# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 9995 | 0 | 1.0000 | Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon. Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect β-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization. | 2010 | 20729070 |
| 8184 | 1 | 0.9968 | Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria. The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes. | 2020 | 32523110 |
| 408 | 2 | 0.9967 | Deletion of pcnB affects antibiotic susceptibility in resistant Escherichia coli by reducing copy number of ColE1-family plasmids. Plasmids play a major role in the spread of antibiotic resistance genes in bacteria. Plasmid copy number (PCN) is often tightly regulated. In plasmids of the ColE1-type, this regulation happens by a negative feedback mechanism using an antisense RNA. Here, we employed a sequencing-based method for determining PCN to demonstrate that copy number of different ColE1-family plasmids harboring antibiotic resistance genes increases during antibiotic treatment. Further, we show that deletion of the gene pcnB reduces the copy number of ColE1-family plasmids in E. coli MG1655, which in turn results in a reduced resistance to antimicrobials of the classes aminoglycosides, β-lactams and tetracyclines. In the absence of antibiotic selection, the deletion of pcnB also decreased the number of ColE1-type plasmids in a bacterial population. Hence, PcnB, which polyadenylates RNA, marking it for decay, represents a potential drug and helper-drug target that could be used to reduce PCN to re-sensitize bacteria with multi-copy-number resistance-plasmids to treatment with different antimicrobials. | 2025 | 40069245 |
| 5070 | 3 | 0.9966 | Sequence-specific DNA solid-phase extraction in an on-chip monolith: Towards detection of antibiotic resistance genes. Antibiotic resistance of bacteria is a growing problem and presents a challenge for prompt treatment in patients with sepsis. Currently used methods rely on culturing or amplification; however, these steps are either time consuming or suffer from interference issues. A microfluidic device was made from black polypropylene, with a monolithic column modified with a capture oligonucleotide for sequence selective solid-phase extraction of a complementary target from a lysate sample. Porous properties of the monolith allow flow and hybridization of a target complementary to the probe immobilized on the column surface. Good flow-through properties enable extraction of a 100μL sample and elution of target DNA in 12min total time. Using a fluorescently labeled target oligonucleotide related to Verona Integron-Mediated Metallo-β-lactamase it was possible to extract and detect a 1pM sample with 83% recovery. Temperature-mediated elution by heating above the duplex melting point provides a clean extract without any agents that interfere with base pairing, allowing various labeling methods or further downstream processing of the eluent. Further integration of this extraction module with a system for isolation and lysis of bacteria from blood, as well as combining with single-molecule detection should allow rapid determination of antibiotic resistance. | 2017 | 28734608 |
| 9981 | 4 | 0.9966 | High-contrast imaging of cellular non-repetitive drug-resistant genes via in situ dead Cas12a-labeled PCR. In situ imaging of genes of pathogenic bacteria can profile cellular heterogeneity, such as the emergence of drug resistance. Fluorescence in situ hybridization (FISH) serves as a classic approach to image mRNAs inside cells, but it remains challenging to elucidate genomic DNAs and relies on multiple fluorescently labeled probes. Herein, we present a dead Cas12a (dCas12a)-labeled polymerase chain reaction (CasPCR) assay for high-contrast imaging of cellular drug-resistant genes. We employed a syncretic dCas12a-green fluorescent protein (dCas12a-GFP) to tag the amplicons, thereby enabling high-contrast imaging and avoiding multiple fluorescently labeled probes. The CasPCR assay can quantify quinolone-resistant Salmonella enterica in mixed populations and identify them isolated from poultry farms. | 2024 | 39229640 |
| 259 | 5 | 0.9966 | Dual-Plasmid Mini-Tn5 System to Stably Integrate Multicopy of Target Genes in Escherichia coli. The efficiency of valuable metabolite production by engineered microorganisms underscores the importance of stable and controllable gene expression. While plasmid-based methods offer flexibility, integrating genes into host chromosomes can establish stability without selection pressure. However, achieving site-directed multicopy integration presents challenges, including site selection and stability. We introduced a stable multicopy integration method by using a novel dual-plasmid mini-Tn5 system to insert genes into Escherichia coli's genome. The gene of interest was combined with a removable antibiotic resistance gene. After the selection of bacteria with inserted genes, the antibiotic resistance gene was removed. Optimizations yielded an integration efficiency of approximately 5.5 × 10(-3) per recipient cell in a single round. Six rounds of integration resulted in 19 and 5 copies of the egfp gene in the RecA(+) strain MG1655 and the RecA(-) strain XL1-Blue MRF', respectively. Additionally, we integrated a polyhydroxybutyrate (PHB) synthesis gene cluster into E. coli MG1655, yielding an 8-copy integration strain producing more PHB than strains with the cluster on a high-copy plasmid. The method was efficient in generating gene insertions in various E. coli strains, and the inserted genes were stable after extended culture. This stable, high-copy integration tool offers potential for diverse applications in synthetic biology. | 2024 | 39418641 |
