Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria. - Related Documents




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504201.0000Multiplex loop-mediated isothermal amplification (multi-LAMP) assay for rapid detection of mcr-1 to mcr-5 in colistin-resistant bacteria. Purpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria. Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection. Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay. Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.201931308708
504110.9999Development and Validation of a Clinical Laboratory Improvement Amendments-Compliant Multiplex Real-Time PCR Assay for Detection of mcr Genes. Increased use of colistin in both human and veterinary medicine has led to the emergence of plasmid-mediated colistin resistance (mcr genes). In this study, we report the development of a real-time PCR assay using TaqMan probe-based chemistry for detection of mcr genes from bacterial isolates. Positive control isolates harboring mcr-1 and mcr-2 yielded exponential amplification curves with the assay, and the amplification efficiency was 98% and 96% for mcr-1 and mcr-2, respectively. Each target gene could be reproducibly detected from a sample containing 10(3) cfu/mL of mcr-harboring bacteria, and there was no cross-reactivity with DNA extracted from several multidrug-resistant bacteria harboring other resistance genes, but lacking mcr genes. Both sensitivity and specificity of the mcr real-time PCR assay were 100% in a method validation performed with a set of 25 previously well-characterized bacterial isolates containing mcr-positive and -negative bacteria. This newly developed assay is a rapid and sensitive tool for detecting emerging mcr genes in cultured bacterial isolates. The assay was successfully validated according to quality standards of the Clinical Laboratory Improvement Amendments (CLIA).201930942652
504020.9999Rapid detection and differentiation of mobile colistin resistance (mcr-1 to mcr-10) genes by real-time PCR and melt-curve analysis. BACKGROUND: The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount. AIM: To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes. METHODS: All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results. FINDINGS: In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed. CONCLUSIONS: The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.202133485969
508830.9998A 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
508940.9998A 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
182450.9998Subtype Screening of bla(IMP) Genes Using Bipartite Primers for DNA Sequencing. Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of bla(IMP) genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all bla(IMP) genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for bla(IMP-81), 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of bla(IMP), which is useful for the surveillance of bacteria with bla(IMP) in clinical and public health settings or environmental fields.202133790070
159860.9997A method to detect Escherichia coli carrying the colistin-resistance genes mcr-1 and mcr-2 using a single real-time polymerase chain reaction and its application to chicken cecal and porcine fecal samples. Colistin is one of the last-resort antibiotics for the treatment of multidrug-resistant infections in humans, but transmissible colistin-resistance genes have emerged in bacteria from animals. The rapid and sensitive detection among animals of colonization with bacteria carrying these genes is critical in helping to control further spread. Here we describe a method for broth enrichment of colistin-resistant Escherichia coli from animal fecal and cecal samples followed by real-time polymerase chain reaction (PCR) for the simultaneous detection of two of the main colistin-resistance genes, mcr-1 and mcr-2. The PCR uses a single set of nondegenerative primers, and mcr variants can be differentiated by melt-curve analysis. Overnight culture enrichment was effective for amplifying colistin-resistant E. coli, even when initially present in numbers as low as 10 bacteria per gram of sample. The mcr-1 and mcr-2 genes were not found in any of the Ontario swine and poultry samples investigated.201830363381
182370.9997Finding 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
222880.9997Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
