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568900.9989A 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
492710.9989Optical 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
512520.9988Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies. The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected >96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.202438354391
512430.9988Oxford 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
259940.9988Evaluation of whole-genome sequencing protocols for detection of antimicrobial resistance, virulence factors and mobile genetic elements in antimicrobial-resistant bacteria. Introduction. Antimicrobial resistance (AMR) poses a critical threat to global health, underscoring the need for rapid and accurate diagnostic tools. Methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae (ESBL-Kp) are listed among the World Health Organization's priority pathogens.Hypothesis. A rapid nanopore-based protocol can accurately and efficiently detect AMR genes, virulence factors (VFs) and mobile genetic elements (MGEs) in MRSA and ESBL-Kp, offering performance comparable to or superior to traditional sequencing methods.Aim. Evaluate whole-genome sequencing (WGS) protocols for detecting AMR genes, VFs and MGEs in MRSA and ESBL-Kp, to identify the most accurate and efficient tool for pathogen profiling.Methodology. Five distinct WGS protocols, including a rapid nanopore-based protocol (ONT20h) and four slower sequencing methods, were evaluated for their effectiveness in detecting genetic markers. The protocols' performances were compared across AMR genes, VFs and MGEs. Additionally, phenotypic antimicrobial susceptibility testing was performed to assess concordance with the genomic findings.Results. Compared to four slower sequencing protocols, the rapid nanopore-based protocol (ONT20h) demonstrated comparable or superior performance in AMR gene detection and equivalent VF identification. Although MGE detection varied among protocols, ONT20h showed a high level of agreement with phenotypic antimicrobial susceptibility testing.Conclusion. The findings highlight the potential of rapid WGS as a valuable tool for clinical microbiology, enabling timely implementation of infection control measures and informed therapeutic decisions. However, further studies are required to optimize the clinical application of this technology, considering costs, availability of bioinformatics tools and quality of reference databases.202540105741
493850.9988Optical 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
503160.9988Rapid Tracing of Resistance Plasmids in a Nosocomial Outbreak Using Optical DNA Mapping. Resistance to life-saving antibiotics increases rapidly worldwide, and multiresistant bacteria have become a global threat to human health. Presently, the most serious threat is the increasing spread of Enterobacteriaceae carrying genes coding for extended spectrum β-lactamases (ESBL) and carbapenemases on highly mobile plasmids. We here demonstrate how optical DNA maps of single plasmids can be used as fingerprints to trace plasmids, for example, during resistance outbreaks. We use the assay to demonstrate a potential transmission route of an ESBL-carrying plasmid between bacterial strains/species and between patients, during a polyclonal outbreak at a neonatal ward at Sahlgrenska University Hospital (Gothenburg, Sweden). Our results demonstrate that optical DNA mapping is an easy and rapid method for detecting the spread of plasmids mediating resistance. With the increasing prevalence of multiresistant bacteria, diagnostic tools that can aid in solving ongoing routes of transmission, in particular in hospital settings, will be of paramount importance.201627627201
493970.9987Identification 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
501980.9987Extended-spectrum beta-lactamases: definition, history, an update on their genetic environment and detection methods. Bacterial resistance remains a major challenge in the therapeutic field. Beta-lactam antibiotics are widely used to treat Enterobacteriaceae, especially third-generation cephalosporins (3GCs), which are used in infections caused by bacteria resistant to first- and second-line antibiotics. However, these bacteria have been able to develop resistance against the used antibiotics through the production of extended-spectrum beta-lactamase (ESBL) enzymes. These enzymes inactivate 3GCs and are sensitive to beta-lactamase inhibitors such as clavulanic acid. This resistance is acquired by plasmids (IncF, IncI, IncK…) which carry mobile genetic elements (insertion sequence, transposon…) with genes coding for these enzymes, namely, the bla (CTX-M), bla (SHV) and bla (TEM), which code for the most frequent types of ESBL (CTX-M, SHV and TEM). Unfortunately, when ESBLs are not identified in time, appropriate treatment is delayed, reducing the chances of cure. Current data highlight the spread and dangerousness of ESBL-producing bacteria worldwide and confirm the priority given to these bacteria by the World Health Organization, which insists on vigilance in identifying them, both in patients and through surveillance studies. The aim of the current review is to provide a better understanding of ESBLs, to highlight their historical evolution and to show the importance of their genetic environment in the dissemination and spread of these enzymes worldwide, as well as the techniques used to detect them in laboratory studies. Current data demonstrate the degree of danger posed by ESBL-producing bacteria and confirm the priority given to these bacteria by the World Health Organization for the development of new antimicrobial agents.202540554694
