Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Rapid Detection of blaKPC-9 Allele from Clinical Isolates.

ABSTRACT: The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the blaKPC-2 from the blaKPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several blaKPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions.

SUBMITTER: Gartzonika K

PROVIDER: S-EPMC8072647 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Rapid Detection of blaKPC-9 Allele from Clinical Isolates.

Gartzonika Konstantina K Bozidis Petros P Priavali Ephthalia E Sakkas Hercules H

Pathogens (Basel, Switzerland) 20210417 4

The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digesti ...[more]

PMID: 33920533

Similar Datasets

mcr-1 in Multidrug-Resistant blaKPC-2-Producing Clinical Enterobacteriaceae Isolates in Singapore.

Project description: Not available

| S-EPMC5038244 | biostudies-literature

Real-time detection of blaKPC in clinical samples and surveillance specimens.

Project description:A real-time PCR assay was developed targeting the bla(KPC) responsible for Klebsiella pneumoniae carbapenemase (KPC)-mediated carbapenem resistance and was validated for testing colonies or enrichment broth cultures. The assay accurately detects KPC-containing strains with high analytical specificity and sensitivity.

| S-EPMC3165576 | biostudies-literature

Rapid Nanopore Sequencing of Plasmids and Resistance Gene Detection in Clinical Isolates.

Project description:Recent advances in nanopore sequencing technology have led to a substantial increase in throughput and sequence quality. Together, these improvements may permit real-time benchtop genomic sequencing and antimicrobial resistance gene detection in clinical isolates. In this study, we evaluated workflows and turnaround times for a benchtop long-read sequencing approach in the clinical microbiology laboratory using the Oxford Nanopore Technologies MinION sequencer. We performed genomic and plasmid sequencing of three clinical isolates with both MinION and Illumina MiSeq, using different library preparation methods (2D and rapid 1D) with the goal of antimicrobial resistance gene detection. We specifically evaluated the advantages of using plasmid DNA for sequencing and the value of supplementing MinION sequences with MiSeq reads for increasing assembly accuracy. Resequencing of three plasmids in a reference Klebsiella pneumoniae isolate demonstrated ∼99% accuracy of draft MinION-only assembly and >99.9% accuracy of assembly polished with MiSeq reads. Plasmid DNA sequencing of previously uncharacterized clinical extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and K. pneumoniae isolates using MinION allowed successful identification of antimicrobial resistance genes in the draft assembly corresponding to all classes of observed plasmid-based phenotypic resistance. Importantly, use of plasmid DNA enabled lower depth sequencing, and assemblies sufficient for full antimicrobial resistance gene annotation were obtained with as few as 2,000 to 5,000 reads, which could be acquired in 20 min of sequencing. With a MinION-only workflow that balances accuracy against turnaround time, full annotation of plasmid resistance gene content could be obtained in under 6 h from a subcultured isolate, less time than traditional phenotypic susceptibility testing.

| S-EPMC5703817 | biostudies-literature

Rapid and simultaneous detection of blaKPC and blaNDM by use of multiplex real-time PCR.

Project description:The increasing incidence of carbapenem nonsusceptibility among clinically important species is of global concern. Identification of the molecular mechanisms underlying carbapenem nonsusceptibility is critical for epidemiological investigations. In this report, we describe a real-time PCR-based assay capable of simultaneously detecting blaKPC and blaNDM, two of the most important carbapenemases, directly from culture in less than 90 min. The assay was validated with blaKPC- and blaNDM-carrying clinical isolates and demonstrated 100% concordance with the Carba NP test.

| S-EPMC3666810 | biostudies-literature

New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates.

Project description:In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.

| S-EPMC538849 | biostudies-literature

Pyrosequencing for rapid detection of extensively drug-resistant Mycobacterium tuberculosis in clinical isolates and clinical specimens.

Project description:Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.

| S-EPMC3911348 | biostudies-literature

Detection of blaKPC and blaNDM carbapenemase genes among Klebsiella pneumoniae isolates in Addis Ababa, Ethiopia: Dominance of blaNDM.

Project description:BackgroundInfections caused by Klebsiella pneumoniae have been difficult to control because of the worldwide emergence of carbapenem-resistant isolates mainly due to carbapenemase production. Information regarding carbapenemase-producing K. pneumoniae is still scarce in Ethiopia. Therefore, the current study aimed to determine the prevalence of carbapenemase-producing K. pneumoniae and to assess the occurrence of blaNDM and blaKPC carbapenemase genes.MethodsA cross-sectional study was conducted from September 2018 to February 2019 at Tikur Anbessa Specialized Hospital, Addis Ababa, Ethiopia. A total of 132 non-duplicate K. pneumoniae isolates were studied. Phenotypic confirmation of carbapenemase production was done by modified Carbapenem Inactivation Method (mCIM). Multiplex PCR was performed for the detection of carbapenemase-encoding genes blaKPC, and blaNDM.ResultsOut of the total 132 K. pneumoniae isolates, 39 (29.6%) were non-susceptible to one or more carbapenems. The prevalence of carbapenemase-producing isolates from the total was 28 (21.2%) with mCIM of which the most dominant gene was blaNDM 26 (92.9%) and one isolate carried blaKPC concomitantly. Carbapenemase-producing K. pneumoniae isolates were 100% non-susceptible to half of the antimicrobials used in the study, including meropenem and ertapenem. Previous use of carbapenems was associated with carbapenemase production (P = 0.004).ConclusionsThe prevalence of carbapenemase-producing K. pneumoniae isolates was worrying in the study area. To our knowledge, the study described the emergence of blaNDM and blaKPC gene carrying K. pneumoniae in Ethiopia for the first time. Further large-scale molecular-based studies, including other carbapenemase genes and sequencing of K. pneumoniae, are warranted to have a clear awareness about the presence of antimicrobial resistance high-risk clones in Ethiopia.