298890.9997Establishment of a Multiplex Loop-Mediated Isothermal Amplification Method for Rapid Detection of Sulfonamide Resistance Genes (sul1, sul2, sul3) in Clinical Enterobacteriaceae Isolates from Poultry. Antimicrobial resistance genes play an important role in mediating resistance to sulfonamide in Gram-negative bacteria. While PCR is the current method to detect sulfonamide resistance genes (sul1, sul2, sul3), it is time-consuming and costly and there is an urgent need to develop a more convenient, simpler and rapid test for the sul. In this study, we describe a multiplex loop-mediated isothermal amplification (m-LAMP) assay we developed for the rapid and simultaneous detection of three sul. This m-LAMP assay successfully detected seven reference strains with different sul genotypes, but was negative for nine sul-negative reference strains. The m-LAMP products were verified by HinfI restriction enzyme digestion and the detection limit of the test was 0.5 pg genomic DNA per reaction. Testing 307 sulfonamide-resistant Enterobacteriaceae clinical isolates with the m-LAMP revealed all were positive for the sul with sul2 (79.5%) and sul1 (64.5%) being most prevalent, and sul3 the least (12.1%). Of the Enterobacteriaceae isolates tested, the Salmonella Indiana, a newly emerging serovar resistant to numerous antimicrobials, were most commonly positive with 33% having sul3.201829708802
2249100.9997Tracking Multidrug Resistance in Gram-Negative Bacteria in Alexandria, Egypt (2020-2023): An Integrated Analysis of Patient Data and Diagnostic Tools. BACKGROUND: The rise in carbapenem-resistant Enterobacteriaceae (CRE) in Egypt, particularly in hospital settings, poses a significant public health challenge. This study aims to develop a combined epidemiological surveillance tool utilizing the Microreact online platform (version 269) and molecular microarray technology to track and analyze carbapenem-resistant Escherichia coli strains in Egypt. The objective is to integrate molecular diagnostics and real-time data visualization to better understand the spread and evolution of multidrug-resistant (MDR) bacteria. METHODS: The study analyzed 43 E. coli isolates collected from Egyptian hospitals between 2020 and 2023. Nanopore sequencing and microarray analysis were used to identify carbapenemase genes and other resistance markers, whereas the VITEK2 system was employed for phenotypic antibiotic susceptibility testing. Microreact was used to visualize epidemiological data, mapping the geographic and temporal distribution of resistant strains. RESULTS: We found that 72.09% of the isolates, predominantly from pediatric patients, carried the blaNDM-5 gene, while other carbapenemase genes, including blaOXA-48 and blaVIM, were also detected. The microarray method demonstrated 92.9% diagnostic sensitivity and 87.7% diagnostic specificity compared to whole-genome sequencing. Phenotypic resistance correlated strongly with next-generation sequencing (NGS) genotypic data, achieving 95.6% sensitivity and 95.2% specificity. CONCLUSIONS: This method establishes the utility of combining microarray technology, NGS and real-time data visualization for the surveillance of carbapenem-resistant Enterobacteriaceae, especially E. coli. The high concordance between genotypic and phenotypic data underscores the potential of DNA microarrays as a cost-effective alternative to whole-genome sequencing, especially in resource-limited settings. This integrated approach can enhance public health responses to MDR bacteria in Egypt.202439766575
5773110.9997LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria. BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 μg/mL) and vancomycin (50 μg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.201729169321
4956120.9997Rapid 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
5038130.9997Simple 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
1703140.9997Acinetobacter baumannii clinical isolates from outbreaks in Erbil hospitals after the COVID-19 pandemic. INTRODUCTION: Acinetobacter baumannii is endemic in hospital environments, and since the coronavirus disease 2019 (COVID-19) pandemic, multidrug-resistant A. baumannii has become more potent. This potential evolution is driven by the undetectable numbers of gene resistances it has acquired. We evaluated the antibiotic-resistance genes in isolates from patients in Erbil hospitals. METHODOLOGY: This is the first study to demonstrate the antimicrobial resistance epidemic in Erbil, Iraq. A total of 570 patients, including 100 COVID-19 patients were tested. Isolate identification, characterization, antibiotics susceptibility test, polymerase chain reaction (PCR) amplification of the antibiotic resistance genes in both bacterial chromosome and plasmid, 16S-23S rRNA gene intergenic spacer (ITS) sequencing using the Sanger DNA sequencing, and phylogenetic analysis were used in this study. RESULTS: Only 13% of A. baumannii isolates were from COVID-19 patients. All isolates were multi-drug resistant due because of 24 resistance genes located in both the bacterial chromosome or the plasmid. blaTEM gene was detected in the isolates; however, aadB was not detected in the isolated bacteria. New carbapenemase genes were identified by Sanger sequencing and resistance genes were acquired by plasmids. CONCLUSIONS: The study identified metabolic differences in the isolates; although all the strains used the coumarate pathway to survive. Several resistance genes were present in the isolates' plasmids and chromosome. There were no strong biofilm producers. The role of the plasmid in A. baumannii resistance development was described based on the results.202439499748