182390.9987Finding the Missing IMP Gene: Overcoming the Imipenemase IMP Gene Drop-Out in Automated Molecular Testing for Carbapenem-Resistant Bacteria Circulating in Latin America. Carbapenem resistance is considered one of the greatest current threats to public health, particularly in the management of infections in clinical settings. Carbapenem resistance in bacteria is mainly due to mechanisms such as the production of carbapenemases (such as the imipenemase IMP, or other enzymes like VIM, NDM, and KPC), that can be detected by several laboratory tests, including immunochromatography and automated real-time PCR (qPCR). Methods: As part of local studies to monitor carbapenem-resistant bacteria in Costa Rica, two cases were initially identified with inconsistent IMP detection results. A possible gene drop-out in the automated qPCR test was suggested based on the negative result, contrasting with the positive result by immunochromatography and whole-genome sequencing. We hypothesized that molecular testing could be optimized through the development of tailored assays to improve the detection of IMP genes. Thus, using IMP gene sequences from the local isolates and regional sequences in databases, primers were redesigned to extend the detection of IMP alleles of regional relevance. Results: The tailored qPCR was applied to a local collection of 119 carbapenem-resistant isolates. The genomes of all 14 positive cases were sequenced, verifying the results of the custom qPCR, despite the negative results of the automated testing. Conclusions: Guided by whole-genome sequencing, it was possible to extend the molecular detection of IMP alleles circulating in Latin America using a tailored qPCR to overcome IMP gene drop-out and false-negative results in an automated qPCR.202540867967
5123100.9987Ultrafast and Cost-Effective Pathogen Identification and Resistance Gene Detection in a Clinical Setting Using Nanopore Flongle Sequencing. Rapid bacterial identification and antimicrobial resistance gene (ARG) detection are crucial for fast optimization of antibiotic treatment, especially for septic patients where each hour of delayed antibiotic prescription might have lethal consequences. This work investigates whether the Oxford Nanopore Technology's (ONT) Flongle sequencing platform is suitable for real-time sequencing directly from blood cultures to identify bacteria and detect resistance-encoding genes. For the analysis, we used pure bacterial cultures of four clinical isolates of Escherichia coli and Klebsiella pneumoniae and two blood samples spiked with either E. coli or K. pneumoniae that had been cultured overnight. We sequenced both the whole genome and plasmids isolated from these bacteria using two different sequencing kits. Generally, Flongle data allow rapid bacterial ID and resistome detection based on the first 1,000-3,000 generated sequences (10 min to 3 h from the sequencing start), albeit ARG variant identification did not always correspond to ONT MinION and Illumina sequencing-based data. Flongle data are sufficient for 99.9% genome coverage within at most 20,000 (clinical isolates) or 50,000 (positive blood cultures) sequences generated. The SQK-LSK110 Ligation kit resulted in higher genome coverage and more accurate bacterial identification than the SQK-RBK004 Rapid Barcode kit.202235369431
5038110.9987Simple and quick detection of extended-spectrum β-lactamase and carbapenemase-encoding genes using isothermal nucleic acid amplification techniques. The spread of plasmid-mediated antibiotic-resistant bacteria must be controlled; to this end, developing kits for simple and rapid detection in food and clinical settings is desirable. This review describes the detection of antibiotic resistance genes in extended-spectrum β-lactamase （ESBL）- and carbapenemase-producing bacteria. Loop-mediated isothermal amplification （LAMP）, a technique developed in Japan, is a useful diffusion amplification method that does not require equipment like thermal cyclers, and amplifies the target gene in 30 min at about 65℃. Although most reports targeting ESBL and carbapenemase genes are intended for clinical use, environmental and food samples have also been targeted. Recombinase polymerase amplification （RPA） has recently been developed; in RPA, the reaction proceeds under the human skin with reaction conditions of 30 min at 37℃. Detection of ESBL and carbapenemase-encoding genes in food and clinical samples using RPA has been reported in limited studies. However, research on RPA has just begun, and further development is expected.202338233166
5020120.9987Detection of expanded-spectrum β-lactamases in Gram-negative bacteria in the 21st century. Emerging β-lactamase-producing-bacteria (ESBL, AmpC and carbapenemases) have become a serious problem in our community due to their startling spread worldwide and their ability to cause infections which are difficult to treat. Diagnosis of these β-lactamases is of clinical and epidemiological interest. Over the past 10 years, several methods have been developed aiming to rapidly detect these emerging enzymes, thus preventing their rapid spread. In this review, we describe the range of screening and detection methods (phenotypic, molecular and other) for detecting these β-lactamases but also whole genome sequencing as a tool for detecting the genes encoding these enzymes.201526162631
