Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. - Related Documents




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493901.0000Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. OBJECTIVES: The introduction of metagenomic sequencing to diagnostic microbiology has been hampered by slowness, cost and complexity. We explored whether MinION nanopore sequencing could accelerate diagnosis and resistance profiling, using complicated urinary tract infections as an exemplar. METHODS: Bacterial DNA was enriched from clinical urines (n = 10) and from healthy urines 'spiked' with multiresistant Escherichia coli (n = 5), then sequenced by MinION. Sequences were analysed using external databases and bioinformatic pipelines or, ultimately, using integrated real-time analysis applications. Results were compared with Illumina data and resistance phenotypes. RESULTS: MinION correctly identified pathogens without culture and, among 55 acquired resistance genes detected in the cultivated bacteria by Illumina sequencing, 51 were found by MinION sequencing directly from the urines; with three of the four failures in an early run with low genome coverage. Resistance-conferring mutations and allelic variants were not reliably identified. CONCLUSIONS: MinION sequencing comprehensively identified pathogens and acquired resistance genes from urine in a timeframe similar to PCR (4 h from sample to result). Bioinformatic pipeline optimization is needed to better detect resistances conferred by point mutations. Metagenomic-sequencing-based diagnosis will enable clinicians to adjust antimicrobial therapy before the second dose of a typical (i.e. every 8 h) antibiotic.201727667325
493510.9998Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA. Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.202236290058
493620.9998A New Tool for Analyses of Whole Genome Sequences Reveals Dissemination of Specific Strains of Vancomycin-Resistant Enterococcus faecium in a Hospital. A new easy-to-use online bioinformatic tool analyzing whole genome sequences of healthcare associated bacteria was used by a local infection control unit to retrospectively map genetic relationship of isolates of E. faecium carrying resistance genes to vancomycin in a hospital. Three clusters of isolates were detected over a period of 5 years, suggesting transmission between patients. Individual relatedness between isolates within each cluster was established by SNP analyses provided by the system. Genetic antimicrobial resistance mechanisms to antibiotics other than vancomycin were identified. The results suggest that the system is suited for hospital surveillance of E. faecium carrying resistance genes to vancomycin in settings with access to next Generation Sequencing without bioinformatic expertise for interpretation of the genome sequences.202134778297
508930.9997A TaqMan-based multiplex real-time PCR assay for the rapid detection of tigecycline resistance genes from bacteria, faeces and environmental samples. BACKGROUND: Tigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant bacteria. Recently, novel tigecycline resistance genes tet(X3) and tet(X4) have been reported, which pose a great challenge to human health and food security. The current study aimed to establish a TaqMan-based real-time PCR assay for the rapid detection of the tigecycline-resistant genes tet(X3) and tet(X4). RESULTS: No false-positive result was found, and the results of the TaqMan-based real-time PCR assay showed 100% concordance with the results of the sequencing analyses. This proposed method can detect the two genes at the level of 1 × 10(2) copies/μL, and the whole process is completed within an hour, allowing rapid screening of tet(X3) and tet(X4) genes in cultured bacteria, faeces, and soil samples. CONCLUSION: Taken together, the TaqMan-based real-time PCR method established in this study is rapid, sensitive, specific, and is capable of detecting the two genes not only in bacteria, but also in environmental samples.202032571294
224740.9997Metagenomic identification of pathogens and antimicrobial-resistant genes in bacterial positive blood cultures by nanopore sequencing. Nanopore sequencing workflows have attracted increasing attention owing to their fast, real-time, and convenient portability. Positive blood culture samples were collected from patients with bacterial bloodstream infection and tested by nanopore sequencing. This study compared the sequencing results for pathogen taxonomic profiling and antimicrobial resistance genes to those of species identification and phenotypic drug susceptibility using traditional microbiology testing. A total of 37 bacterial positive blood culture results of strain genotyping by nanopore sequencing were consistent with those of mass spectrometry. Among them, one mixed infection of bacteria and fungi was identified using nanopore sequencing and confirmatory quantitative polymerase chain reaction. The amount of sequencing data was 21.89 ± 8.46 MB for species identification, and 1.0 MB microbial strain data enabled accurate determination. Data volumes greater than or equal to 94.6 MB nearly covered all the antimicrobial resistance genes of the bacteria in our study. In addition, the results of the antimicrobial resistance genes were compared with those of phenotypic drug susceptibility testing for Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. Therefore, the nanopore sequencing platform for rapid identification of causing pathogens and relevant antimicrobial resistance genes complementary to conventional blood culture outcomes may optimize antimicrobial stewardship management for patients with bacterial bloodstream infection.202338192400
