized via click reaction as the carrier with cationic PEI, capable of delivering the CAR and packaging plasmids to co-transfect Jurkat cells and undergo expression. The PEBP and mPEG parts of the co-polymer provide hydrophobic and hydrophilic interfaces and lead to the co-polymer self-assembly into micelles in water and encapsulation of the DNA plasmids. The mPEG-bPEI-PEBP-DNA composites with different N/P ratios were incubated with the CD19 overexpression K562 cells to identify the CAR functions. The obtained CAR-Jurkat cells had the ability to secrete interferon-γ and interleukin-2. The cytotoxic effects to CD19-K562 cells suggest that the induced CAR-Jurkat cells have an excellent targeted antitumor activity.

Genetic analysis of virulence and antimicrobial-resistant plasmid pOU7519 in Salmonella enterica serovar Choleraesuis.

Zoonotic Salmonella enterica serovar Choleraesuis (S. Choleraesuis), causing paratyphoid in pigs and bacteremia in humans, commonly carry a virulence plasmid and sometimes a separate antimicrobial-resistant plasmid or merging together. This study aimed to analyze the likely mechanism of how to form a virulence-resistance chimera of plasmid in S. Choleraesuis. Whole plasmid sequence of pOU7519 in S. Choleraesuis strain OU7519 was determined using shotgun cloning and sequencing. Sequence annotation and comparison were performed to determine the sequence responsible for the formation of a chimeric virulence-resistance pOU7519. Other chimeric plasmids among the collected strains of S. Choleraesuis were also confirmed. The sequence of pOU719, 127,212bp long, was identified to be a chimera of the virulence plasmid pSCV50 and a multidrug-resistant plasmid pSC138 that have been found in S.Choleraesuis strain SC-B67. The pOU7519 is a conjugative plasmid carrying various mobile DNAs, including prophages, insertion sequences, integrons and transposons, especially a Tn6088-like transposon. By dissecting the junction site of the pSCV50-pSC138 chimera in pOU7519, defective sequences at integrase gene scv50 (int) and its attachment site (att) were found, and that likely resulted in a stable chimera plasmid due to the failure of excision from the pSCV50-pSC138 chimera. Similar structure of chimera was also found in other large plasmids. The deletion of both the int and att sties could likely block chimera excision, and result in an irreversible, stable pSCV50-pSC138 chimera. The emergence of conjugative virulence and antimicrobial-resistant plasmids in S. Choleraesuis could pose a threat to health public.

Simultaneous Gene Delivery and Tracking through Preparation of Photo-Luminescent Nanoparticles Based on Graphene Quantum Dots and Chimeric Peptides

Designing suitable nano-carriers for simultaneous gene delivery and tracking is in the research priorities of the molecular medicine. Non-toxic graphene quantum dots (GQDs) with two different (green and red) emission colors are synthesized by Hummer’s method and characterized by UV-Vis, Photoluminescence (PL), Fourier Transform Infrared (FTIR) and Raman spectroscopies, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The GQDs are conjugated with MPG-2H1 chimeric peptide and plasmid DNA (pDNA) by non-covalent interactions. Following conjugation, the average diameter of the prepared GQDs increased from 80 nm to 280 nm in complex structure, and the ζ-potential of the complex increased (from −36.87 to −2.56 mV). High transfection efficiency of the nano-carrier and results of confocal microscopy demonstrated that our construct can be considered as a nontoxic carrier with dual functions for gene delivery and nuclear targeting.