4926130.9987Complete Assembly of Escherichia coli Sequence Type 131 Genomes Using Long Reads Demonstrates Antibiotic Resistance Gene Variation within Diverse Plasmid and Chromosomal Contexts. The incidence of infections caused by extraintestinal Escherichia coli (ExPEC) is rising globally, which is a major public health concern. ExPEC strains that are resistant to antimicrobials have been associated with excess mortality, prolonged hospital stays, and higher health care costs. E. coli sequence type 131 (ST131) is a major ExPEC clonal group worldwide, with variable plasmid composition, and has an array of genes enabling antimicrobial resistance (AMR). ST131 isolates frequently encode the AMR genes bla(CTX-M-14), bla(CTX-M-15), and bla(CTX-M-27), which are often rearranged, amplified, and translocated by mobile genetic elements (MGEs). Short DNA reads do not fully resolve the architecture of repetitive elements on plasmids to allow MGE structures encoding bla(CTX-M) genes to be fully determined. Here, we performed long-read sequencing to decipher the genome structures of six E. coli ST131 isolates from six patients. Most long-read assemblies generated entire chromosomes and plasmids as single contigs, in contrast to more fragmented assemblies created with short reads alone. The long-read assemblies highlighted diverse accessory genomes with bla(CTX-M-15), bla(CTX-M-14), and bla(CTX-M-27) genes identified in three, one, and one isolates, respectively. One sample had no bla(CTX-M) gene. Two samples had chromosomal bla(CTX-M-14) and bla(CTX-M-15) genes, and the latter was at three distinct locations, likely transposed by the adjacent MGEs: ISEcp1, IS903B, and Tn2 This study showed that AMR genes exist in multiple different chromosomal and plasmid contexts, even between closely related isolates within a clonal group such as E. coli ST131.IMPORTANCE Drug-resistant bacteria are a major cause of illness worldwide, and a specific subtype called Escherichia coli ST131 causes a significant number of these infections. ST131 bacteria become resistant to treatments by modifying their DNA and by transferring genes among one another via large packages of genes called plasmids, like a game of pass-the-parcel. Tackling infections more effectively requires a better understanding of what plasmids are being exchanged and their exact contents. To achieve this, we applied new high-resolution DNA sequencing technology to six ST131 samples from infected patients and compared the output to that of an existing approach. A combination of methods shows that drug resistance genes on plasmids are highly mobile because they can jump into ST131's chromosomes. We found that the plasmids are very elastic and undergo extensive rearrangements even in closely related samples. This application of DNA sequencing technologies illustrates at a new level the highly dynamic nature of ST131 genomes.201931068432
4935140.9987Three 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
1883150.9987Whole genome sequence of colistin-resistant Escherichia coli from western India. BACKGROUND: With virtually dried out new antibiotic discovery pipeline, emergence and spread of antimicrobial resistance is a cause for global concern. Colistin, a cyclic polypeptide antibiotic, often regarded as last resort for multi drug resistance gram-negative bacteria, is also rendered ineffective by horizontal transfer of resistance genes. Surveillance of colistin resistance in GNB is essential to ascertain molecular epidemiology. METHODS: Whole genome sequencing (WGS) of an unusual colistin resistant urinary isolate of Escherichia coli was performed using Illumina MiSeq platform using 2x250bp V2 chemistry by following the manufactures protocol (Illumina Inc. USA). Multiple web-based bio-informatic tools were utilized to ascertain antibiotic resistant genes. RESULTS: An approximate 5.4 Mb of genome of the urinary isolate AFMC_UC19 was sequenced successfully. Mobile colistin resistance gene (mcr) on the plasmid responsible for horizontal spread was absent in the isolate. CONCLUSION: Colistin resistance has been reported previously in Klebsiella pneumoniae and it is a rare occurrence in Escherichia coli in Indian setting. Although the isolate lack mcr mediated colistin resistance, emergence and spread of colistin resistant in gram-negative bacteria pose a threat.202134305283
5018160.9987Multidrug-resistant Gram-negative bacteria: a product of globalization. Global trade and mobility of people has increased rapidly over the last 20 years. This has had profound consequences for the evolution and the movement of antibiotic resistance genes. There is increasing exposure of populations all around the world to resistant bacteria arising in the emerging economies. Arguably the most important development of the last two decades in the field of antibiotic resistance is the emergence and spread of extended-spectrum β-lactamases (ESBLs) of the CTX-M group. A consequence of the very high rates of ESBL production among Enterobacteriaceae in Asian countries is that there is a substantial use of carbapenem antibiotics, resulting in the emergence of plasmid-mediated resistance to carbapenems. This article reviews the emergence and spread of multidrug-resistant Gram-negative bacteria, focuses on three particular carbapenemases--imipenem carbapenemases, Klebsiella pneumoniae carbapenemase, and New Delhi metallo-β-lactamase--and highlights the importance of control of antibiotic use.201525737092
