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998100.9936High-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.202439229640
508410.9934Cloth-based hybridization array system for the identification of antibiotic resistance genes in Salmonella. A simple macroarray system based on the use of polyester cloth as the solid phase for DNA hybridization has been developed for the identification and characterization of bacteria on the basis of the presence of various virulence and toxin genes. In this approach, a multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP is used to simultaneously amplify different marker genes, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. As an example of the applicability of this cloth-based hybridization array system (CHAS) in the characterization of foodborne pathogens, a method has been developed enabling the detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. The CHAS is a simple, cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the identification of key pathogen-specific marker genes in bacterial isolates.200718363231
247620.9930A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa. The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.202337888028
974330.9927Simultaneous Detection of Antibiotic Resistance Genes on Paper-Based Chip Using [Ru(phen)(2)dppz](2+) Turn-on Fluorescence Probe. Antibiotic resistance, the ability of some bacteria to resist antibiotic drugs, has been a major global health burden due to the extensive use of antibiotic agents. Antibiotic resistance is encoded via particular genes; hence the specific detection of these genes is necessary for diagnosis and treatment of antibiotic resistant cases. Conventional methods for monitoring antibiotic resistance genes require the sample to be transported to a central laboratory for tedious and sophisticated tests, which is grueling and time-consuming. We developed a paper-based chip, integrated with loop-mediated isothermal amplification (LAMP) and the "light switch" molecule [Ru(phen)(2)dppz](2+), to conduct turn-on fluorescent detection of antibiotic resistance genes. In this assay, the amplification reagents can be embedded into test spots of the chip in advance, thus simplifying the detection procedure. [Ru(phen)(2)dppz](2+) was applied to intercalate into amplicons for product analysis, enabling this assay to be operated in a wash-free format. The paper-based detection device exhibited a limit of detection (LOD) as few as 100 copies for antibiotic resistance genes. Meanwhile, it could detect antibiotic resistance genes from various bacteria. Noticeably, the approach can be applied to other genes besides antibiotic resistance genes by simply changing the LAMP primers. Therefore, this paper-based chip has the potential for point-of-care (POC) applications to detect various gene samples, especially in resource-limited conditions.201829323478
507140.9926Versatile 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.202539605319
922850.9926In 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.202438277772
508260.9925A quantitative real-time PCR assay for the detection of tetR of Tn10 in Escherichia coli using SYBR Green and the Opticon. Bacteria of implant infections are extremely resistant to antibiotics. One reason for this antibiotic resistance are transposons; the well-known transposon Tn10, for example, mediates tetracycline resistance to Escherichia coli. Two genes of Tn10, tetA and tetR, are essential for the mechanism of resistance. These genes encode a drug-specific efflux protein and a tetracycline repressor protein, respectively. Tn10 is also widely used in molecular biology. For example, tTA, a recombinant derivate of tetR, has been utilised for a highly efficient gene regulation system in mammalian cells. We have examined E. coli isolates from implant infections for tetracycline resistance and for the presence of tetR. A real-time PCR assay was developed for detection of tetR with SybrGreen using the Opticon PCR machine of MJ Research. This method offers a quick, sensitive, efficient, and reliable approach to the detection and quantification of genes. Clinical isolates of E. coli were examined successfully for tetracycline resistance and for the presence of tetR. The real-time PCR is effective using a variety of templates including isolated E. coli DNA, pure colonies, or liquid culture sources. Using quantified standard DNA, this assay can accurately detect as few as 15 copies. Moreover, this assay has the ability to quantify the number of tetR genes in the presence of contaminating mammalian DNA. In conclusion, the tetR real-time PCR offers new methods for detection and quantification of tetracycline-resistant bacteria and tTA in transfected cell-lines or transgenic animals.200415165753