| 387 | 6 | 0.9965 | Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid-containing Escherichia coli. Plasmid pBR322 and its numerous derivatives are used extensively for research and in biotechnology. The tetracycline-resistance (TcR) genes in these plasmids are expressed constitutively and cells carrying these plasmids are resistant to tetracycline. We have shown that expression of the TcR gene has an adverse effect on the reproductive fitness of plasmid-containing bacteria in both glucose-limited batch and chemostat cultures. If the TcR genes are inactivated at any one of three different restriction sites, mixed cultures of plasmid-free and plasmid-containing bacteria grow at the same rate. | 1985 | 3005111 |
| 4741 | 7 | 0.9965 | Detection of antimicrobial resistance-associated proteins by titanium dioxide-facilitated intact bacteria mass spectrometry. Titanium dioxide-modified target plates were developed to enhance intact bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The plates were designed to photocatalytically destroy the bacterial envelope structure and improve the ionization efficiency of intracellular components, thereby promoting the measurable mass range and the achievable detection sensitivity. Accordingly, a method for rapid detection of antimicrobial resistance-associated proteins, conferring bacterial resistance against antimicrobial drugs, was established by mass spectrometric fingerprinting of intact bacteria without the need for any sample pre-treatment. With this method, the variations in resistance proteins' expression levels within bacteria were quickly measured from the relative peak intensities. This approach of resistance protein detection directly from intact bacteria by mass spectrometry is useful for fast discrimination of antimicrobial-resistant bacteria from their non-resistant counterparts whilst performing species identification. Also, it could be used as a rapid and convenient way for initial determination of the underlying resistance mechanisms. | 2018 | 29719694 |
| 9228 | 8 | 0.9965 | In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria. In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level. | 2024 | 38277772 |
| 264 | 9 | 0.9965 | The Tn5 bleomycin resistance gene confers improved survival and growth advantage on Escherichia coli. The bleomycin resistance gene (ble) of transposon Tn5 is known to decrease the death rate of Escherichia coli during stationary phase. Bleomycin is a DNA-damaging agent and bleomycin resistance is produced by improved DNA repair which also requires the host genes aidC and polA coding, respectively, for an alkylation-inducible gene product and DNA polymerase I. In the absence of the drug, this DNA repair system is believed to cause the slower death rate of bleomycin-resistant bacteria. In this study, the effect of ble and aidC genes on the viability of bacteria and their growth rate in chemostat competitions was studied. The results indicate, that bleomycin-resistant bacteria display greater fitness under these conditions. Another beneficial effect of transposon Tn5 had been previously attributed to the insertion sequence IS 50 R. We were not able to reproduce this result with IS 50 R, however, the complete transposon was beneficial under similar conditions. Moreover, we showed the Tn5 fitness effect to be aidC-dependent. The ble gene was discovered after the fitness effect of IS 50 R had been established; it has not previously been considered to mediate the beneficial effect of Tn5. This possibility is discussed based on the molecular mechanism of bleomycin resistance. | 1994 | 7510018 |
| 5071 | 10 | 0.9964 | Versatile and Portable Cas12a-mediated Detection of Antibiotic Resistance Markers. Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 10(2) CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results redout (Fluorescence vs FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup. | 2025 | 39605319 |
| 9297 | 11 | 0.9964 | Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria. Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract. | 2022 | 35601876 |
| 450 | 12 | 0.9964 | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells. | 2000 | 10829079 |
| 6312 | 13 | 0.9964 | D-serine deaminase is a stringent selective marker in genetic crosses. The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source. The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed. | 1995 | 7814336 |
| 286 | 14 | 0.9964 | Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells? The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tcr region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed. | 1984 | 6323922 |