569450.9997Multiplex characterization of human pathogens including species and antibiotic-resistance gene identification. The efficient medical treatment of infections requires detailed information about the pathogens involved and potential antibiotic-resistance mechanisms. The dramatically increasing incidence of multidrug-resistant bacteria especially highlights the importance of sophisticated diagnostic tests enabling a fast patient-customized therapy. However, the current molecular detection methods are limited to either the detection of species or only a few antibiotic-resistance genes.In this work, we present a human pathogen characterization assay using a rRNA gene microarray identifying 75 species comprising bacteria and fungi. A statistical classifier was developed to facilitate the automated species identification. Additionally, the clinically most important β-lactamases were identified simultaneously in a 100-plex reaction using padlock probes and the same microarray. The specificity and sensitivity of the combined assay was determined using clinical isolates. The detection limit was 10(5) c.f.u. ml(-1), recovering 89 % of the detectable β-lactamase-encoding genes specifically. The total assay time was less than 7 hand the modular character of the antibiotic-resistance detection allows the easy integration of further genetic targets. In summary, we present a fast, highly specific and sensitive multiplex pathogen characterization assay.201626489938
493860.9997Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: a case study of an outbreak in a rural Ethiopian hospital. OBJECTIVES: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements. We believe that this method has the potential to be a tool of pivotal importance for MDR control. METHODS: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. RESULTS: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. CONCLUSIONS: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low- and middle-income countries.202032653928
509270.9997Rapid 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
495680.9997Rapid Identification of Plasmid Replicon Type and Coexisting Plasmid-Borne Antimicrobial Resistance Genes by S1-Pulsed-Field Gel Electrophoresis-Droplet Digital Polymerase Chain Reaction. Bacterial drug resistance is a significant food safety problem and public health threat. Plasmids carrying drug resistance genes may result in the rapid spread of resistance among different bacteria, hosts, and environments; therefore, antibiotic resistance monitoring and continuing research into the mechanisms of drug resistance are urgently needed. Southern blotting with probes for antibiotic resistance genes and even next-generation sequencing have been used previously to detect plasmid-borne resistance genes, but these approaches are complex and time-consuming. The next-generation sequencing requires strict laboratory conditions and bioinformatics analysis ability. In this study, we developed a simplified and sensitive method to detect plasmid-borne antimicrobial resistance genes and plasmid replicon types. Salmonella strains carrying plasmids of three different replicon types that contained mcr-1 and two ESBL-producing genes were used to verify the new method. The plasmids harbored by the Salmonella strains were separated by S1 nuclease treatment and pulsed-field gel electrophoresis (PFGE), then recovered and used as the templates for droplet digital polymerase chain reaction (ddPCR) to identify target genes. The target genes were present in significantly higher copy numbers on the plasmids than the background noise. These results were consistent with the plasmid sequencing results. This S1-PFGE-ddPCR method was less time-consuming to perform than Southern blot and complete plasmid sequencing. Therefore, this method represents a time-saving alternative for detecting plasmid-borne genes, and is likely to be a valuable tool for detecting coexisting plasmid-borne drug resistance genes.202133661029
493790.9997Fast and Accurate Large-Scale Detection of β-Lactamase Genes Conferring Antibiotic Resistance. Fast detection of β-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scaleblaFinder detected almost all clinically available bla genes. Notably, the large-scaleblaFinder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scaleblaFinder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.201526169415