2512170.9987Understanding and addressing β-lactam resistance mechanisms in gram-negative bacteria in Lebanon: A scoping review. BACKGROUND: A growing threat to public health is the worldwide problem of antimicrobial resistance (AMR), in which gram-negative organisms are playing a significant role. Antibiotic abuse and misuse, together with inadequate monitoring and control protocols, have contributed to the emergence of resistant strains. This global scenario prepares us to look more closely at the situation in Lebanon. The aim of this review is to investigate in detail the resistance mechanisms and related genes that are displayed by gram-negative organisms in Lebanon. METHODS: A comprehensive analysis was carried out to pinpoint and gather information regarding gram-negative bacteria displaying resistance to antibiotics. To contribute to a complete understanding of the current state of antibiotic resistance in gram-negative strains, it was intended to collect and evaluate data on these organisms' resistance patterns in a comprehensive manner. RESULTS: Several studies have emphasized the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) in Lebanon, specifically noting Escherichia coli and Klebsiella pneumoniae as the most frequent culprits, with OXA-48 and NDM-1 being the primary carbapenemases discovered. Furthermore, the TEM β-lactamase families are the primary source of extended-spectrum β-lactamases (ESBLs) in Shigella and Salmonella. Additionally, resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa have been linked to nosocomial infections in the country. CONCLUSION: There is a considerable frequency of antibiotic overuse and misuse in Lebanon, based to the limited data available on antibiotic consumption. In conclusion, antibiotic stewardship initiatives and additional research beyond the confines of single-center studies in Lebanon are needed.202539981361
4942180.9986Nanopore-based enrichment of antimicrobial resistance genes - a case-based study. Rapid screening of hospital admissions to detect asymptomatic carriers of resistant bacteria can prevent pathogen outbreaks. However, the resulting isolates rarely have their genome sequenced due to cost constraints and long turn-around times to get and process the data, limiting their usefulness to the practitioner. Here we used real-time, on-device target enrichment ("adaptive") sequencing as a highly multiplexed assay covering 1,147 antimicrobial resistance genes. We compared its utility against standard and metagenomic sequencing, focusing on an isolate of Raoultella ornithinolytica harbouring three carbapenemases (NDM, KPC, VIM). Based on this experimental data, we then modelled the influence of several variables on the enrichment results and predicted the large effect of nucleotide identity (higher is better) and read length (shorter is better). Lastly, we showed how all relevant resistance genes are detected using adaptive sequencing on a miniature ("Flongle") flow cell, motivating its use in a clinical setting to monitor similar cases and their surroundings.202336949817
2497190.9986Rapid Simultaneous Detection of the Clinically Relevant Carbapenemase Resistance Genes blaKPC, blaOXA48, blaVIM and blaNDM with the Newly Developed Ready-to-Use qPCR CarbaScan LyoBead. Antibiotic resistance, in particular the dissemination of carbapenemase-producing organisms, poses a significant threat to global healthcare. This study introduces the qPCR CarbaScan LyoBead assay, a robust, accurate, and efficient tool for detecting key carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaVIM. The assay utilizes lyophilized beads, a technological advancement that enhances stability, simplifies handling, and eliminates the need for refrigeration. This feature renders it particularly well-suited for point-of-care diagnostics and resource-limited settings. The assay's capacity to detect carbapenemase genes directly from bacterial colonies without the need for extensive sample preparation has been demonstrated to streamline workflows and enable rapid diagnostic results. The assay demonstrated 100% specificity and sensitivity across a diverse range of bacterial strains, including multiple allelic variants of target genes, facilitating precise identification of resistance mechanisms. Bacterial strains of the species Acinetobacter baumannii, Citrobacter freundii, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae and Pseudomonas aeruginosa were utilized as reference material for assay development (n = 9) and validation (n = 28). It is notable that the assay's long shelf life and minimal operational complexity further enhance its utility for large-scale implementation in healthcare, food safety, and environmental monitoring. The findings emphasize the necessity of continuous surveillance and the implementation of rapid diagnostic methods for the effective detection of resistance genes. Furthermore, the assay's potential applications in other fields, such as toxin-antitoxin system research and monitoring of resistant bacteria in the community, highlight its versatility. In conclusion, the qPCR CarbaScan LyoBead assay is a valuable tool that can contribute to the urgent need to combat antibiotic resistance and improve global public health outcomes.202539940986