| 6197 | 15 | 0.9964 | Structure and location of tellurium deposited in Escherichia coli cells harbouring tellurite resistance plasmids. The plasmids RP4Ter and pHH1508a, which belong to the P and HII incompatibility groups, respectively, confer resistance to potassium tellurite (K2TeO3) on Escherichia coli. The genes for tellurite resistance were cloned from each plasmid onto the vector pUC8 to create pDT1366 and pD1364, respectively. Unstained, unfixed bacteria carrying these plasmids contained black intracellular deposits when grown on media containing tellurite. Thin sections of these bacteria fixed with glutaraldehyde were prepared and examined by electron microscopy. The black deposits were located inside the cell and were frequently associated with the inner membrane of the bacterium. Bacteria containing pDT1366 or pDT1364, and therefore a higher gene dosage of the Ter determinant, contained more black deposits, but had a decreased resistance, as measured by the minimum inhibitory concentration using the agar dilution method. Using the technique of electron spectroscopic imaging, the black intracellular deposits were shown to contain predominantly reduced metallic tellurium, and significant amounts of oxygen or carbon, thereby confirming earlier results using X-ray diffraction analysis of whole cells. | 1988 | 3042886 |
| 9739 | 16 | 0.9963 | Au-Fe(3)O(4) nanozyme coupled with CRISPR-Cas12a for sensitive and visual antibiotic resistance diagnosing. The accumulation and spread of antibiotic resistance bacteria (ARB) in the environment may accelerate the formation of superbugs and seriously threaten the health of all living beings. The timeliness and accurate diagnosing of antibiotic resistance is essential to controlling the propagation of superbugs in the environment and formulating effective public health management programs. Herein, we developed a speedy, sensitive, accurate, and user-friendly colorimetric assay for antibiotic resistance, via a synergistic combination of the peroxidase-like property of the Au-Fe(3)O(4) nanozyme and the specific gene identification capability of the CRISPR-Cas12a. Once the CRISPR-Cas12a system recognizes a target resistance gene, it activates its trans-cleavage activity and subsequently releases the Au-Fe(3)O(4) nanozymes, which oxidizes the 3,3,5,5-tetramethylbenzidine (TMB) with color change from transparent to blue. The diagnosing signals could be captured and analyzed by a smartphone. This method detected kanamycin-resistance genes, ampicillin-resistance genes, and chloramphenicol-resistance genes by simple operation steps with high sensitivity (<0.1 CFU μL(-1)) and speediness (<1 h). This approach may prove easy for the accurate and sensitive diagnosis of the ARGs or ARB in the field, thus surveilling and controlling the microbial water quality flexibly and efficiently. | 2023 | 36925313 |
| 426 | 17 | 0.9963 | Plasmid-determined resistance to serum bactericidal activity: a major outer membrane protein, the traT gene product, is responsible for plasmid-specified serum resistance in Escherichia coli. Resistance to the bactericidal activity of serum appears to be an important virulence property of invasive bacteria. The conjugative multiple-antibiotic-resistance plasmid R6-5 was found to confer upon Escherichia coli host bacteria increased resistance against rabbit serum. Gene-cloning techniques were used to localize the serum resistance determinant of R6-5 to a segment of the plasmid that encodes conjugal transfer functions, and a pACYC184 hybrid plasmid, designated pKT107, that contains this segment was constructed. The generation and analysis of deletion and insertion mutant derivatives of the pKT107 plasmid that no longer specify serum resistance permitted precise localization of the serum-resistance cistron on the R6-5 map and demonstrated that this locus is coincident with that of traT, one of the two surface exclusion genes of R6-5. Examination of the proteins synthesized in E. coli minicells of pKT107 and its serum-sensitive mutant derivative plasmids confirmed that the serum-resistance gene product of R6-5 is the traT protein and showed that this protein is a major structural component (about 21,000 copies per cell) of the bacterial outer membrane. | 1980 | 6995306 |
| 315 | 18 | 0.9963 | Phosphorothioate DNA as an antioxidant in bacteria. Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H(2)O(2)) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H(2)O(2). DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H(2)O(2). Resistance to H(2)O(2) was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H(2)O(2) than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H(2)O(2). These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria. | 2012 | 22772986 |
| 420 | 19 | 0.9963 | Transferable nitrofuran resistance conferred by R-plasmids in clinical isolates of Escherichia coli. A high proportion of nitrofuran-resistant strains has been found in a collection of antibiotic-resistant Gram-negative bacteria isolated from patients with urinary tract infections. Some of the Escherichia coli carried R-plasmids that conferred resistance to nitrofurantoin and nitrofurazone. The mechanism of resistance is not clear; only in lactose non-fermenting recipients was there a decrease in the nitrofuran-reducing ability of whole-cell suspensions. One of the plasmids conferred enhanced resistance to UV light on DNA repair defective mutants but not on repair efficient strains. In some resistant strains, the total resistance was apparently the result of a combination of chromosomal and plasmid-borne genes. The presence of the plasmid may allow the development of higher resistance levels by mutation of chromosomal genes. | 1983 | 6368515 |