5820100.9997Sequencing Methods to Study the Microbiome with Antibiotic Resistance Genes in Patients with Pulmonary Infections. Various antibiotic-resistant bacteria (ARB) are known to induce repeated pulmonary infections and increase morbidity and mortality. A thorough knowledge of antibiotic resistance is imperative for clinical practice to treat resistant pulmonary infections. In this study, we used a reads-based method and an assembly-based method according to the metagenomic next-generation sequencing (mNGS) data to reveal the spectra of ARB and corresponding antibiotic resistance genes (ARGs) in samples from patients with pulmonary infections. A total of 151 clinical samples from 144 patients with pulmonary infections were collected for retrospective analysis. The ARB and ARGs detection performance was compared by the reads-based method and assembly-based method with the culture method and antibiotic susceptibility testing (AST), respectively. In addition, ARGs and the attribution relationship of common ARB were analyzed by the two methods. The comparison results showed that the assembly-based method could assist in determining pathogens detected by the reads-based method as true ARB and improve the predictive capabilities (46% > 13%). ARG-ARB network analysis revealed that assembly-based method could promote determining clear ARG-bacteria attribution and 101 ARGs were detected both in two methods. 25 ARB were obtained by both methods, of which the most predominant ARB and its ARGs in the samples of pulmonary infections were Acinetobacter baumannii (ade), Pseudomonas aeruginosa (mex), Klebsiella pneumoniae (emr), and Stenotrophomonas maltophilia (sme). Collectively, our findings demonstrated that the assembly-based method could be a supplement to the reads-based method and uncovered pulmonary infection-associated ARB and ARGs as potential antibiotic treatment targets.202439113195
5823110.9997Comparing Patient Risk Factor-, Sequence Type-, and Resistance Locus Identification-Based Approaches for Predicting Antibiotic Resistance in Escherichia coli Bloodstream Infections. Rapid diagnostic tests for antibiotic resistance that identify the presence or absence of antibiotic resistance genes/loci are increasingly being developed. However, these approaches usually neglect other sources of predictive information which could be identified over shorter time periods, including patient epidemiologic risk factors for antibiotic resistance and markers of lineage. Using a data set of 414 Escherichia coli isolates recovered from separate episodes of bacteremia at a single academic institution in Toronto, Ontario, Canada, between 2010 and 2015, we compared the potential predictive ability of three approaches (epidemiologic risk factor-, pathogen sequence type [ST]-, and resistance gene identification-based approaches) for classifying phenotypic resistance to three antibiotics representing classes of broad-spectrum antimicrobial therapy (ceftriaxone [a 3rd-generation cephalosporin], ciprofloxacin [a fluoroquinolone], and gentamicin [an aminoglycoside]). We used logistic regression models to generate model receiver operating characteristic (ROC) curves. Predictive discrimination was measured using apparent and corrected (bootstrapped) areas under the curves (AUCs). Epidemiologic risk factor-based models based on two simple risk factors (prior antibiotic exposure and recent prior susceptibility of Gram-negative bacteria) provided a modest predictive discrimination, with AUCs ranging from 0.65 to 0.74. Sequence type-based models demonstrated strong discrimination (AUCs, 0.83 to 0.94) across all three antibiotic classes. The addition of epidemiologic risk factors to sequence type significantly improved the ability to predict resistance for all antibiotics (P < 0.05). Resistance gene identification-based approaches provided the highest degree of discrimination (AUCs, 0.88 to 0.99), with no statistically significant benefit being achieved by adding the patient epidemiologic predictors. In summary, sequence type or other lineage-based approaches could produce an excellent discrimination of antibiotic resistance and may be improved by incorporating readily available patient epidemiologic predictors but are less discriminatory than identification of the presence of known resistance loci.201930894438