973870.9925Detection and Quantification of Antimicrobial-Resistant Cells in Aquatic Environments by Bioorthogonal Noncanonical Amino Acid Tagging. Aquatic environments are important reservoirs of antibiotic wastes, antibiotic resistance genes, and bacteria, enabling the persistence and proliferation of antibiotic resistance in different bacterial populations. To prevent the spread of antibiotic resistance, effective approaches to detect antimicrobial susceptibility in aquatic environments are highly desired. In this work, we adopt a metabolism-based bioorthogonal noncanonical amino acid tagging (BONCAT) method to detect, visualize, and quantify active antimicrobial-resistant bacteria in water samples by exploiting the differences in bacterial metabolic responses to antibiotics. The BONCAT approach can be applied to rapidly detect bacterial resistance to multiple antibiotics within 20 min of incubation, regardless of whether they act on proteins or DNA. In addition, the combination of BONCAT with the microscope enables the intuitive characterization of antibiotic-resistant bacteria in mixed systems at single-cell resolution. Furthermore, BONCAT coupled with flow cytometry exhibits good performance in determining bacterial resistance ratios to chloramphenicol and population heterogeneity in hospital wastewater samples. In addition, this approach is also effective in detecting antibiotic-resistant bacteria in natural water samples. Therefore, such a simple, fast, and efficient BONCAT-based approach will be valuable in monitoring the increase and spread of antibiotic resistance within natural and engineered aquatic environments.202236251006
508380.9925Multiplex Microarrays in 96-Well Plates Photoactivated with 4-Azidotetrafluorobenzaldehyde for the Identification and Quantification of β-Lactamase Genes and Their RNA Transcripts. Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin-peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.202338275665
450790.9925Occurrence and antibiotic susceptibility profiles of Burkholderia cepacia complex in coastal marine environment. During an environmental study of bacterial resistance to antibiotics in coastal waters of the Kaštela Bay, Adriatic Sea, Croatia, 47 Burkholderia cepacia complex (Bcc) isolates were recovered from seawater and mussel (Mytilus galloprovincialis) samples. All isolates showed multiple antibiotic resistance. Among the isolates, two Burkholderia cenocepacia isolates produced chromosomally encoded TEM-116 extended-spectrum β-lactamase (ESBL). Analysis of outer membrane proteins revealed that decreased expression of a 36-kDa protein could be associated with a high level of β-lactam resistance in both isolates. Phenotypic study of efflux system also indicated an over-expression of Resistance-Nodulation-Cell Division (RND) efflux-mediated mechanism in one of the isolates. This study demonstrated the presence of Bcc in seawater and M. galloprovincialis, which gives evidence that coastal marine environment, including mussels, could be considered as a reservoir for Bcc species. Detection of ESBL-encoding genes indicates the potential role of these bacteria in the maintenance and dispersion of antibiotic resistance genes.201222428949
4741100.9924Detection 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.201829719694
9742110.9924BOCS: DNA k-mer content and scoring for rapid genetic biomarker identification at low coverage. A single, inexpensive diagnostic test capable of rapidly identifying a wide range of genetic biomarkers would prove invaluable in precision medicine. Previous work has demonstrated the potential for high-throughput, label-free detection of A-G-C-T content in DNA k-mers, providing an alternative to single-letter sequencing while also having inherent lossy data compression and massively parallel data acquisition. Here, we apply a new bioinformatics algorithm - block optical content scoring (BOCS) - capable of using the high-throughput content k-mers for rapid, broad-spectrum identification of genetic biomarkers. BOCS uses content-based sequence alignment for probabilistic mapping of k-mer contents to gene sequences within a biomarker database, resulting in a probability ranking of genes on a content score. Simulations of the BOCS algorithm reveal high accuracy for identification of single antibiotic resistance genes, even in the presence of significant sequencing errors (100% accuracy for no sequencing errors, and >90% accuracy for sequencing errors at 20%), and at well below full coverage of the genes. Simulations for detecting multiple resistance genes within a methicillin-resistant Staphylococcus aureus (MRSA) strain showed 100% accuracy at an average gene coverage of merely 0.515, when the k-mer lengths were variable and with 4% sequencing error within the k-mer blocks. Extension of BOCS to cancer and other genetic diseases met or exceeded the results for resistance genes. Combined with a high-throughput content-based sequencing technique, the BOCS algorithm potentiates a test capable of rapid diagnosis and profiling of genetic biomarkers ranging from antibiotic resistance to cancer and other genetic diseases.201931173943