| S-EPMC9045624 | biostudies-literature

Polyphasic characterization of carbapenem-resistant Klebsiella pneumoniae clinical isolates suggests vertical transmission of the blaKPC-3 gene.

Project description:Carbapenem-resistant Klebsiella pneumoniae are a major global threat in healthcare facilities. The propagation of carbapenem resistance determinants can occur through vertical transmission, with genetic elements being transmitted by the host bacterium, or by horizontal transmission, with the same genetic elements being transferred among distinct bacterial hosts. This work aimed to track carbapenem resistance transmission by K. pneumoniae in a healthcare facility. The study involved a polyphasic approach based on conjugation assays, resistance phenotype and genotype analyses, whole genome sequencing, and plasmid characterization by pulsed field gel electrophoresis and optical DNA mapping. Out of 40 K. pneumoniae clinical isolates recovered over two years, five were carbapenem- and multidrug-resistant and belonged to multilocus sequence type ST147. These isolates harboured the carbapenemase encoding blaKPC-3 gene, integrated in conjugative plasmids of 140 kbp or 55 kbp, belonging to replicon types incFIA/incFIIK or incN/incFIIK, respectively. The two distinct plasmids encoding the blaKPC-3 gene were associated with distinct genetic lineages, as confirmed by optical DNA mapping and whole genome sequence analyses. These results suggested vertical (bacterial strain-based) transmission of the carbapenem-resistance genetic elements. Determination of the mode of transmission of antibiotic resistance in healthcare facilities, only possible based on polyphasic approaches as described here, is essential to control resistance propagation.

| S-EPMC7909683 | biostudies-literature

Rapid detection of the CYP2A6*12 hybrid allele by Pyrosequencing technology.

Project description:BackgroundIdentification of CYP2A6 alleles associated with reduced enzyme activity is important in the study of inter-individual differences in drug metabolism. CYP2A6*12 is a hybrid allele that results from unequal crossover between CYP2A6 and CYP2A7 genes. The 5' regulatory region and exons 1-2 are derived from CYP2A7, and exons 3-9 are derived from CYP2A6. Conventional methods for detection of CYP2A6*12 consist of two-step PCR protocols that are laborious and unsuitable for high-throughput genotyping. We developed a rapid and accurate method to detect the CYP2A6*12 allele by Pyrosequencing technology.MethodsA single set of PCR primers was designed to specifically amplify both the CYP2A6*1 wild-type allele and the CYP2A6*12 hybrid allele. An internal Pyrosequencing primer was used to generate allele-specific sequence information, which detected homozygous wild-type, heterozygous hybrid, and homozygous hybrid alleles. We first validated the assay on 104 DNA samples that were also genotyped by conventional two-step PCR and by cycle sequencing. CYP2A6*12 allele frequencies were then determined using the Pyrosequencing assay on 181 multi-ethnic DNA samples from subjects of African American, European Caucasian, Pacific Rim, and Hispanic descent. Finally, we streamlined the Pyrosequencing assay by integrating liquid handling robotics into the workflow.ResultsPyrosequencing results demonstrated 100% concordance with conventional two-step PCR and cycle sequencing methods. Allele frequency data showed slightly higher prevalence of the CYP2A6*12 allele in European Caucasians and Hispanics.ConclusionThis Pyrosequencing assay proved to be a simple, rapid, and accurate alternative to conventional methods, which can be easily adapted to the needs of higher-throughput studies.

| S-EPMC2736934 | biostudies-literature

Universal antimicrobial resistance detection from clinical bacterial isolates using proteomics

Project description:Antimicrobial resistance (AMR) is an increasing challenge for therapy and management of bacterial infections. Currently, antimicrobial resistance detection relies on phenotypic assays, which are performed independently of species identification. On the contrary, phenotypic prediction from molecular data using genomics is gaining interest in clinical microbiology and might become a serious alternative in the future. Although, in general protein analysis should be superior to genomics for phenotypic prediction, no untargeted proteomics workflow specifically related to AMR detection has been proposed so far. In this study, we present a universal proteomics workflow to detect the bacterial species and antimicrobial resistance related proteins in the absence of secondary antibiotic cultivation in less than 4 h from a primary culture. The method was validated using a sample cohort of 7 bacterial species and 11 AMR determinants represented by 13 protein isoforms which resulted in a sensitivity of 92 % (100 % with vancomycin inference) and a specificity of 100 % with respect to AMR determinants. This proof-of concept study demonstrates the high potential of untargeted proteomics for clinical microbiology.

2021-11-25 | PXD022425 | Pride