5087120.9997Sensitive colorimetric detection of antibiotic resistant Staphylococcus aureus on dairy farms using LAMP with pH-responsive polydiacetylene. Rapidly and accurately detecting antibiotic-resistant pathogens in agriculture and husbandry is important since these represent a major threat to public health. While much attention has been dedicated to detecting now-common resistant bacteria, such as methicillin-resistant Staphylococcus aureus, fewer methods have been developed to assess resistance against macrolides in Staphylococcus aureus (SA). Here, we report a visual on-site detection system for macrolide resistant SA in dairy products. First, metagenomic sequencing in raw milk, cow manure, water and aerosol deposit collected from dairy farms around Tianjin was used to identify the most abundant macrolide resistance gene, which was found to be the macB gene. In parallel, SA housekeeping genes were screened to allow selective identification of SA, which resulted in the selection of the SAOUHSC_01275 gene. Next, LAMP assays targeting the above-mentioned genes were developed and interpreted by agarose gel electrophoresis. For on-site application, different pH-sensitive colorimetric LAMP indicators were compared, which resulted in selection of polydiacetylene (PDA) as the most sensitive candidate. Additionally, a semi-quantitative detection could be realized by analyzing the RGB information via smartphone with a LOD of 1.344 × 10(-7) ng/μL of genomic DNA from a milk sample. Finally, the proposed method was successfully carried out at a real farm within 1 h from sample to result by using freeze-dried reagents and portable devices. This is the first instance in which PDA is used to detect LAMP products, and this generic read-out system can be expanded to other antibiotic resistant genes and bacteria.202336327562
5124130.9997Oxford nanopore long-read sequencing enables the generation of complete bacterial and plasmid genomes without short-read sequencing. INTRODUCTION: Genome-based analysis is crucial in monitoring antibiotic-resistant bacteria (ARB)and antibiotic-resistance genes (ARGs). Short-read sequencing is typically used to obtain incomplete draft genomes, while long-read sequencing can obtain genomes of multidrug resistance (MDR) plasmids and track the transmission of plasmid-borne antimicrobial resistance genes in bacteria. However, long-read sequencing suffers from low-accuracy base calling, and short-read sequencing is often required to improve genome accuracy. This increases costs and turnaround time. METHODS: In this study, a novel ONT sequencing method is described, which uses the latest ONT chemistry with improved accuracy to assemble genomes of MDR strains and plasmids from long-read sequencing data only. Three strains of Salmonella carrying MDR plasmids were sequenced using the ONT SQK-LSK114 kit with flow cell R10.4.1, and de novo genome assembly was performed with average read accuracy (Q > 10) of 98.9%. RESULTS AND DISCUSSION: For a 5-Mb-long bacterial genome, finished genome sequences with accuracy of >99.99% could be obtained at 75× sequencing coverage depth using Flye and Medaka software. Thus, this new ONT method greatly improves base-calling accuracy, allowing for the de novo assembly of high-quality finished bacterial or plasmid genomes without the need for short-read sequencing. This saves both money and time and supports the application of ONT data in critical genome-based epidemiological analyses. The novel ONT approach described in this study can take the place of traditional combination genome assembly based on short- and long-read sequencing, enabling pangenomic analyses based on high-quality complete bacterial and plasmid genomes to monitor the spread of antibiotic-resistant bacteria and antibiotic resistance genes.202337256057
4927140.9997Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak. The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the bla(CTX-M-15) gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the bla(CTX-M-15) gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying bla(CTX-M) group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.IMPORTANCE This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.201931289171
4943150.9997Targeted sequencing of Enterobacterales bacteria using CRISPR-Cas9 enrichment and Oxford Nanopore Technologies. Sequencing DNA directly from patient samples enables faster pathogen characterization compared to traditional culture-based approaches, but often yields insufficient sequence data for effective downstream analysis. CRISPR-Cas9 enrichment is designed to improve the yield of low abundance sequences but has not been thoroughly explored with Oxford Nanopore Technologies (ONT) for use in clinical bacterial epidemiology. We designed CRISPR-Cas9 guide RNAs to enrich the human pathogen Klebsiella pneumoniae, by targeting multi-locus sequence type (MLST) and transfer RNA (tRNA) genes, as well as common antimicrobial resistance (AMR) genes and the resistance-associated integron gene intI1. We validated enrichment performance in 20 K. pneumoniae isolates, finding that guides generated successful enrichment across all conserved sites except for one AMR gene in two isolates. Enrichment of MLST genes led to a correct allele call in all seven loci for 8 out of 10 isolates that had depth of 30× or more in these regions. We then compared enriched and unenriched sequencing of three human fecal samples spiked with K. pneumoniae at varying abundance. Enriched sequencing generated 56× and 11.3× the number of AMR and MLST reads, respectively, compared to unenriched sequencing, and required approximately one-third of the computational storage