5092120.9924Rapid detection of antibiotic resistance genes in lactic acid bacteria using PMMA-based microreactor arrays. The emergence of lactic acid bacteria (LABs) resistant to existing antimicrobial drugs is a growing health crisis. To decrease the overuse of antibiotics, molecular diagnostic systems that can rapidly determine the presence of antibiotic resistance (AR) genes in LABs from yogurt samples are needed. This paper describes a fully integrated, miniaturized plastic chip and closed-tube detection chemistry that performs multiplex nucleic acid amplification. High-throughput identification of AR genes was achieved through this approach, and six AR genes were analyzed simultaneously in < 2 h. This time-to-result included the time required for the extraction of DNA. The detection limit of the chip was 10(3) CFU mL(-1), which was consistent with that of tube LAMP. We detected and identified multiple DNAs, including streptomycin, tetracycline, and vancomycin resistance-associated genes, with complete concordance to the Kirby-Bauer disk diffusion method.Key Points• A miniaturized chip was presented, and multiplex nucleic acid amplification was performed.• The device can be integrated with LAMP for rapid detection of antibiotic resistance genes.• The approach had a high throughput of AR gene analysis in lactic acid bacteria.202032488313
9740130.9923Nitrogen-Doped Carbon Dots Facilitate CRISPR/Cas for Reducing Antibiotic Resistance Genes in the Environment. The continued acquisition and propagation of antibiotic resistance genes (ARGs) in the environment confound efforts to manage the global rise in antibiotic resistance. Here, CRISPR-Cas9/sgRNAs carried by nitrogen-doped carbon dots (NCDs) were developed to precisely target multi-"high-risk" ARGs (tet, cat, and aph(3')-Ia) commonly detected in the environment. NCDs facilitated the delivery of Cas9/sgRNAs to Escherichia coli (E. coli) without cytotoxicity, achieving sustained elimination of target ARGs. The elimination was optimized using different weight ratios of NCDs and Cas9 protein (1:1, 1:20, and 1:40), and Cas9/multi sgRNAs were designed to achieve multi-cleavage of ARGs in either a single strain or mixed populations. Importantly, NCDs successfully facilitated Cas9/multi sgRNAs for resensitization of antibiotic-resistant bacteria in soil (approaching 50%), whereas Cas9/multi sgRNAs alone were inactivated in the complex environment. This work highlights the potential of a fast and precise strategy to minimize the reservoir of antibiotic resistance in agricultural system.202438335532
9984140.9923Multiplex base editing to convert TAG into TAA codons in the human genome. Whole-genome recoding has been shown to enable nonstandard amino acids, biocontainment and viral resistance in bacteria. Here we take the first steps to extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and highly multiplex editing in mammalian cells.202235918324
3280150.9923Optimization of five qPCR protocols toward the detection and the quantification of antimicrobial resistance genes in environmental samples. Here, we describe the optimization and validation of five quantitative PCR (qPCR) assays by employing the SYBRGreen chemistry paired with melting curve analysis to detect and quantify clinically relevant antimicrobial resistance genes (ARGs) (i.e. ermB, bla(CTXM1-like), bla(CMY-2), qnrA and qnrS) from environmental samples (i.e. soil and manure). These five protocols accurately detected and quantified the aforementioned ARGs in complex environmental matrices and represent useful tools for both diagnostic and monitoring activities of resistant bacteria and ARGs into the environment.202134754761
5074160.9923Cas14VIDet: A visual instant method free from PAM restriction for antibiotic resistance bacteria detection. A personalized treatment strategy that selects sensitive antibiotics based on Helicobacter pylori (H. pylori) resistance genes is currently the most effective approach to address the challenge of H. pylori eradication. However, the widespread adoption of this strategy is hindered by the long processing times and high costs associated with traditional resistance gene detection methods. In this study, we combined ultra-fast PCR with CRISPR/Cas14 into a single reaction system, establishing a rapid, one-pot visual platform named Cas14VIDet (Cas14-based Visual Instant Detection) for detecting H. pylori resistance genes. Cas14VIDet does not require a PAM sequence and excels in identifying single nucleotide polymorphisms, with the detection sensitivity approaching the level of a single bacterial colony (10(0) CFU/mL). The entire detection process can be completed within 10 min, and results are directly observable with the naked eye. We validated Cas14VIDet by testing 50 clinical samples and compared it with Sanger sequencing. The results showed that Cas14VIDet achieved 100% sensitivity, 100% specificity, and 100% accuracy in detecting H. pylori resistance genes to levofloxacin. Therefore, we believe this method holds great potential for rapid detection of H. pylori resistance, potentially supporting personalized treatment of H. pylori infections in the future.202539527901