5090150.9997A TaqMan real-time PCR assay for detection of qacEΔ1 gene in Gram-negative bacteria. The transfer of biocide and antibiotic resistance genes by mobile genetic elements is the most common mechanism for rapidly acquiring and spreading resistance among bacteria. The qacEΔ1 gene confers the resistance to quaternary ammonium compounds (QACs). It has also been considered a genetic marker for the presence of class 1 integrons associated with multidrug-resistant (MDR) phenotypes in Gram-negative bacteria. In this study, a TaqMan real-time PCR assay was developed to detect the qacEΔ1 gene in Gram-negative bacteria. The assay has a detection limit of 80 copies of the qacEΔ1 gene per reaction. No false-positive or false-negative results have been observed. Simultaneous amplification and detection of the 16S rRNA gene is performed as an endogenous internal amplification control (IAC). The TaqMan real-time PCR assay developed is a rapid, sensitive, and specific method that could be used to monitor resistance to QACs, the spread of class 1 integrons, and the prediction of associated MDR phenotypes in Gram-negative bacteria.202439395725
5039160.9997Analytical 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
5827170.9997Duplex dPCR System for Rapid Identification of Gram-Negative Pathogens in the Blood of Patients with Bloodstream Infection: A Culture-Independent Approach. Early and accurate detection of pathogens is important to improve clinical outcomes of bloodstream infections (BSI), especially in the case of drug-resistant pathogens. In this study, we aimed to develop a culture-independent digital PCR (dPCR) system for multiplex detection of major sepsiscausing gram-negative pathogens and antimicrobial resistance genes using plasma DNA from BSI patients. Our duplex dPCR system successfully detected nine targets (five bacteria-specific targets and four antimicrobial resistance genes) through five reactions within 3 hours. The minimum detection limit was 50 ag of bacterial DNA, suggesting that 1 CFU/ml of bacteria in the blood can be detected. To validate the clinical applicability, cell-free DNA samples from febrile patients were tested with our system and confirmed high consistency with conventional blood culture. This system can support early identification of some drug-resistant gram-negative pathogens, which can help improving treatment outcomes of BSI.202134528911
2225180.9997Evaluation of the DNA microarray "AMR Direct Flow Chip Kit" for detection of antimicrobial resistance genes from Gram-positive and Gram-negative bacterial isolated colonies. INTRODUCTION: The AMR Direct Flow Chip assay allows the simultaneous detection of a large variety of antibiotic resistance genetic markers. To assess this kit's performance, we use isolated colonies as starting material. The assay has been approved by the European Economic Area as a suitable device for in vitro diagnosis (CE IVD) using clinical specimens. METHODS: A total of 210 bacterial isolates harbouring either one or more antimicrobial resistance genes including plasmid-encoded extended-spectrum β-lactamases (SHV, CTX-M) and carbapenemases (GES, SME, KPC, NMC/IMI, SIM, GIM, SPM, NDM, VIM, IMP, and OXA), mecA, vanA and vanB, and 30 controls were included. RESULTS: The assay displayed a sensitivity and specificity of 100% for all target genes included in the array. CONCLUSION: The AMR Direct Flow Chip Kit is an accurate assay for detecting genes which commonly confer resistance to β-lactams and vancomycin from isolated colonies in culture of Gram-positive and Gram-negative bacteria.201930857832
2229190.9997A pentaplex real-time PCR assay for rapid identification of major beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 directly from bacteria in blood. Introduction. Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3-5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes (Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia.Aim. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant Enterobacteriaceae bacteria (CREs) from the United States.Hypothesis/Gap Statement. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40-60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes.Methodology. Primers and probes were created which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay.Results. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4-8 c.f.u. ml(-1).Conclusion. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.202134878374