space. Targeting the intI1 gene often led to detection of 10-20 proximal resistance genes due to the long reads produced by ONT sequencing. We demonstrated that CRISPR-Cas9 enrichment combined with ONT sequencing enabled improved genomic characterization outcomes over unenriched sequencing of patient samples. This method could be used to inform infection control strategies by identifying patients colonized with high-risk strains. IMPORTANCE: Understanding bacteria in complex samples can be challenging due to their low abundance, which often results in insufficient data for analysis. To improve the detection of harmful bacteria, we implemented a technique aimed at increasing the amount of data from target pathogens when combined with modern DNA sequencing technologies. Our technique uses CRISPR-Cas9 to target specific gene sequences in the bacterial pathogen Klebsiella pneumoniae and improve recovery from human stool samples. We found our enrichment method to significantly outperform traditional methods, generating far more data originating from our target genes. Additionally, we developed new computational techniques to further enhance the analysis, providing a thorough method for characterizing pathogens from complex biological samples.202539772804
4975160.9997Comprehensive genomic and plasmid characterization of multidrug-resistant bacterial strains by R10.4.1 nanopore sequencing. The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum β-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens.202438460283
5689170.9997A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales. Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (bla(NDM)) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry bla(NDM) and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of bla(NDM)-(1) carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 10(0) CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.202438667187
5088180.9997A Multiplex SYBR Green Real-Time PCR Assay for the Detection of Three Colistin Resistance Genes from Cultured Bacteria, Feces, and Environment Samples. The aim of the study was to develop a multiplex assay for rapid detection of mcr-1, mcr-2, and mcr-3, a group of genes of conferring resistance to colistin mediated by plasmid in Enterobacteriaceae. A SYBR Green based real-time PCR assay has been designed to detect the mcr genes, and applied to cultured bacteria, feces and soil samples. All three mcr genes could be detected with a lower limit of 10(2) cultured bacteria. This test was highly specific and sensitive, and generated no false-positive results. The assay was also conclusive when applied to feces and soil samples containing mcr-1-positive Escherichia coli, which could facilitate the screening of mcr genes not only in the bacteria, but also directly from the environment. This simple, rapid, sensitive, and specific multiplex assay will be useful for rapid screening of the colistin resistance in both clinical medicine and animal husbandry.201729163387
5663190.9997Development of multiplex Luminex assays for the surveillance of antimicrobial resistance genes in nasal samples. Bovine respiratory disease (BRD) is the major cause of morbidity and mortality in feedlot cattle. It is the major driver for the therapeutic use of antimicrobials in feedlot cattle with their continued use and effectiveness being underpinned through the implementation of stewardship programs that include monitoring of resistance levels. To enable these programs, rapid and user-friendly assays are needed to detect antimicrobial resistance genes (ARG) for efficient monitoring. This study developed multiplex Luminex assays targeting 34 ARGs and validated them using reference strains of Pasteurellaceae and other bacteria, as well as field samples from nasal swabs of cattle (n = 94) undergoing BRD treatment at an Australian feedlot. One swab was collected from each nostril of every animal, with one being used for bacterial culture and conventional PCR analyses for ARGs, while the DNA extracted from the second swab was analyzed using the novel Luminex assays for the presence or absence of the ARGs of interest. The pathogens isolated by culture were tested for macrolide resistance genes erm(42), mph(E) and msr(E); sulfonamide resistance genes, sul1 and sul2; florfenicol resistance gene floR; β-lactam resistance gene bla(Rob-1) and tetracycline resistance genes tet(Q) and tet(Y), by conventional PCR. Kappa statistics suggested a moderate agreement between the tests in detecting the macrolide resistance genes. Luminex based analyses identified more resistance genes than PCR on cultured organisms, revealing the presence of a broader array of these genes than previously reported. In addition to detecting more genes, Luminex assays could process a higher number of samples in a single day, making them well-suited for ongoing surveillance of antimicrobial resistance in BRD affected cattle. This capability is essential for optimising therapeutic use and detecting emerging resistance patterns.202540848749