5086170.9923Detection of genetically modified microorganisms in soil using the most-probable-number method with multiplex PCR and DNA dot blot. The principal objective of this study was to detect genetically modified microorganisms (GMMs) that might be accidentally released into the environment from laboratories. Two methods [plate counting and most-probable-number (MPN)] coupled with either multiplex PCR or DNA dot blots were compared using genetically modified Escherichia coli, Pseudomonas putida, and Acinetobacter oleivorans harboring an antibiotic-resistance gene with additional gfp and lacZ genes as markers. Alignments of sequences collected from databases using the Perl scripting language (Perl API) and from denaturing gradient gel electrophoresis analysis revealed that the gfp, lacZ and antibiotic-resistance genes (kanamycin, tetracycline, and ampicillin) in GMMs differed from the counterpart genes in many sequenced genomes and in soil DNA. Thus, specific multiplex PCR primer sets for detection of plasmid-based gfp and lacZ antibiotic-resistance genes could be generated. In the plate counting method, many antibiotic-resistant bacteria from a soil microcosm grew as colonies on antibiotic-containing agar plates. The multiplex PCR verification of randomly selected antibiotic-resistant colonies with specific primers proved ineffective. The MPN-multiplex PCR method and antibiotic-resistant phenotype could be successfully used to detect GMMs, although this method is quite laborious. The MPN-DNA dot blot method screened more cells at a time in a microtiter plate containing the corresponding antibiotics, and was shown to be a more efficient method for the detection of GMMs in soil using specific probes in terms of labor and accuracy.201121810467
5039180.9923Analytical validation of a novel high multiplexing real-time PCR array for the identification of key pathogens causative of bacterial ventilator-associated pneumonia and their associated resistance genes. OBJECTIVES: Rapid diagnosis and appropriate empirical antimicrobial therapy before the availability of conventional microbiological results is of pivotal importance for the clinical outcome of ventilator-associated pneumonia (VAP). We evaluated the VAPChip, a novel, closed cartridge molecular tool aiming to identify directly from clinical samples and within a working day the principal bacteria causative of VAP as well as clinically relevant β-lactam resistance genes. METHODS: The Real-time Array PCR for Infectious Diseases (RAP-ID) is a novel technology that combines multiplex PCR with real-time microarray detection. The VAPChip is a closed cartridge kit adapted to the RAP-ID instrument that targets 13 key respiratory pathogens causative of VAP and 24 relevant antimicrobial resistance genes that mediate resistance to β-lactam agents, including extended-spectrum cephalosporins and carbapenems. Analytical validation of the VAPChip was carried out blindly on a collection of 292 genotypically characterized bacterial reference and clinical isolates, including 225 isolates selected on the basis of their species identification and antimicrobial resistance profiles and 67 bacterial isolates belonging to the oropharyngeal flora not targeted by the array. RESULTS: The limit of detection of the assay lies between 10 and 100 genome copies/PCR and the dynamic range is five orders of magnitude permitting at least semi-quantitative reporting of the results. Sensitivity, specificity and negative and positive predictive values ranged from 95.8% to 100% for species identification and detection of resistance genes. CONCLUSIONS: VAPChip is a novel diagnostic tool able to identify resistant bacterial isolates by RAP-ID technology. The results of this analytical validation have to be confirmed on clinical specimens.201323065698
3959190.9922Antibiotic resistance. How wild are wild mammals? In bacteria associated with humans, antimicrobial resistance is common, both in clinical isolates and in the less-studied commensal flora, and it is thought that commensal and environmental bacteria might be a hidden reservoir of resistance. Gilliver et al. have reported that resistance is also prevalent in faecal bacteria from wild rodents living in northwest England. Here we test the faeces of moose, deer and vole in Finland and find an almost complete absence of resistance in enterobacteria. Resistance is thus not a universal property of enterobacterial populations, but may be a result of the human use of antibiotics.200111343104